complications—hypoglycemia ada.pdf

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COMPLICATIONS—HYPOGLYCEMIA


Guided Audio Tour: Understanding Hypoglycemia in Diabetes (Posters 378-Pto 385-P), see page 15.

& 378-PHistory of Severe Hypoglycemia and Score on Clarke Questionnaire is Associated With Blunted Counterregulatory Response to Experi-mental Hypoglycemia in Patients With Type 1 DiabetesAMIR MOHEET, ANJALI KUMAR, LISA CHOW, LYNN E. EBERLY, ELIZABETH R. SEAQUIST, Minneapolis, MN

Clarke questionnaire is widely used to identify patients with type 1 dia-betes (T1DM) who have impaired hypoglycemia awareness (IHA). It consists of 8 questions about hypoglycemia(HG) symptoms and past episodes of HG; scores of 4 or more imply IHA. While the accuracy of CQ has been exam-ined using patient reported HG and HG associated symptom scores, the relationship between HG-induced counterregulatory (CR) responses, which are responsible for the generation of many HG associated symptoms, and CQ scores has not been examined. In this study we tested the hypothesis that there is a signifi cant relationship between high CQ scores and reduced CR response to HG.18 subjects (8F/10M, 42 ± 12 yrs, BMI 26.7 ± 3.9 kg/m2,mean ± SD) with T1DM and IHA (defi ned as score >4 on CQ) were examined. They underwent a 2 hour hyperinsulinemic (2.0 mU/kg/min) clamp with a glucose target of 50 mg/dl. During HG, mean glucose was maintained at 48 ± 5 mg/dl. Blood was collected at baseline and every 10 minutes during HG for measurement of CR hormones. During HG, epinephrine (Epi) ranged within 15-584 pg/dl and norepinephrine (Nepi) 38.3-528.5 pg/dl.Using Spearman’s correlation, a signifi cant inverse relationship was found between CQ score and mean Epi (rs = -0.52, p =0.03) and mean Nepi (rs = -0.42, p = 0.08) dur-ing HG. Using Wilcoxon Two-Sample tests, a signifi cant inverse relationship was found for a history of severe HG within the last year (question 4 on CQ) with mean Epi (p = 0.009) and with mean Nepi (p = 0.003) responses, but not with other CR hormones. History of moderate HG (question 3 on CQ) was not found to be associated with CR responses. These results demonstrate that history of severe HG and high total score on CQ is signifi cantly related to re-duced CR response to HG in patients with T1DM. Therefore, such responses on the CQ may indicate those patients with the most profound IHA, which can be of value in both the research and the clinical setting.

& 379-PAntecedent Hypoglycemia does not Impair the Glycemia-Increasing Effect and Counterregulatory Responses of a 10-Second Sprint in People With Type 1 DiabetesPAUL A. FOURNIER, RAYMOND J. DAVEY, NIRU PARAMALINGAM, ELIZABETH A. DAVIS, TIMOTHY W. JONES, Perth, Australia

A 10-second sprint can not only increase blood glucose concentrations when plasma insulin are at basal levels, but also prevent blood glucose lev-els from falling post-exercise if performed after moderate intensity exer-cise in people with type 1 diabetes mellitus (T1D), thus providing a means of reducing the risk of exercise-mediated hypoglycemia. Since an episode of hypoglycemia attenuates the counterregulatory responses to a sub-sequent bout of moderate-intensity exercise, the aim of this study was to investigate whether antecedent hypoglycemia inhibits the counterregula-tory responses and glycemia-increasing effect of a 10-second sprint. Eight individuals with T1D (6M, 2F; age 21.3±2.7 years; BMI of 25.9±5.1 kg.m-2;HbA1c of 7.7±1.0%; mean±SD) were subjected in the morning of different days to either a one-hour episode of hypoglycemia or euglycemia by infus-ing insulin at a constant rate of 80 mU.m-2.min-1 and adjusting the infusion rate of a 20% dextrose solution. Immediately after,all participants were ex-posed to a three-hour insulin-only euglycemic clamp before they performed a 10-second sprint on a cycle ergometer under basal insulin conditions. During the morning clamps, blood glucose levels were signifi cantly differ-ent between the hypoglycemic (2.8±0.3 mmol/L) and euglycemic (5.4±0.5 mmol/L; p<0.05) conditions. In response to the afternoon sprint, blood glu-cose increased signifi cantly, reaching similar maximal levels at 45 minutes post-sprint (6.5±1.1 and 6.5±0.7 mmol/L in the hypoglycemia and euglycemia treatments, respectively). Sprinting resulted in a signifi cant post-exercise increase in plasma catecholamines, cortisol and growth hormone levels in both treatments (p<0.05). In conclusion, antecedent hypoglycemia does not affect the glycemia-increasing effect of a 10-second sprint in individuals

with T1D, thus supporting further the benefi ts of sprinting in hypoglycemia prevention.

Supported by: National Health and Medical Research Council of Australia

& 380-PPrediction of Severe Inpatient Hypoglycemia Using Blood Glucose TrendsRANEE R. LLEVA, SANDI-JO GALATI, KARRIE C. HENDRICKSON, JANIS E. BOZZO, ZHENQIU LIN, SILVIO E. INZUCCHI, New Haven, CT, New York, NY

Inpatient hypoglycemia (hypo) is associated with higher mortality. An AACE/ADA consensus on hospital glucose management suggests reassess-ment of anti-hyperglycemic regimens with blood glucose (BG) <100mg/dL. The value of point-of-care (POC) BG monitoring in predicting inpatient hypo is not known.We examined POC BGs in adult DM patients (pts) on general med-ical-surgical wards at our hospital over 1 year. Of the 480 pts with at least 1 BG<50mg/dL (‘severe hypo’), 368 experienced their event after hospital day 2, had at least 2 BGs recorded during those 48 hours, and were treated with insulin/sulfonylureas. DM controls (2379) were on similar therapies and had no documented BG<50mg/dL during their stays. Pts were matched for age, length of stay, service, and number of BG tests; 211 pairs were identi-fi ed. All BGs 48 hrs prior to the hypo event in the 211 cases, as well as a randomly selected 48-hr period of BGs in controls, were categorized into fi ve glycemic windows: <60, <70, <80, <90, & <100mg/dL. Chi-square tests were then used to compare the 2 groups.The proportion with BG<100mg/dL was higher in cases vs. controls. The relative frequencies became increasingly disparate as antecedent BGs fell <90mg/dL; values <60mg/dL occurred >10 times more frequently in cases vs. controls.We conclude that inpatients with severe hypo frequently have BGs in the low-normal/mild-moderately hypo range in the 48 hours preceding their events. BG <100mg/dL should serve as a marker to consider adjustment of the anti-hyperglycemic regimen, sup-porting the AACE/ADA recommendations. The need to adjust therapy seems more compelling the further the BG falls <100mg/dL.

Percent of pts with low-normal/mildly hypo BGs preceding severe hypo eventAntecedent BGs (mg/dL)

<100 <90 <80 <70 <60Severe hypo 60.2% 46.0% 33.7% 22.8% 11.4%No severe hypo 50.2% 25.1% 16.1% 6.2% 1.0%P-value 0.0398 <0.0001 <0.0001 <0.0001 <0.0001

& 381-PHypoglycemia Alert Dogs—Innovative Assistance for People With Type 1 DiabetesDANA S. HARDIN, DUSTIN HILLMAN, JENNIFER CATTET, Indianapolis, IN, West Lafayette, IN

Hypoglycemia (hypo) is the most common side effect of insulin therapy in people with type 1 diabetes (T1D). Despite education directed to prevent and treat it, hypo continues to cause disruption in lives of people with T1D. For the past 10 years, the Indiana Canine Assistance Network (ICAN) has trained dogs for mobility assistance for people with disabilities. This abstract chronicles results of training our fi rst hypo alert dog. A 2-year-old mobility assistance Labrador/Golden Retriever dog was trained to recognize hypo. Perspiration samples were collected from patients during hypo (BG <65 mg/dL) and normoglycemia. Samples were blinded in containers placed on a Lazy Susan apparatus. The dog was rewarded for appropriate recognition of the hypo samples and trained to alert by nudging the trainer’s arm. After the dog was introduced to the potential owner, the owner’s perspiration samples were used to complete training before placement. A questionnaire consist-ing of validated and nonvalidated components was used to assess frequency and severity of hypo as well as emotional responses at 2 weeks pre- and 6 months post-dog placement.Figure 1 displays 2 of the questionnaire results. The correlation between alerts and hypo (by self-monitored blood glucose) was greater than 98% by 3 months post-dog placement.We demonstrated clear impact on hypo in a patient with T1D. These preliminary qualitative (n=1) fi ndings support our continued training (10 additional dogs are currently in training) and monitoring. Additional studies are planned to try to elucidate the specifi c biomarkers scented by the dogs.

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Supported by: Eli Lilly and Company

& 382-PEffects of Antecedent GABA A Receptor Activation on Counterregu-latory Responses to Subsequent Exercise in Type 1 DiabetesMAKA S. HEDRINGTON, MAIA MIKELADZE, NINO G. JOY, CHERYL YOUNG, DONNA B. TATE, LINDSAY PULLIAM, LISA M. YOUNK, IAN C. DAVIS, CHELSEA YOUNK, STEPHEN N. DAVIS, Baltimore, MD

The effects of the Benzodiazepine - GABA(A) receptor agonist alprazolam (Alp) on physiologic responses during next day exercise in type 1 diabetes (T1DM) is unknown. To test the hypothesis that Alp would blunt counter-regulatory responses during subsequent exercise, 30 non-obese (16M / 14F) T1DM (HBA1c 7.5±0.3) were studied during separate 2 day protocols. Day 1 consisted of morning and afternoon 2 hr euglycemic (eugly) or hypoglycemic (hypo) clamps with or without 1 mg Alp given 30 min before each clamp. Day 2 consisted of 90 min euglycemic cycling exercise at 50% VO2 max. Tritiated glucose was used to measure glucose kinetics. Despite equivalent day 2 insulin (13±1 μU/ml) and glucose levels (95±1.6 mg/dl), plasma epinephrine, norepinephrine, glucagon, cortisol and growth hormone responses (fi nal 15 minutes) were reduced similarly following either Alp or hypo as compared to eugly control. Lipolysis (glycerol, NEFA) and endogenous glucose produc-tion (EGP) were also reduced during exercise similarly following Alp or hypo (Table 1). In summary, selective activation of GABA(A) receptors with Alp can reduce key neuroendocrine, autonomic nervous system and metabolic counterregulatory responses to subsequent exercise in patients with T1DM. We conclude that activation of GABA (A) receptors can result in widespread counterregulatory failure similar to antecedent hypo during subsequent ex-ercise in individuals with T1DM.

Table 1 Mean ±SEM *p<0.05 compared to eugly controlDay 1eugly+Alp Day 1eugly Day 1hypo

Epinephrine (pg/mL) 60±9* 112±18 58±10*Norepinephrine (pg/mL) 321±63* 670±98 416±48*Glucagon (pg/mL) 4±2* 9±2 4±1*Growth Hormone (ng/mL) 5±2* 9±1 3±1*Glycerol (μmol/L) 61±12* 106±17 63±9*NEFA (mmol/L) 83±56* 240±59 71±38*

EGP (mg/kg/min) 2.5±0.4* 3.9±0.5 3.1±0.2

& 383-PRecurrent Hypoglycemia Reduces Adrenal Epinephrine Release Evoked by Splanchnic Nerve StimulationBRANLY O. ORBAN, VANESSA H. ROUTH, BARRY E. LEVIN, JOSHUA R. BERLIN, Newark, NJ, East Orange, NJ

Tight glycemic control with intensive insulin therapy is used to prevent diabetic complications due to hyperglycemia. However, intensive insulin therapy can lead to insulin induced hypoglycemia (IIH). Recurrent hypogly-cemia (RH) blunts the body’s counterregulatory response (CRR) to subse-quent hypoglycemia leading to decreased epinephrine (Epi) release and an impaired ability to restore euglycemia. Most studies of RH havefocused on the contribution of the brain to reduced Epi release, but whether RH affects adrenal medullary function has not been investigated.In this study, IIH (56±5 mg/dL) was produced (1 U/kg, sc) in 8-wk old male Sprague Dawley rats daily for 3 days (RH). Control rats (Con) received saline injections. In one ex-periment, all animals received insulin on day 4 and plasma glucose, Epi, and corticosterone (Cort) were monitored. The time course of glucose decline was identical, but the elevations in plasma Epi at 60 and 90 min and CORT at 90 and 120 min after insulin injection were signifi cantly smaller in RH rats (P<0.05). Thus, RH blunted the CRR in this protocol.In a second experi-ment, on day 4, rats were anesthetized, and an electrode was placed on the

splanchnic nerve branch innervating the left adrenal gland. The left renal vein was catheterized and the outfl ow of this gland was isolated from the systemic circulation to determine Epi release in response to nerve electrical stimulation. Before and during stimulation, mean arterial pressure and heart rate in Con and RH groups were similar. Epi release produced by sustained (5 min) electrical stimulation at 30Hz was less in the RH animals (P<0.05). Epi release produced by short stimulations (30 sec) over a range of frequencies (1Hz to 100Hz) tended to be less in the RH group. Total Epi content, measured in right adrenal glands, was similar in Con and RH groups. These results suggest that RHimpairs intrinsic adrenal medullary Epi release in addition to blunting central stimulation of sympathetic outfl ow.

Supported by: NIH (5F31DK086681-02)

& 384-PTo What Extent Does Exercise Reduce Sympathoadrenal and Symp-tomatic Response to Subsequent Hypoglycemia in Humans?NADIA KHOURY, W. TODD CADE, AMY C. HAUCH, TAMARA HERSHEY, ANA MA-RIA ARBELAEZ, St. Louis, MO

Episodes of hypoglycemia following play and exercise in patients with type I diabetes are frequent clinical complaints. Exercise (~50% V02 max x 90 min x 2) has been reported to reduce sympathoadrenal and symptomatic responses to hypoglycemia the following day in nondiabetic adults (Am J Physiol 280: E908, 2001) and adults with type 1 diabetes (Diabetes 53:1798, 2004). However, we found that higher intensity exercise at ~70% V02 max x 90 min x 2 did not reduce the plasma epinephrine or symptomatic responses to hypoglycemia at 55mg/dL or 45 mg/dL the following day in healthy young nondiabetic adults (n=9). In this study, we observed that an intravenous glu-cose infusion of an average of ~10 mg·kg-1·min-1 during the second bout of exercise was required to maintain plasma glucose concentrations at 90 mg/dL. Thus, to test the hypothesis that this glucose infusion prevented attenua-tion of the responses to hypoglycemia on Day 2, we repeated the study with-out glucose infusion during exercise (n=8). Although mean plasma glucose concentrations declined to a nadir of 46 + 2 mg/dL during the second bout of exercise (rising to an average of 69 + 2 mg/dL), the plasma epinephrine and symptomatic responses to hypoglycemia were not reduced the follow-ing day. Thus, in 17 studies, by repeated measures ANOVA, there was no signifi cant difference in plasma epinephrine and symptomatic responses to hypoglycemia following antecedent exercise or rest.

Day 1 Day 2Condition Glucose

(mg/dL + SE)Epinephrine(pg/mL + SE)

SymptomScore

Rest 91 + 1 35 + 7 2 + 1Exercise 90 + 1 72 + 22 3 + 1Rest 56 + 1 233 + 32 8 + 2Exercise 56 + 1 230 + 41 8 + 2Rest 47 + 1 539 + 66 17 + 3Exercise 47 + 1 477 + 62 15 + 2

These data indicate that in healthy individuals any effect of exercise to reduce sympathoadrenal and symptomatic responses to subsequent hypo-glycemia is small.

Supported by: Children’s Discov Inst 1KL2RR024994-01 and WU Mallinckrodt Inst Radiol Pilot

& 385-PBrain Glycogen Is Important for Motor Learning during HypoglycemiaSTACI A. WEAVER, SHANNON N. SHARPE, BARTHOLOMEW A. PEDERSON, Muncie, IN

Hypoglycemia is the major obstacle preventing the benefi ts associated with intensive insulin therapy in individuals with diabetes. An acute effect of hypoglycemia is compromised cognitive function, including impaired mem-ory. Brain glycogen, the only signifi cant energy store in the brain, is utilized during hypoglycemia and has been hypothesized to be important for learning under normal conditions. The objective of this study was to utilize geneti-cally engineered mice (MGSKO/GSL30) with defi cient brain glycogen stores to determine the importance of brain glycogen for learning under hypogly-cemic conditions. Our hypothesis was that if brain glycogen is important for learning during hypoglycemia, mice defi cient in brain glycogen would exhibit impaired learning during hypoglycemia. Wild type and MGSKO/GSL30 mice were subjected to a rotarod motor learning protocol for 4 trials over a course of 8 days. Latency to fall was the measurement of learning. For trial 1 (day 1), mice were trained in the fed state. For trial 2 (day 2) and trial 3 (day 6),

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mice were fasted and injected with either vehicle or with insulin, to induce hypoglycemia (blood glucose ~3.5 mM). In trials 1, 2 and 3, fed and vehicle in-jected wild type and MGSKO/GSL30 mice performed similarly to each other. During trials 2 and 3, hypoglycemia impaired performance similarly in both genotypes. For trial 4 (day 8), mice were tested in the fed state. Wild type mice that had been hypoglycemic during trials 2 and 3 had similar latency in trial 4 as compared to wild type mice that were previously fed or vehicle-injected. However, MGSKO/GSL30 mice that had been hypoglycemic during trials 2 and 3 had decreased latency in trial 4 as compared to both wild type mice subjected to the same conditions and MGSKO/GSL30 mice not previ-ously hypoglycemic. These data suggest that brain glycogen is not required for motor learning in normoglycemic conditions but is important for motor learning during hypoglycemia.

Supported by: DK078370

Guided Audio Tour: Stop Hypoglycemia (Posters 386-P to 393-P), see page 15.

& 386-PEffect of Antecedent Exercise on the Glycemia-Increasing Effect and Counterregulatory Responses of a Short SprintPAUL A. FOURNIER, TARA D. JUSTICE, GRETA L. HAMMER, RAYMOND J. DAVEY, NIRU PARAMALINGAM, KYM J. GUELFI, ELIZABETH A. DAVIS, TIMOTHY W. JONES, Perth, Australia

Performing a short sprint under basal insulin conditions in individuals with type 1 diabetes mellitus (T1D) not only increases blood glucose levels, but also prevents glycemia from falling post-exercise if performed immediately after moderate-intensity exercise, thus reducing the risk of exercise-mediat-ed hypoglycemia. Since moderate-intensity exercise can blunt the counter-regulatory hormone responses to an identical bout of exercise performed hours later in the day, the aim of this study was to investigate whether ante-cedent exercise can inhibit the counterregulatory responses and glycemia-increasing effect of sprinting in non-diabetic individuals. Eight non-diabetic males were subjected to a familiarization session and two trials (adminis-tered following a counterbalanced design) during which they either rested (CON) or cycled at 65% VO2peak for 60 min (EX) before being infused with [6,6-2H]glucose. Three hours later, they performed a 30-sec maximal sprint on a cycle ergometer. In response to sprinting, blood glucose increased signifi -cantly and reached similar peak levels at 10 min of recovery in EX and CON, but fell more rapidly afterward in EX. This rise in glycemia may be explained by the higher glucose rate of appearance (Ra) compared with glucose rate of disappearance (Rd; p < 0.05). Plasma insulin levels peaked in both trials after 30 min of recovery, but were lower in EX at 45 min (p = 0.042) of recovery. Growth hormone levels increased during recovery, but to a lesser extent in EX (p<0.05). The post-exercise rise in cortisol and norepinephrine levels did not differ between trials. In conclusion, antecedent exercise does not at-tenuate the glycemia-increasing effect of a sprint performed hours later, but accelerates the subsequent decline in blood glucose levels. It remains to be seen whether antecedent exercise affects the glycaemia-increasing effect of sprinting in individuals with T1D.

Supported by: National Health and Medical Research Council of Australia

& 387-PProspectively Planned Meta-Analysis Comparing Hypoglycemia Rates of Insulin Degludec With Those of Insulin GlargineROBERT E. RATNER, STEPHEN GOUGH, CHANTAL MATHIEU, STEFANO DEL PRATO, BRUCE W. BODE, HENRIETTE MERSEBACH, LARS ENDAHL, BERNARD ZINMAN, Hyattsville, MD, Oxford, United Kingdom, Leuven, Belgium, Pisa, Italy, Atlanta, GA, Søborg, Denmark, Toronto, ON, Canada

Insulin degludec (IDeg), a new basal insulin that forms soluble multi-hexamers after s.c. injection, has an ultra-long and stable glucose-lowering effect, with low day-to-day variability. The objective of this pre-specifi ed analysis was to compare the rate of hypoglycemia with IDeg vs. insulin glargine (IGlar) across trials. Hypoglycemia was defi ned as rates of self-reported confi rmed hypoglycemia (PG<56 mg/dL or severe hypoglycemia requiring assistance) and nocturnal confi rmed hypoglycemia (00:01-05:59 incl.). Rates were analyzed with a negative binomial regression model on patient level data. All open-labeled, randomized, treat-to-target phase 3a trials of 26 or 52 weeks were included in which once-daily IDeg (n=2899) was compared to IGlar (n=1431) in T1DM (2 trials) and T2DM (5 trials). IDeg resulted in statistically signifi cantly lower rates of overall and nocturnal hy-poglycemia compared to IGlar in T2DM, and for nocturnal hypoglycemia in T1DM (Table). Signifi cant benefi ts of IDeg were also evident for the pooled

population (T1DM+T2DM). Analyses of the maintenance period (stable gly-cemia and insulin dose [obtained from 16 weeks onward]), demonstrated more pronounced benefi ts of IDeg. Additional heterogeneity analysis re-vealed signifi cantly less risk of severe hypoglycemia with IDeg vs. IGlar in insulin-naïve T2DM (rate ratio 0.14 [0.03; 0.70]95%CI). In conclusion, this meta-analysis demonstrates that treatment with IDeg provides important clini-cal advantages with signifi cantly lower rates of both overall and nocturnal hypoglycemia than IGlar at similar levels of A1c.

Table 1: Meta-analysis of confi rmed hypoglycemiaHypoglycemiarate ratio

(IDeg/IGlar)Nocturnal hypoglycemiarate

ratio(IDeg/IGlar)

Totalperiod

Maintenanceperiodb

Total period

Maintenanceperiodb

T2DM 0.83 [0.74; 0.94]*

0.75[0.66; 0.87]*

0.68[0.57; 0.82]*

0.62[0.49; 0.78]*

T2DM-insulin-naïve 0.83 [0.70; 0.98]*

0.72[0.58; 0.88]*

0.64[0.48; 0.86]*

0.51[0.36; 0.72]*

T1DM 1.10 [0.96; 1.26]

1.020.88; 1.19]

0.83[0.69; 1.00]*

0.75[0.60; 0.94]*

T1DM+T2DMa 0.91[0.83; 0.99]*

0.84[0.75; 0.93]*

0.74[0.65; 0.85]*

0.68[0.58; 0.80]*

a The pooled analysis was the primary endpoint; b From 16 weeks onwards; [ ] = 95% confi dence intervals; * p<0.05

Supported by: Novo Nordisk A/S

& 388-PType of Insulin and Age are Predictors of Hospitalization due to Se-vere Hypoglycemia: The EpiHypo StudySARI MÄKIMATTILA, PASI KORHONEN, JARI HAUKKA, FABIAN HOTI, PIA PA-JUNEN, TERO SAUKKONEN, Espoo, Finland

Incidence and recurrence of severe hypoglycemic (SH) events among patients with diabetes mellitus (DM) was evaluated in a retrospective nation-wide reg-ister-based linkage study in Finland. SH was defi ned as a hospitalization or a secondary health care visit due to DM with severe hypoglycemia (ICD E10.00 or E11.00). Total population (n=140,035) comprised patients who purchased insulin during 2000-2009 and were followed-up for SH events until end of year 2009 or death. The present analysis comprised those 77,046 patients who had not used insulin glargin (IGla), insulin detemir (IDet) or NPH insulin (NPH) before year 2000. Stratifi ed incidence rates with 95% CIs were calculated. Hazard ratios (HR) were estimated by Cox’s proportional hazards model. 9,716 SH events were identifi ed. Type of DM (type 1 or 2) was not associated with risk of SH. Compared to IGla, risk of SH was lower during use of IDet (HR 0.76, CI 0.67-0.87), and higher during use of NPH (HR 1.19, CI 1.11-1.28) (Fig. 1 upper panel). Female gender predicted lower risk (HR 0.93, CI 0.88-0.98), and increasing age predicted higher risk of SH (Fig.1 lower panel). Risk of SH recurrence was lower during IDet (HR 0.60, CI 0.52-0.69), and higher during NPH (HR 1.58, CI 1.46-1.71) compared to IGla.In conclusion, our data shows that increasing age and type of long-acting insulin are predictors of hospitalization due to SH. Risk of hospitalization due to SH could potentially be modifi ed by selection of long-acting insulin.

Figure 1: Occurrence of the fi rst hospitalization or secondary care visit due to SH by type of insulin (upper panel) and by age group (lower panel).

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& 389-PHypoglycemia in Diabetes Requiring Emergency Services Interven-tion: Patient Characteristics and MortalityALICE C. HUSKINSON, MATTHEW J. BOTTOMLEY, LINDA CLAPHAM, CATHRYN JAMES, SAAD H. ALZAHRANI, RHODRI KING, PETER J. GRANT, RAMZI A. AJ-JAN, Leeds, United Kingdom

Hypoglycemia is a common complication of diabetes treatment and can be life-threatening. The aim of this work was to characterize individuals with diabetes sustaining severe hypoglycemia requiring emergency services inter-vention, covering a population of approximately 28,000 diabetes subjects over a period of 5.5 years.A total of 1312 episodes were recorded in 858 subjects, with 55% occurring in individuals with type 1 diabetes (T1DM), 39% in type 2 diabetes (T2DM) and a minority unclassifi ed. Two third of T1DM subjects were males, whereas gender distribution was equal amongst T2DM individu-als. Mean age (range) of T1DM subjects was49 yrs (8-95), whereas T2DM subjects were older at 73 yrs (32-97). Of the T2DM subjects, 27% were not on insulin treatment with the majority receiving a sulphonylurea (90%). Mean capillary blood glucose (±SEM) at arrival of emergency services was lower in T1DM compared with T2DM subjects (1.71±0.03 mmol/L and 1.96±0.04 mmol/L, respectively; p<0.001). In T2DM individuals, the severity of hypogly-cemia in insulin users and non-insulin users was similar at 1.93±0.05 mmol/L and 1.98±0.06 mmol/L, respectively (p=0.51). HbA1c was higher in T1DM than T2DM subjects (66±1 mmol/mol and 60±1 mmol/mol, respectively; p<0.001), and insulin users in T2DM had signifi cantly higher HbA1c compared with those not on this therapy. Around one third of patients self-reported hypoglycemic unawareness. Mortality within one year of the severe hypoglycemic event was 17.1% in the whole population, which is comparable to the one year mor-tality of 15.6% following myocardial infarction in diabetes subjects in our area.This observational study demonstrates that severe hypoglycemia is a common complication in both T1DM and T2DM, and frequently occurs in non-insulin treated individuals. We show high mortality in the fi rst year following severe hypoglycemia, although it is unclear at present whether low blood glucose directly contributes to death in these individuals.

& 390-PSpontaneous Versus Insulin Induced Hypoglycemia and Mortality in Hospitalized PatientsRAJESH GARG, APOORVA TRIVEDI, SHELLEY HURWITZ, ALEXANDER TURCHIN, Boston, MA

Hypoglycemia is a risk factor for increased mortality in hospitalized pa-tients. However, most studies suggest it to be a marker rather than a cause of increased mortality. Therefore, insulin induced hypoglycemia may have lower mortality than spontaneous hypoglycemia. We investigated the re-lationship between hypoglycemia and mortality and the impact of insulin treatment on this relationship. Data were obtained from electronic medical records of patients admitted between 4/1/2008 and 11/30/2010. Patients with one or more blood glucose values 50 mg/dl from the point-of-care glucose testing were considered hypoglycemic. Patients treated with insulin were assumed to have insulin induced hypoglycemia. No patient was re-ceiving any non-insulin anti-diabetic agents. Age, gender and race matched patients with blood glucose values >70 mg/dl were included as the non-hypoglycemic control group. Thus, we had four groups of patients: 1) non-insulin treated hypoglycemia (NIH) (N=135); 2) insulin treated hypoglycemia

(IH) (N=961); 3) non-insulin treated control (NIC) (N=1058) and 4) insulin treated control (IC) (N=736). Insulin treated patients were older: age 58 ± 15, 60 ± 15, 58 ± 16 and 63 ± 14 years in NIH, IH, NIC and IC groups respectively. Death during hospitalization was 35 %, 20%, 1% and 5 % in the four groups respectively. Death rate was signifi cantly higher in hypoglycemic compared with control groups and in NIH compared with IH (p <0.0001) but lower in NIC compared with IC group (P <0.0001). After controlling for age, gender and Charlson comorbidity score all pair-wise comparisons for death rate were still signifi cant (p<0.001). On multivariable logistic regression analysis, odd ratio of death in IH relative to NIH was 0.48 [95% CI: (0.33, 0.72), p<0.001]. These data suggest a higher mortality rate among patients who develop hypoglycemia, spontaneous as well insulin induced, although patients with insulin induced hypoglycemia have lower mortality than those with sponta-neous hypoglycemia.

& 391-PThe Impact of Insulin Type on Severe Hypoglycemia Events Requir-ing Inpatient and Emergency Department Care in Patients With Type 2 DiabetesMATTHEW SOLOMON, SANDEEP VIJAN, FELICIA FORMA, RYAN CONRAD, NICK SUMMERS, DARIUS LAKDAWALLA, Stanford, CA, Ann Arbor, MI, Bridgewater, NJ, Santa Monica, CA, Los Angeles, CA

Although clinical trials suggest that basal analogues have a lower risk of hypoglycemia than other forms of insulin, conclusive data are lacking. We evaluated the risk from different insulin types on severe hypoglycemia (SHG) events requiring inpatient (IP) or emergency department (ED) care in patients with Type 2 diabetes (DM).Using a large database of privately-insured work-ing-age US adults from 1998-2009, we conducted a retrospective cohort study of DM patients newly started on insulin after at least 6 months of treatment with oral anti-diabetic medications (OADs). We measured SHG events after insulin initiation occurring in the IP or ED setting. Patients were classifi ed into an insulin group based on the most frequently used insulin type prior to the SHG event. Multivariate Cox models assessed the association between insu-lin type and the risk of subsequent SHG events, controlling for demographics, comorbidities, and the number and type of concomitantly prescribed OADs.The cohort included 8,626 patients (age 53.5 yrs; 55% female) with DM followed for an average of 4.0 yrs after insulin initiation. Of these, 161 (1.9%) had a SHG event at an average of 3.1 yrs after insulin initiation. Patients with SHG events were slightly older (56.1 yrs vs 53.4 yrs), used a similar number of OADs (1.1 vs 1.2), and were more likely to have multiple comorbidities compared to those without SHG events. In multivariate Cox models, premixed insulin (HR 2.07; p<0.01), isophane insulin (NPH) (HR 2.01; p<0.01), and rapid acting insulin (HR 2.78; p< 0.01) had a higher risk of SHG events compared to glargine, and detemir showed a non-signifi cant trend toward more SHG events (HR 1.20; p=0.73). These results were unchanged in sensitivity analyses of more inclu-sive defi nitions of SHG events.Among patients with DM who initiate insulin therapy, we found glargine users had lower rates of SHG events requiring IP or ED care compared to users of other insulin formulations.

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& 393-PHigher Levels of Cytokines and Glycemic Variability are Associated With Hypoglycemia and Increased Mortality in Medical Intensive Care Unit PatientsFRANCISCO PASQUEL, CHRISTOPHER NEWTON, FARNOOSH FARROKHI, SAU-METH CARDONA, DAWN SMILEY, LIMIN PENG, GUILLERMO UMPIERREZ, Atlanta, GA

The prognostic importance of increased levels of infl ammatory cytokines on glycemic variability and outcome in critically ill patients with hypergly-cemia is not known. Accordingly, we determined the association between infl ammatory cytokines (IL6, TNF- ), oxidative stress markers (DCF, TBA), glycemic variability (determined by standard deviation of BG measures), hy-poglycemic events and mortality in 150 medical ICU patients (age: 58±12 yr; male: 54%; known diabetes: 54%, [mean ±SD]) treated with continuous insu-lin infusion (CII). The mean BG before CII was 189±60 mg/dL and decreased to a mean BG of 121±23 mg/dL during CII. The overall mortality was 24%. Non-survivors had higher levels of baseline IL-6 and TNF- compared to survivors (both, p=0.01). We found mortality to be strongly associated with log IL6 values (died vs. lived: 3.9±1.3 vs. 3.1±1.6 log pg/mL, p=0.01) and log TNF- (3.0±1.0 vs 2.6±0.6 log pg/mL, p=0.01), but not with admission or hos-pital BG concentrations (P=NS). Glycemic variability correlated with plasma IL-6 (p=0.001) and TNF- (p=0.028) levels but not with oxidative stress mark-ers (p=NS). Patients with hypoglycemia (<70mg/dL) during CII had a higher average log TNF- levels during CII (2.9±0.8 vs 2.7±0.6 log pg/mL; p=0.029), and a longer length of stay (23±18 days vs 17±25 days, p=0.005) compared to patients without hypoglycemia. Patients with more hypoglycemic events (0 vs 1-2 vs >2) had higher average log TNF- during CII levels (2.7±0.6 vs 2.9±0.95 vs 2.9±0.4 log pg/mL; p=0.036). In addition, non-survivors with hypoglycemia had higher glycemic variability than survivors with hypoglyce-mia (24.4±8 vs 31.1±12 mg/dL; p=0.023). In summary, higher plasma levels of infl ammatory cytokines (IL6 and TNF- ) and glycemic variability were posi-tively correlated with increased risk of hypoglycemic events and hospital mortality in critically ill patients with hyperglycemia in the ICU.

Supported by: sanofi -aventis

COMPLICATIONS—MACROVASCULAR—ATHEROSCLEROTIC CARDIOVASCULAR DISEASE AND

HUMAN DIABETES

Guided Audio Tour: Diabetes and the Macrovascular Tree—Pathology, Function, and Assessment (Posters 394-P to 401-P), see page 17.

& 394-PThe Detection of Ischemia in Asymptomatic Diabetics (DIAD) Study Risk ScoreDEBORAH A. CHYUN, CHARLES M. CLELAND, LAWRENCE H. YOUNG, SILVIO E. INZUCCHI, JANICE E. DAVEY, FRANS J. WACKERS, FOR THE DIAD STUDY INVES-TIGATORS, New York, NY, New Haven, CT

Coronary heart disease (CHD) is the leading cause of death in type 2 diabetes (T2DM). Current risk score calculators typically do not include diabetes-specifi c prognostic factors and do not perform well in this popula-tion. Validated diabetes-specifi c risk calculators are needed to estimate CHD risk in this population. We analyzed data from the Detection of Ischemia in Asymptomatic Diabetics (DIAD) study in order to identify factors associ-ated with cardiac events (cardiac death, acute coronary syndromes, heart failure, and revascularization) and to develop and validate a risk score for older adults with T2DM. The mean age of the 1,119 subjects (54% male) was 60.7±6.6 years, with mean T2DM duration of 8.5±7 years, mean HbA1c, 7.1±1.5% and 23% using insulin. During 5 years of follow-up, there were 94 (8.4%) cardiac events. Cox proportional hazards regression revealed the fol-lowing independent predictors of events: black ethnicity (HR 0.37, p=.01); T2DM duration (HR 1.07 per year, p<.0001); HbA1c (HR 1.26 per 1% increase, p=.0008); insulin use (HR 0.51; p=.01); extremity numbness (HR 1.87, p=.003); the lowest quartile of Valsalva ratio (VR) (HR 1.58, p=.04); highest quartile of pulse pressure (HR 2.04, p=.001); family history of CHD (HR 1.61, p=.05); waist:hip ratio (HR 1.04 per each .01 increase; p=.003); no regular physi-cal activity (HR 1.53, p=.05); and minor ST-T-wave abnormalities on ECG (HR 1.74, p=.03). A risk score equation was developed based on comparison to a low risk subject; equations were developed with and without VR and ECG data as these are often not routinely available. Internal validation of the model, using 250 bootstrap resamples, revealed adequate predictive ability of the models (Somer’s D rank correlation and concordance index). These

results suggest that incorporation of diabetes-related factors is important in cardiac risk stratifi cation in older patients with T2DM. Accurate CHD risk prediction in the T2DM population may enhance care delivery and improve outcomes.

& 395-PUpdated UKPDS Risk Engine that Estimates Primary and Secondary Cardiovascular Disease Risk in People With Recently-Diagnosed or Established Type 2 DiabetesRUTH L. COLEMAN, RICHARD J. STEVENS, RURY R. HOLMAN, Oxford, United Kingdom

The UKPDS Risk Engine is a diabetes-specifi c risk calculator that uses the 20-year UK prospective diabetes study (UKPDS) trial data to estimate future coronary heart disease (CHD) and stroke risk. Recognized limitations are that it does not estimate total cardiovascular disease (CVD) risk, can only be used in individuals with newly-diagnosed type 2 diabetes and does not take account of albuminuria.We have produced a new UKPDS Risk Engine that incorporates the 10-year UKPDS post-trial monitoring data. It utilizes over 40,000 patient-years of data with 1,115 cardiovascular disease (CVD) events, defi ned as: CVD death, non-fatal myocardial infarction (MI), non-fatal stroke or ischaemic heart disease. Patients were entered into the model at ran-dom intervals after diagnosis of diabetes so that risks could be estimated in people with varying degrees of diabetes duration, and with a history of previous MI or stroke where these had occurred prior to model entry.New statistically-independent risk factors, in addition to those in the old model (age, gender, ethnicity, smoking status, HbA1c, systolic blood pressure, total-to-HDL cholesterol ratio and atrial fi brillation), were: previous MI or stroke, macroalbuminuria, microalbuminuria, duration of diagnosed diabetes and body mass index, with hazard ratios of 3.06, 1.56, 1.32, 1.05 and 1.03 respec-tively in the CVD equations. CHD risk estimates generated by the new model were more precise than the old model, providing 22% narrower 95% Confi -dence Intervals.The updated UKPDS Risk Engine provides a more precise and more generalizable diabetes-specifi c risk calculator that estimates the risk of CVD and CVD death, in addition to CHD and stroke. It can estimate risks for primary or secondary CV events, and in people who have established or newly-diagnosed diabetes.

& 396-PAcute Hyperglycemia Reduces Cerebral Vasomotor Reactivity in Insulin-Resistant SubjectsPAOLA PALAZZO, ILARIA GIORDANI, ILARIA MALANDRUCCO, FABIANA PIC-CONI, FRANCESCO PASSARELLI, FABRIZIO VERNIERI, RICCARDO ALTAVILLA, SIMONA FRONTONI, Rome, Italy

Cerebral Vasomotor Reactivity (CVR), a reliable method to measure cerebral hemodynamics, is reduced in type 2 diabetic patients, thus con-tributing to cerebrovascular morbility and mortality. This study aimed to investigate the impact of acute hyperglycemia on cerebral reactivity, in a condition of insulin-resistance, before the onset of overt hyperglycemia.We, therefore, studied seventeen patients with metabolic syndrome (MS) and 3 age-matched controls. Metabolic syndrome was defi ned according to the International Diabetes Federation criteria. All subjects underwent a 2-hour hyperglycaemic clamp (HC), at a blood glucose level of 13.9 mmol/l. In order to avoid the possible confounding effect of hyperinsulinemia, endogenous insulin secretion was inhibited by octreotide. At baseline, 60 and 120 min of the HC, and 2 hours after the end, CVR was evaluated, by means of bi-lateral Transcranial Doppler measurement of middle cerebral artery mean fl ow velocity, and expressed as percentage change during inhalation of a mixture of air and 7% CO2.In MS patients, but not in controls, CVR was signifi cantly reduced after 1 hour and 2 hours of stable hyperglycemia vs.baseline (50.23% vs. 62.38%, p=0.002 and 54.27% vs. 62.38%, p=0.015 re-spectively). Two hours after the end of HC, CVR value returned to baseline.The fi nding that acute hyperglycemia worsened CVR only in patients with metabolic syndrome, but not in controls, suggests that insulin resistance per se plays a role in CVR response to acute hyperglycemia, before the onset of diabetes.This observation has many clinical implications, particularly on the possible prognostic meaning of the stress-induced hyperglycemia, in terms of cerebrovascular disease in patients with metabolic syndrome, but with-out type 2 diabetes.

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& 397-PPost-Operative Glycemic Control and Survival 1 Year after Cardiac Surgery: Ten Year Experience With Intensive Glycemic Manage-ment at the Pittsburgh VAR.H. RAO, PETER L. PERREIAH, CANDACE A. CUNNINGHAM, Pittsburgh, PA

Intensive glycemic management was implemented in 2111 patients under-going cardiac surgery (924 with diabetes) at our hospital, initially to a 90-150 mg/dl target blood glucose [BG] with a formula-based algorithm, tightened further to 80-120 mg/dl after 2005, using an adaptive basal-bolus Glycemic Expert system for Nurse-Implemented Euglycemia (GENIE). We analyzed the impact of postoperative glycemic exposure (Time-Weighted Excess Glucose [TWEG], defi ned as the integrated area under the curve of BG >120 mg/dl over time) and severe hypoglycemia (BG<40mg/dl) on mortality 1 year after surgery.One year mortality showed a clear separation, akin to a ‘threshold effect’, between patients with TWEG 4000 mg.dl-1.hr (equivalent to main-taining BG ~140 mg/dl for 200h) and those with lower levels of glycemic exposure (Odds Ratio, OR, 4.5 [95% CI 3.2- 6.5] p<0.001), there being no mor-tality differences between various subgroups with TWEG <4000. Increased 1 year mortality was also associated with the occurrence of 1 or more severe hypoglycemic events (OR 12.04 [CI 6.5-22.3] p<0.001) but, when glycemic ex-posure was taken into account, the effect of hypoglycemia on mortality was seen only in association with TWEG 4000 (OR 8.2 [CI 3.8-18] p<0.001), not with TWEG <4000 (OR 3.6 [CI 0.8-17] p NS). Finally, patients without a prior diagnosis of diabetes at the time of surgery had a signifi cantly higher 1 year mortality than those with a prior diagnosis of diabetes (OR 1.6 [CI 1.1-2.3] p<0.01).Our results show that decreased long-term survival after cardiac surgery is associated with (a) relatively modest levels of glycemic exposure, and (b) the occurrence of severe hypoglycemia, but only in the context of higher glycemic exposure. On the other hand, (c) survival is better in patients with a prior diagnosis of diabetes compared to those who develop postop-erative (stress) hyperglycemia without a prior diagnosis of diabetes.

& 398-PSexual Dysfunction as a Marker of CVD in Males With 50 or More Years of Type 1 DiabetesSARA J. TUREK, STEPHANIE M. HASTINGS, JENNIFER K. SUN, GEORGE L. KING, HILLARY A. KEENAN, Boston, MA

Increasing rates of diabetes and associated cardiovascular (CV) compli-cations have a substantial adverse effect on quality of life and fi nancial burden on the healthcare system. There is increasing evidence that sexual dysfunction may be a predictor of increased cardiometabolic risk in aging men. The goal of this analysis was to investigate the association of sexual dysfunction with clinical markers of vascular disease among males using data from the 50 Year Medalist Study who have documented 50+ years of type 1 diabetes. Males (48.8%, n=316/658) in this group had mean age of 71.3 (±8.41) y, mean diabetes duration of 59.1 (±7.22) y, mean BMI of 26.3 (±3.74) kg/m2 and mean HbA1c of 7.04% (±0.89). Prevalence of sexual dys-function was characterized by a “yes” response to the question: “Have you ever had sexual problems?”(69.8%). Risk factors associated with sexual dysfunction included elevated HbA1c (7.1± 0.9% v. 6.8 ±0.8%, p<0.001), BMI (26.7 ±3.9 v. 25.2 ±3.1 kg/m2, p=0.02), higher total cholesterol (159.9 ±31.8 v. 150.1 ±30.6 mg/dL, 0.02), and lower HDL (54.9 ±16.1 v. 61.7 ±17.4 mg/dL, p<0.01). Age and duration (71.4 ±8.1 v. 71.2 ±9.1 y, p=0.7; 59.3 ±7.8 v. 59.0 ±7.0 y, p=1.0, respectively) were not associated with dysfunction. In this group, sexual dysfunction was not signifi cantly associated with any microvascular complication (p>0.05); however, dysfunction was associated with CVD OR: 1.7 (95% CI:1.02, 2.7) independent of age (CV OR adjusted for age 1.7 (1.02, 2.8)) and BMI (CV OR adjusted for BMI 2.3 (1.1, 4.6)). Congruent with its association with CV, levels of infl ammatory markers including IL-6 (median 0.1 [Q2 0.06, Q3 0.3] v. 0.09 [0.04, 0.19] pg/mL, p=0.03) and PAI-1 (204.1 [ 106.4, 246.5] v. 160.0 [99.0, 203.5], pg/mL p=0.08) were also higher in those reporting dysfunction. These fi ndings indicate that sexual dysfunction may be a marker for CV in those with extreme duration diabetes. This fi nding is of clinical importance as sexual dysfunction is a more overt sign of CV risk than clinical markers of the disease.

& 399-PEffects of Insulin and Combined Hyperglycemia and Elevated FFA on Endothelial Function, Adhesion Molecules and Platelet Aggrega-tion in Healthy ManNINO G. JOY, CHERYL YOUNG, DONNA B. TATE, MAKA S. HEDRINGTON, MAIA MIKELADZE, IAN C. DAVIS, LINDSAY PULLIAM, LISA M. YOUNK, CHELSEA YOUNK, STEPHEN N. DAVIS, Baltimore, MD

The acute vascular effects of combined hyperglycemia and elevated FFA in the presence of clamped hyperinsulinemia are largely unknown. This study tested the hypothesis that, hyperinsulinemia could rescue endothelial func-tion; reduce platelet aggregation and pro atherogenic mechanisms during moderate hyperglycemia and hyperlipidemia in healthy humans. Eighteen individuals (9M/9F), age 40±2 years, BMI 28.3±1 kg/m2 were studied during 4 separate randomized protocols. The pancreatic clamp was combined with a 20 % Intralipid infusion and 4 hour glucose clamps consisting of either; 1) euinsulinemic/ euglycemia (90±1mg/dl), 2) euinsulinemic /hyperglycemia (200±2mg/dl), 3) hyperinsulinemic /euglycemia or 4) hyperinsulinemic/hyper-glycemia. Two D doppler ultrasound was used to determine fl ow mediated di-lation (FMD) of the brachial artery.Results mean ± SEM from the fi nal 30 min of clamps are shown in table 1.In Summary, in the presence of moderately increased hyperglycemia and FFA; insulin can signifi cantly reduce P-Selectin, ICAM, VCAM levels and improve Nitric Oxide mediated endothelial function. Additionally insulin can reverse the effects of FFA to impair endothelial func-tion and elevate P-Selectin and adhesion molecules. We conclude that acute hyperinsulinemia can reverse the deleterious vascular effects of combined FFA and hyperglycemia or FFA alone on endothelial function, platelet aggre-gation and pro atherogenic mechanisms in healthy man.

Table 1: *p<0.05 to euins/hypergly; † p<0.05 to euins/eugly

Euins/eugly

Hyperins/eugly

Euins/hypergly

Hyperins/hypergly

P-selectin pg/mL 43±6 -4±5* † 55±7 15±5*†FMD % change -1.9±1.3 -0.1±1.9* -4.8±1.8 -1.2±2*VCAM ng/mL -2±22 -31±42*† 110±30 -128±-46*†ICAM ng/mL 8±6 -16±6*† 14.±8 -9±-5*†

FFA μmol/L 993±157 837±157 866±225 940±195Insulin μU/mL 25±6 23±9 113±19 105±20

& 400-PAn Alternative Pathway for Hypofi brinolysis in Type 2 Diabetes: The Role of Complement C3K. HESS, SAAD ALZAHRANI, JACKIE PRICE, MARK STRACHAN, NATALIE OX-LEY, FLADIA PHOENIX, NIKOLAUS MARX, VERENA SCHROEDER, RHODRI KING, RAMZI AJJAN, Aachen, Germany, Leeds, United Kingdom, Edinburgh, United King-dom, United Kingdom, Bern, Switzerland

Plasminogen activator inhibitor (PAI)-1 is regarded as the main antifi brin-olytic protein in diabetes but recent work indicates that complement C3, an infl ammatory protein, directly modulates fi brinolysisin type 1 diabetes (T1DM). Therefore, we investigated the role of complement C3 in fi brinolysis in a large cohort of T2DM subjects.Fibrin clot lysis was determined in 875 patients enrolled on the Edinburgh type 2 diabetes study using a turbidime-tric assay. Plasma levels of complement C3, C-reactive protein (CRP), PAI-1 andfi brinogen were analysed by ELISA.Clot lysis time showed a highly sig-nifi cant correlation with C3 and PAI-1 plasma levels (r=0.25, p<0.0001 and r=0.15, p<0.0001;respectively). In contrast, a relatively weak correlation was detected with CRP (r=0.08, p=0.02) and fi brinogen (r=0.08, p=0.01). Plasma levels of C3, CRP, fi brinogen or PAI-1 did not differ in the presence of previ-ous history of myocardial infarction or cerebrovascular disease. Plasma lev-els of all four proteins correlated with body mass index, but only fi brinogen showed an interaction with age and duration of diabetes. Ina regression model involving these proteins, C3 was a predictor of lysis time [predictive value for 0.1 mg/ml change in plasma levels 14.4 (95% CI 7.9, 21.0) p<0.001], as was PAI-1 (predictive value for 0.1 ng/ml change in plasma levels 8.2 (4.2, 12.1; p<0.001) with a smaller effect shown for 0.1 mg/ml change in fi brinogen [5.3 (0.01-10.5); p=0.049] and none detected for CRP [-2.5 (-9.7, 4.8); p=0.50]. No correlation was demonstrated between C3 and PAI-1 plasma levels in-dicating the two proteins affect different pathways in fi brin clot lysis. In multivariable analysis, drug therapies failed to predict C3 plasma levels, although a trend was observed with antiplatelet treatment in men. C3 is at least as strong as PAI-1 at predicting fi brin clot lysis in subjects with T2DM.

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Therefore, future studies should analyse C3 plasma levels as a surrogate marker of fi brinolysis potential in this population.

Supported by: NIHR and the BHF

& 401-PAssessment of Asymptomatic Coronary Artery Diseases in Chinese Adults With Different Glycaemic StatusGUANG NING, YU XU, YUFANG BI, MIN XU, MIAN LI, TIANGE WANG, YU LIU, JIELI LU, YUHONG CHEN, WEIQING WANG, Shanghai, China

To evaluate the risks of subclinical coronary artery disease (CAD) in early diabetes and prediabetes, the non-invasive dual-source computed tomogra-phy coronary artery angiography (DSCT-CA) was used to visualize the coro-nary artery lumen, identifying the location, severity, and composition of ath-erosclerotic plaques. A total of 401 age- and sex-matched participants with normal glucose regulation (NGR), prediabetes or diabetes diagnosed less than 5 years were randomly selected from a community-dwelling population aged 40-60 years and asymptomatic of CAD to undergo a comprehensive examination including a detailed questionnaire, anthropometric measure-ments, biochemical evaluations and a DSCT-CA examination. Obstructive CAD was defi ned as more than 50% narrowing of vessel lumen. Coronary artery characteristics of the study population according to glucose meta-bolic status are shown in table 1. Multivariate logistic regression analysis revealed few independent risk factors for obstructive CAD. Women were strongly protected from having obstructive CAD (OR [95% confi dence inter-val (CI)]: 0.33 (0.14-0.81), P = 0.016). Prediabetes was not associated with an increased risk of obstructive CAD, whereas diabetes was associated with a signifi cant 1.34-fold elevated risk (OR (95% CI): 2.34 (1.01-5.43), P =0.047) compared with NGR after adjusted for other conventional cardiometabolic risk factors. In conclusion, using DSCT-CA to anatomically assess subclini-cal coronary atherosclerosis in a real world Chinese population, we found a markedly increased risk of obstructive CAD in early diabetes.

Table 1. Coronary artery characteristics of study population by glucose meta-bolic statusCharacteristicsof coronary arteries

NGR(n=135)

Prediabetes (n=132)

Diabetes(n=134)

Age-and sex-adjustedP values

(Prediabetesvs. NGR)


(Diabetesvs. NGR)


(Diabetes vs. Prediabetes)

Age (years) 52.3 ± 4.4 52.6 ± 4.2 52.8 ± 4.4 0.53 0.34 0.75

Male n (%) 56 (41.5) 67 (50.8) 67 (50.0) 0.13 0.16 0.90

Family history of CAD n (%)

22 (16.3) 20 (15.2) 26 (19.4) 0.90 0.44 0.36

Current smoker n (%)

40 (29.6) 43 (32.6) 46 (34.3) 0.47 0.74 0.61

Coronary cal-cium score > 0 n (%)

15 (11.1) 15 (11.4) 30 (22.4) 0.98 0.024 0.021

Any coronary plaques n (%)

58 (43.0) 77 (58.3) 74 (55.2) 0.026 0.067 0.58

ObstructiveCAD n (%)

10 (7.4) 10 (7.6) 22 (16.4) 0.90 0.042 0.027

Guided Audio Tour: Diabetes and Cardiovascular Disease—From Coro-nary Calcifi cation to Cardiovascular Disease Mortality (Posters 402-P to 407-P), see page 17.

& 402-PThe Association of Vascular Atherosclerotic Plaque and Cardiovas-cular Mortality in the Diabetes Heart StudySUBHASHISH AGARWAL, AMANDA J. COX, DAVID M. HERRINGTON, NEAL JORGENSEN, JIANZHAO XU, J.J. CARR, BARRY I. FREEDMAN, DONALD W. BOWDEN, Novi, MI, Winston-Salem, NC, Seattle, WA

Coronary artery calcium (CAC), carotid artery calcium (CAAC), and abdomi-nal aorta calcium (AAC) are noninvasive measures of atherosclerosis that predict mortality. In a prior study we demonstrated that CAC is a powerful predictor of all-cause mortality (odds of death six fold higher) and cardiovas-cular (CVD) mortality (odds of death eleven fold higher) in type 2 diabetes in CAC > 1000 compared to CAC < 10 beyond traditional risk factors. Here we compared the predictive value of atherosclerotic plaque across multiple vascular beds for CVD mortality. 854 participants, ages 39-86, with com-

plete data on diabetes, imaging and covariates in the Diabetes Heart Study (DHS) were followed for an average of 7.4 years. Atherosclerosis imaging was performed utilizing fast-gated helical CT scan at baseline to obtain mea-sures of coronary, carotid, and abdominal aorta Agatston calcium scores. Seven-year risk estimates for CVD mortality were obtained using logistic regression models adjusted for age, gender, smoking, systolic blood pres-sure, antihypertensive medication use, total and high-density lipoprotein cholesterol, and race/ethnicity including log transformed (CAC+1), (CAAC+1), and (AAC+1).8% (68/854) of participants died during follow-up. CAC was as-sociated more strongly than either carotid or abdominal calcium with mortal-ity. After adjusting for confounders, and each other (CAC, CAAC, and AAC), the odds ratio of CVD mortality increased 1.67 fold (95% CI, 1.06-2.72) for each 1-standard deviation (SD) increment of log transformed CAC score, 1.50 fold (95% CI, 0.99-2.32) for each 1-SD increment of log CAAC, and 1.21 fold (95% CI, 0.67-2.30) for each 1-SD increment of log AAC. A receiver operating characteristic curve analysis also suggested that CAC score was a better predictor of mortality than was CAAC and AAC, with AUC’s of 0.78, 0.77, and 0.77, respectively.In this study CAC score is a better predictor of CVD mortal-ity than carotid or abdominal aorta vascular beds in diabetes.

& 403-PPatients With Type 2 Diabetes Demonstrate Reduced Cardiac Ener-getics and Increased LV Torsion Compared to Healthy Age Matched ControlsMICHAEL KUEHL, MARTIN J. STEVENS, MUHAMMAD AZAM, ROGER BEADLE, Birmingham, United Kingdom

T2DM induced heart failure can occur in the absence of coronary artery disease and might be related to the altered cardiac substrate metabolism. Magnetic resonance spectroscopy (MRS) allows the measurement of car-diac energetics (CE) non-invasively. Our aim was to determine CE and cardiac function in a cohort of otherwise healthy patients with longstanding T2DM and to compare the fi ndings to sex and age matched healthy controls (HC).A cross-sectional study was conducted in 27 subjects with T2DM free of known coronary heart disease attending the outpatient clinic of the Birming-ham Heartlands Hospital, UK and compared to 17 HC. Phosphocreatine/ -ATP ratios as marker of CE were measured with a Philips Achieva 3T MRI/MRS scanner (Philips Healthcare, Best, the Netherlands). Speckle-tracking echocardiography was performed using a GE Vivid 7 ultrasound (GE Vingmed Ultrasound, Horten, Norway).27 subjects (age, 53 ± 12 years, diabetes dura-tion 11 ± 2 years, female, 42%, HbA1c, 8.4 ± 1.1%, BMI 30.6 ± 4.5%) and 17 control age and sex matched subjects (age, 48 ± 11, female 50%, BMI 24.1 ± 3.7%) have been recruited so far. The phosphocreatine/ -ATP ratio were reduced in patients with T2DM compared to HC (1.07 ± 0.37 vs 1.54 ± 0.53, p<0.01 respectively). In concert, left ventricular (LV) torsion was increased in patients with T2DM compared to HC (2.4 ± 0.76 vs 1.4 ± 0.7°/cm, p=<0.001). There was however no signifi cant correlation between torsion and cardiac energetics.We demonstrate in patients with T2DM free of coronary artery disease that cardiac energetics are reduced and LV torsion is increased. These fi ndings are consistent with our previous results in patients with T1DM, suggesting that impaired cardiac energetics and increased LV torsion are early complications of otherwise healthy asymptomatic diabetes and refl ect the consequences of metabolic dysfunction. The role of these early defi cits in the development of cardiac failure warrants further investigation.

Supported by: Eli Lilly

& 404-PImpact of Heart Failure on Hypoglycemic Complications in Patients With Type 2 Diabetes—An Analysis of the DiaRegis RegistryDIETHELM TSCHÖPE, PETER BRAMLAGE, CHRISTIANE BINZ, MICHAEL KREKLER, KRISTIAN LÖBNER, EVELIN DEEG, ANSELM K. GITT, Bad Oeynhausen, Germany, Mahlow, Germany, München, Germany, Wedel, Germany, Ludwigshafen, Germany

Heart failure and type-2 diabetes is a high risk disease constellation. We hypothesized that hypoglycaemia might be more frequent in patients with diabetes and heart failure than in those without heart failure and may worsen prognosis. Analyses were based on DiaRegis which is a prospective registry in Germany including 3810 patients with type-2 diabetes receiving antidiabetic treatment, oral mono- or oral dual combination therapy. For 3,746 patients data on the presence of heart failure were available. Patients with heart failure (n=370; 9.9%) had a considerable cardiovascular disease burden with coronary artery disease (OR 7.02; 95%CI 5.59-8.82), peripheral arterial disease (3.55; 2.56-4.91) and blindness (12.29; 2.74-55.12) showing the highest increase over patients without heart failure. Hypoglycaemia within 12 months prior to baseline was almost twice as frequent in diabetic patients with heart failure (17.8%) than in those without (10.0%; OR 1.96;

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95%CI 1.47-2.61). Associations were signifi cant for the incidence of symp-tomatic hypoglycaemia with (OR 4.05; 95%CI 1.91-8.58) or without help (OR 1.79; 95% CI 1.27-2.53) as well as for those with need of medical assistance (OR 7.93; 95%CI 2.65-23.73). 61 of 325 patients with heart failure at baseline (18.8%) had any episode of hypoglycaemia during the 12 months follow-up. Myocardial infarction (1.6 vs. 0%), stroke/TIA (OR 4.59; 95%CI 1.29-16.39) and depression (OR 2.98; 95%CI 1.33-6.70) were more frequent in patient with heart failure and incident hypoglycaemia than in those without. We conclude that hypoglycaemia was almost twice as frequent in diabetic pa-tients with heart failure than in those without and was associated with a worse prognosis. The fi ndings suggest that particular attention should be given to an appropriate treatment strategy in patients with type-2 diabetes and overt heart failure to improve patient outcomes.

Supported by: Bristol-Myers Squibb/AstraZeneca

& 405-PPrevalence and Incidence of Peripheral Artery Disease is Higher in Indian WomenJAYASHEEL O. ESHCOL, RAJENDRA PRADEEPA, SIVASANKARAN SUBHASHINI, SARAVANAN JEBARANI, RANJIT M. ANJANA, RANJIT I. UNNIKRISHNAN, VISWANATHAN MOHAN, Iowa City, IA, Chennai, India

Peripheral artery disease (PAD) is a common complication of diabetes associated with increased cardiovascular morbidity but the disease is not well characterized in India. The available research shows a lower prevalence than the west, but studies are few and none have assessed the incidence and progression of PAD. We sought to determine the factors associated with the incidence and progression of PAD in the Asian Indian Type 2 diabetic population.We retrospectively examined all patients who had an ankle bra-chial index (ABI) measurement at a diabetes centre in 2001. The baseline clinical and biochemical characteristics and all follow up ABI measurements were abstracted from the electronic medical record. PAD was defi ned as an ABI <0.9 and incidence was identifi ed as the fi rst occurrence of an ABI <0.9. A change in ABI >0.15 was defi ned as signifi cant progression. Risk factors for prevalent PAD and for progression were analysed by logistic regression and by time-to-event analysis for incident PAD.Two thousand fi ve hundred and twelve patients were followed for an average of 7 years. 7.6% of the study population had PAD in 2001, (11.8% in women and 5.1% in men, adjusted OR 2.7 [CI 1.7-4.2]). Prevalent PAD was associated with high mortality, adjusted HR 3.3[1.4-7.7]. There were 280 new cases of PAD identifi ed resulting in a crude incidence rate of 17 per 1000 patient years. Females had a 2-fold in-creased rate of PAD, adjusted HR 1.94 [CI 1.4-2.7]. Other adjusted variables that predicted incident PAD were increasing age, duration of diabetes and LDL cholesterol. A drop in ABI > 0.15 was seen in 5.5% of patients and this was associated with increasing age, hypertension and LDL.Among Asian In-dian type 2 diabetic patients seen at a tertiary diabetic centre in southern India there is a striking female predominance in incident and prevalent PAD. Age and duration of diabetes are the strongest predictors of both incident and prevalent PAD. This study suggests that in the Indian diabetic popula-tion, women are at higher risk for PAD.

& 406-PThe Association Between Ideal Cardiovascular Health and Coronary Artery Calcifi cation in Adults With and Without Type 1 DiabetesAMY ALMAN, DAVID M. MAAHS, MARIAN REWERS, JANET K. SNELL-BER-GEON, Aurora, CO

In 2010, the American Heart Association identifi ed national 7 goals for ideal cardiovascular health (ICH). The purpose of this study is to examine the prevalence of attaining ICH goals in a population of adults with and without type 1 diabetes (T1D) and to examine the association of ICH with coronary artery calcifi cation (CAC). Since the goal of a fasting plasma glucose <100 mg/dL is not relevant in T1D, this analysis focused on the remaining 6 metrics (see Table).The CACTI study has followed adults with (n=652) and without T1D (n=764) for 6.1±0.5 years. At baseline, diet, smoking, and physical ac-tivity were assessed, and BMI, cholesterol, and blood pressure were mea-sured. Complete ICH data were available on 1177 subjects, and 856 had CAC progression data from the follow-up exam. Descriptive analyses and mul-tivariate logistic regression were performed to examine the prevalence of ICH and its association with CAC presence and progression.The table shows the prevalence of the ICH metrics. Adults with T1D were more likely to meet cholesterol goals than non-diabetics. . A greater number of ICH goals met was associated with a signifi cant reduction in the odds of any CAC at base-line (OR=0.71 (0.62, 0.82)) and the progression of CAC (OR=0.82 (0.70, 0.96)), adjusted for age, sex, diabetes, hemoglobin A1c, triglycerides, and baseline CAC score.In conclusion, the prevalence of ICH metrics is similar in T1D

adults and non-diabetics, and is low in both groups for physical activity and diet. An increasing number of ICH metrics was associated with signifi cantly reduced odds of CAC presence and progression.

Prevalence of Ideal Cardiovascular Health Metrics by Diabetes StatusICH Metric Type 1 diabetes

(n=546)Non-diabetics

(n=631)p-value

Never smoking 364 (66.7) 435 (68.9 0.41BMI < 25 kg/m2 238 (43.6) 294 (46.6) 0.30Physical activity* 54 (9.9) 51 (8.1) 0.28Healthy diet** 8 (1.5) 13 (2.1) 0.44Total cholesterol <200 mg/dl 425 (77.8) 284 (45.0) <.0001Blood pressure <120/80 mm Hg 254 (46.5) 284 (45.0) 0.60Data are presented as N(%). * at least 150 min/week moderate intensity or at least 75 min/week vigorous intensity. ** 4-5 of the following: at least 4.5 cups of fruit or vegetables/day, two 3.5 oz servings of fi sh/week, at least 3 oz servings of fi ber-rich whole grains/day

& 407-PComparison of Hospital Admissions in Patients With DM and Non-DM Admitted With Acute Coronary SyndromeVINCENT WOO, AMANDA HAK, Winnipeg, MB, Canada

The objective of this study was to compare cardiac risk factors, treatment, and outcome between DM and non-DM patients (pts) admitted to a tertiary care centre with acute coronary syndrome (ACS). Data was collected from charts of patients who were admitted to the Health Sciences Centre in Win-nipeg, Canada with ACS between August 2007 and July 2009.The charts of 190 DM pts, of which 96% had DM2, and 263 non-DM pts were examined. Of all the pts included in the study, at the time of admission, 64% were male and the average age was 66 ± 13 years. In terms of cardiac risk factors, non-DM pts were more likely to smoke (37.3%) than DM pts (19.6%); but DM pts were more likely to be obese (BMI > 30; 39% vs. 28%), aboriginal (13.2% vs. 3%) or hypertensive (89% vs. 62%) than non-DM pts. DM pts tended to have lower levels of total cholesterol (4.11mmol/L vs. 4.41mmol/L) and LDL (2.12mmol/L vs. 2.47mmol/L), but higher levels of triglycerides (1.59mmol/L vs. 1.29mmol/L) and serum creatinine (157.8μmol/L vs. 97.3μmol/L) than non-DM pts. DM pts tended to have lower peak levels of creatine kinase (621.3U/L vs. 1009.1U/L) and troponin T (1.81ng/mL vs. 2.87ng/mL); but tended to have lower ejection fractions (43% vs. 49%). As for treatment in hospital, DM pts were as likely to receive thrombolytics or CABG as non-DM pts. Although not signifi cant, DM pts were slightly less likely to have an angiogram or stents (p-values 0.08 and 0.06, respectively). DM pts were more likely to be dis-charged on a calcium channel blocker (32% vs. 19%) or a diuretic (37% vs. 17%); but less likely to be discharged on clopidogrel (44% vs. 60%). Finally, 9.5% of DM pts and 6.1% of non-DM pts died because of ACS (p = 0.18). All differences are statistically signifi cant at = 0.05 unless otherwise noted.Risk factors for ACS such as smoking and high total cholesterol were more prevalent among non-DM pts than DM pts; however, obesity and hyperten-sion were more common among DM pts. In conclusion there continues to be important differences between DM and non-DM pts.

Guided Audio Tour: Cardiovascular Disease in Patients with Diabetes—From Characterization to Mechanisms (Posters 408-P to 413-P), see page 13.

& 408-PAssociations between Circulating Leukocyte Subtype Counts and Carotid Intima-Media Thickness in Japanese Subjects With Type 2 DiabetesTAKESHI MATSUMURA, KAYO TAKETA, HIROYUKI MOTOSHIMA, TAKAFUMI SENOKUCHI, NORIO ISHII, HIROYUKI KINOSHITA, KAZUKI FUKUDA, TAKESHI NISHIKAWA, EIICHI ARAKI, Kumamoto, Japan

The increase in leukocyte count is an independent risk factor for car-diovascular events. However, the association between leukocyte subtype counts with carotid atherosclerotic regression in patients with diabetes is unknown. Therefore, we investigated the correlation between leukocyte subtype counts and atherosclerotic markers in subjects with type 2 diabe-tes. Overall, 360 outpatients with type 2 diabetes were enrolled and blood samples were measured. The intima-media thickness of the common carotid artery (CCA-IMT) was measured by high-resolution B-mode ultrasonogra-phy. Brachial ankle pulse wave velocity (baPWV) was measured using an ABI-form device. CCA-IMT was positively correlated with age, systolic blood pressure (SBP), baPWV and duration of diabetes (r = 0.279, P < 0.001; r = 0.113,

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P = 0.032; r = 0.269, P < 0.001; r = 0.147, P < 0.005, respectively), and nega-tively correlated with body mass index and diastolic blood pressure (DBP) (r =-0.158, P = 0.003; r = -0.112, P = 0.034, respectively). baPWV was positively correlated with age, SBP, DBP, CCA-IMT, urinary albumin and duration of diabetes. Although white blood cell, monocyte, neutrophil, lymphocyte and eosinophil counts were all positively correlated with CCA-IMT (r = 0.301, P <0.001; r = 0.698, P < 0.001; r = 0.265, P < 0.001; r = 0.135, P = 0.01; r = 0.115, P =0.03, respectively), only the monocyte count was correlated with baPWV. Multiple regression analyses showed that the monocyte count, age and baPWV explained 95.8% of the total variance in CCA-IMT, with the mono-cyte count alone explained 69.7% of the variance in CCA-IMT. This indicates that the monocyte count is a strong independent predictor of CCA-IMT. In comparison, age, SBP and CCA-IMT explained 85.5% of the total variance in baPWV. In conclusion, leukocyte counts, particularly the monocyte count, may be convenient and valuable markers for detecting the progression of macrovascular complications in patients with type 2 diabetes.

409-P

& 410-PSpecifi city of Gray Matter Aging in Middle-Aged Patients With Type 1 DiabetesTIMOTHY M. HUGHES, Pittsburgh, PA

Adults with Type 1 diabetes (T1D) develop cognitive and structural brain abnormalities similar to those observed in older adults without diabetes. Initial studies indicate that gray matter volume (GMv) loss in T1D patients is focal rather than global. However, the spatial distribution of GMv loss in middle-aged T1D patients is not known. We apply MRI with high spatial reso-lution to compare GMv of middle-aged T1D adults with age, gender matched controls. We hypothesize that between-group differences are stronger in regions known to be affected by aging (frontal lobes, hippocampus, putamen and thalamus) than in other regions.GMv using 3 Tesla MRI was measured in 33 patients with T1D and 33 age, gender matched non-diabetic controls aged 30-50 years. Between-group differences in GMv were quantifi ed by general linear models adjusted for intracranial volume, blood pressure, total choles-terol levels, smoking status, years of education and waist circumference. A Bonferroni threshold (p<0.004) was applied to account for multiple com-parisons (n=20) and minimize type-1 error.Between-group GMv differences met Bonferroni thresholds in areas affected by aging, with smaller GMV seen in the frontal lobes (11% smaller, p<0.001), thalamus (28% smaller, p0.15) or hippocampus (5% larger, p=0.02). Between-group GMv differences

were also signifi cant in areas not related to the aging process, including the paracentral lobule (17% smaller, p<0.001) and postcentral gyri (11% smaller, p=0.002). GMv of these regions was not signifi cantly correlated with dura-tion of T1D, insulin dose and estimated number of months with elevated A1c at p<0.05.Compared to matched controls, T1D patients had smaller GMv in areas affected in the aging process, including the frontal lobes, thalamus and putamen, but not in the temporal lobes or hippocampus. T1D-related factors did not correlate with GMv in these regions. Potential mechanisms underlying lower GMv seen in T1D, such as microvascular disease, need to be further explored.

Supported by: R01 Grants from NIDDK (DK089028, DK034818) and NIMH (HL089850)

& 411-PPlasma Leptin is Associated With Vascular Endothelial Function, but Not With Smooth Muscle Function in Type 2 Diabetic PatientsTOMOAKI MORIOKA, MASANORI EMOTO, YUKO YAMAZAKI, NAOYA KAWANO, HIROMI URATA, SHOKO TSUCHIKURA, KOKA MOTOYAMA, KATSUHITO MORI, SHINYA FUKUMOTO, TETSUO SHOJI, MASAAKI INABA, Osaka, Japan

Leptin plays major roles in the pathogenesis of atherosclerosis. How-ever, the association of leptin with vascular endothelial or smooth muscle function, that is impaired in the early stage of atherosclerosis, is yet to be determined in human subjects with type 2 diabetes. Therefore, we investi-gated the association of plasma leptin levels with endothelium-dependent, fl ow-mediated dilatation (FMD) and endothelium-independent, nitroglyc-erin-induced dilatation (NMD) of the brachial artery in 156 type 2 diabetic patients (age, 63 ± 10 (SD) years, BMI, 24.4 ± 4.7 kg/m2). The mean values of FMD, NMD and plasma leptin in those subjects were 6.6 ± 3.7 %, 14.1 ± 6.4% and 4.9 ± 4.8 ng/ml, with ranges 0.7 - 18.9, 0.5 - 28.9, and 0.2 - 29.5, respectively. In simple regression analyses, FMD was negatively correlated with age (r = -0.266, p = 0.001), systolic blood pressure (SBP, r = -0.211, p = 0.008), and positively with plasma leptin (r = 0.232, p = 0.004), while NMD was negatively correlated with age (r = -0.414, p < 0.001), SBP (r = -0.348, p < 0.001), but not with plasma leptin (r = -0.099, p = 0.269). In multiple regression analyses including age, sex, diabetes duration, BMI, SBP, serum creatinine, smoking habit, glycated hemoglobin, lipid profi le, and plasma lep-tin, signifi cant independent contributors to FMD were age ( = -0.100, p = 0.010), BMI ( = -0.281, p = 0.004), SBP ( = -0.042, p = 0.010), and plasma leptin ( = 0.363, p < 0.001), whereas those to NMD were age ( = -0.368, p < 0.001), BMI ( = -0.440, p = 0.012), SBP ( = -0.097, p = 0.003), but not plasma leptin ( = 0.016, p = 0.928). In conclusion, plasma leptin level is signifi cantly associated with vascular endothelial function, but not with smooth muscle function, independently of obesity and other cardiovascular risk factors in type 2 diabetes. This study suggests that the vasodilatory effect of leptin is able to be demonstrated in type 2 diabetes through vascular endothelium-dependent mechanisms.

& 412-PMT Preservation of Akt2 Function by Inhibition of Akt Negative Reg-ulators Prevents Diabetic Inhibition of Cardiac Insulin SignalingYI TAN, YUEHUI WANG, XIAOKUN LI, LU CAI, Louisville, KY, Wenzhou, China, Changchun, China

Cardiac insulin resistance is a key pathogenic factor for diabetic cardio-myopathy, but its mechanism remains largely unclear. Here we reported that diabetes, induced by streptozocin, signifi cantly inhibited Akt phosphoryla-tion at both sites of Ser473 and Thr308 from 2 wks to 2 months, but not 5 months. In cardiac-specifi c metallothionein-transgenic (MT-TG) diabetic mice, both phosphorylation sites of Akt were preserved normally at all times of diabetes. Analysis of Akt isoforms revealed that Akt2, but not Akt1, ex-pression and phosphorylation were signifi cantly decreased as the progres-sion of diabetes in wide-type (WT) mice, but not MT-TG mice. Diabetes also increased Akt negative regulators, PTEN phosphorylation and TRB3 expres-sion, in WT but not MT-TG mice, at 5 months of diabetes, suggesting that MT preservation of Akt2 expression and function may be due to its suppression of diabetic up-regulation of Akt negative regulators. For mechanistic study, cardiac H9c2 cells with and without forced MT overexpression (MT-H9c2) were treated with tert-butyl hydroperoxide (tBHP, an organic oxidant). tBHP did not affect basal Akt phosphorylation, but signifi cantly inhibited insulin-stimulated total Akt phosphorylation and increased PTEN phosphorylation and TRB3 expression only in H9c2 cells. Furthermore, tBHP reduced Akt2 phosphorylation in the basal and insulin-stimulating conditions, which were signifi cantly, but not completely, attenuated in MT-H9c2 cells. Silencing TRB3 expression with its SiRNA completely blocked tBHP-induced inhibi-tion of insulin-stimulated Akt2 phosphorylation. These results suggest that

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oxidative stress-attenuated cardiac Akt, especially Akt2, function via up-regulating Akt negative regulators plays a critical role in diabetic inhibition of insulin signaling in the heart. MT preservation of Akt2 function by inhibi-tion of Akt negative regulators prevents diabetic inhibition of cardiac insulin signaling.

& 413-PAmelioration of Diabetic Cardiomyopathy By TAT-KMediated En-hanced Delivery of Metallothionein and SODYONGSOO PARK, JAETAEK KIM, YOUNGHYO LIM, LEEJIN PARK, HYUNOK KIM, SANGMO HONG, Seoul, Republic of Korea

Diabetic cardiomyopathy (DCMP) might be resulted from oxidative stress in heart tissues and antioxidant treatment may improve underlying condi-tions. To circumvent their limited access into the biologic membrane, we made Tat-MT and Tat-SOD constructs applying cell penetrating peptide technologies, delivered to primary cultured neonatal cardiomyocytes and studied cell viability in different injury conditions of high glucose, hypoxia and AGE treatment. The protective effect of Tat-MT and Tat-SOD in com-bination against the development of DCMP was also studied in OLETF rats. Tat-MT and Tat-SOD were successfully delivered to cardiomyocytes, and the intracellular activities of MT and SOD increased in line with the amount of protein delivered. These agents inhibited three different oxidative injuries as well as angiotensin II mediated direct injuries, and changes in the expression of the downstream signaling molecules in caridiomyocytes. A single intrap-eritoneal injection of Tat-MT and Tat-SOD resulted in the delivery of these antioxidants to the heart. Continuous treatment decreased expression of ROS and downstream signaling molecules in heart tissues, and delayed the clinical development of DCMP. Sixteen weeks after treatment, Tat-MT and Tat-SOD combination treatment group showed complete recovery of diastol-ic dysfunction compared with Tat-GFP group. Perivascular and/or interstitial fi brosis were signifi cantly decreased in antioxidant treatment group. TGF-immunoreactivity in the interstitial and perivascular tissue was decreased in antioxidant treatment group. The ameliorative effects of these agents against the development of DCMP in vivo might be related to the inhibition of ROS itself and downstream signaling pathway. We conclude that effec-tive delivery of a combination antioxidant treatment may prevent and treat the pathophysiology in patients with DCMP.

Supported by: Basic Science Research Program through the National Research Foundation

414-P

415-PHbA1c Reduction and Risk of Cardiovascular Diseases in Type 2 Dia-betes: An Observational Study from the Swedish NDRKATARINA EEG-OLOFSSON, BJÖRN ELIASSON, BJÖRN ZETHELIUS, ANN-MARIE SVENSSON, SOFFIA GUDBJÖRNSDOTTIR, JAN CEDERHOLM, Göteborg, Sweden, Uppsala, Sweden

The relationship between glycemic control and cardiovascular risk in type 2 diabetes (T2D) remains unclear. The aim of this observational study was to assess the association between improved glycemic control during follow-up and risk for coronary heart disease (CHD), cardiovascular disease (CVD) and total mortality in patients with T2D from the Swedish National Diabetes Register, baseline HbA1c 7-8.9%, aged 30-75 years, with no previous CVD, followed-up from 2004 to 2009 (mean 5.7 years).Two groups of patients were created based on the median for change in HbA1c during follow-up: 8923 patients (mean baseline age 62.0 years, diabetes duration 8.3 years) with HbA1c decreasing 0.1% or more from baseline to follow-up (mean base-line HbA1c 7.8±0.5% and fi nal 7.0±0.6%), and 9112 patients (mean baseline age 61.3 years, duration 9.2 years) with stable or increasing HbA1c 0.1% or more (mean baseline HbA1c 7.7±0.5% and fi nal 8.3±0.9%).Absolute risk of fi rst-incident fatal/nonfatal CVD was 15.1 events per 1000 person-years in patients with decreasing HbA1c and 26.1 in patients with stable/increasing HbA1c. Adjusted hazard ratios at Cox regression analysis for fi rst-incident fatal/nonfatal CHD, fatal/nonfatal CVD and total mortality, with decreasing HbA1c compared to stable/increasing HbA1c, were 0.61 (95% CI 0.54-0.69), 0.63 (0.57-0.70), and 0.55 (0.49-0.63), respectively, all p<0.001. Adjustment was made for a propensity score with stratifi cation by quintiles, including covariates age, sex, diabetes duration, baseline HbA1c and traditional CVD risk factors and treatments, as well as changes in these risk factors and treatments during the study period. Differences in included covariates be-tween the two groups were balanced when adjusted with the propensity score.HbA1c reduction of mean 0.8% from mean 7.8% to 7.0% (to the treat-ment target) was associated with substantially lower risk of CHD, CVD and total mortality, compared to stable/increasing HbA1c of 7.7 to 8.3.

416-PThe Association of Insulin Resistance and Carotid Intima Media Thickness (IMT) With Thigh and Calf Circumference in Patients With Type 2 Diabetes MellitusJONG SUK PARK, MIN KYUNG KIM, JIYOON HA, YOUNGMI LEE, EUN JIN KWON, JIWOON KIM, SHIN AE KANG, CHUL WOO AHN, KYUNG RAE KIM, Seoul, Re-public of Korea

The relationship between body composition parameters such as thigh and calf circumference and insulin resistance or atherosclerosis in type 2 diabetes is poorly understood. The aim of this study was to investigate the relationship between insulin resistance, atherosclerosis, and thigh and calf circumference in type 2 diabetic patients.A total of 4,427 subjects with type 2 diabetes were enrolled in this study. Insulin sensitivity was assessed according the rate constant for plasma glucose disappearance (Kitt) deter-mined via the short insulin tolerance test. Biochemical and anthropometric profi les were measured according to a standardized protocol. Visceral fat thickness and carotid intima media thickness (IMT) were measured by ultra-sonography.Insulin sensitivity index was signifi cantly correlated with weight adjusted thigh and calf circumference (r=0.244, r=0.211 in men, r=0.279, r=0.258 in women, P<0.01). Thigh circumference was inversely associated with IMT in men and women (r= -0.102, r= -0.170, P<0.05) and calf circum-ference was negatively correlated with IMT in women (r= -0.173, P<0.01). Multiple regression analysis revealed that thigh circumference was inde-pendently correlated with insulin sensitivity index (Kitt) ( = 0.128 in men, = 0.141 in women, P<0.05) and IMT after adjusting for age ( = -0.096 in men,

= -0.156 in women, P<0.05).Calf and thigh circumference were correlated with insulin resistance and IMT, and thigh circumference was independently associated with insulin resistance and IMT in type 2 diabetic patients. These results suggest that thigh circumference may be a new anthropometric marker of insulin resistance and carotid atherosclerosis.

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417-PGenetic Variability in Serine/Threonine Kinase 11 (STK11) is Asso-ciated With the Risk of Coronary Artery Disease in Type 2 Diabetes in a Chinese Han PopulationXIAOWEI MA, GE BAI, LINJUAN HUANG, JIANWEI ZHANG, RUIFEN DENG, SHAN DING, NAN GU, XIAOHUI GUO, Beijing, China

Recent studies indicated that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protectingcar-diac muscle from damage during ischemia. This study aimed to investigate whether genetic variations in the STK 11 gene encoding LKB1 affect the risk of coronary artery disease (CAD) in Chinese type 2 diabetics. 5 hap-lotype-tagging single nucleotide polymorphisms (tag-SNPs) were selected from CHB HapMap database ( Phase II, R#27), and total 288 CAD-positive cases and 159 CAD-negative controls with type 2 diabetes were genotyped for the 5 tag-SNPs by PCR-RFLP assay. The frequencies of allele A at SNP rs12979689 were 34.9% in the CAD group and 29.4% in the control group, respectively. The carriers of minor allele A at rs12977689 had a higher risk of CAD compared to the homozygotes of CC (OR =1.572, 95%CI 1.039-2.376, p = 0.035), and the difference was still signifi cant after adjustment for the other known CAD risk factors (OR’ =1.184 , 95%CI’=1.036-1.353, p’ = 0.013)(Tab.1). The male carriers of major allele C at another SNP rs6510599 had a higher risk of CAD compared to the female homozygotes of TT (adjusted OR’=5.075, 95%CI’ 1. 405-18.329, p’ = 0.013). Our fi ndings suggest that ge-netic variation(s) in STK 11 may be associated with CAD risk in type 2 diabe-tes in the Chinese population.

Association between CAD and SNP12977689 at STK 11 locusgenetype CAD+

n(%)CAD-n(%)

OR(95%CI)

p OR’(95%CI’)

p’

AA/AC 161(61.5) 71(50.4) 1.572(1.039-2.376)

0.035 1.184(1.036-1.353)

0.013

CC 101(38.5) 70(49.6) 1 1

Supported by: National 973 Project (2006CB503903, 2006CB503908)

418-PEffect of Liraglutide on Carotid Intima-Media Thickness in Patients With Type-2 Diabetes: A 4-Month Prospective StudyMANFREDI RIZZO, ANGELO MARIA PATTI, VITTORIA DI BARTOLO, ROSARIA VIN-CENZA GIGLIO, GIUSEPPE MONTALTO, ALI A. RIZVI, Palermo, Italy, Columbia, SC

There is currently high interest in the non-glycemic effects of incretin-based therapies, specifi cally glucagon-like peptide 1 (GLP-1) analogues, such as those on cardiovascular risk markers. Liraglutide has been available in the Italian market for the past one year, where it has been approved to be prescribed in combination with oral hypoglycemic agents. Liraglutide has several non-glycemic properties, but its effect on carotid intima-media thickness (IMT), a recognized marker of subclinical atherosclerosis, is still unknown. We evaluated in our Italian center the effect of liraglutide on carotid IMT, as assessed by B-mode real-time ultrasound, in a prospective study of 33 patients with type 2 diabetes (58% males, age: 59±9 years), when added to metformin at a fi xed dose of 1500/daily. Patients were newly diagnosed or previously treated subjects with oral hypoglycemic agents. The dose of liraglutide was 0.6mg/daily for the fi rst 2 weeks, followed by a dose of 1.2mg/daily. Statistical analysis was done using paired t-test and the Spearman correlation method. At baseline patients weighted 82±9 Kg, with average fasting glucose 9.5±1.4 mmol/L and HbA1c 8.3±0.6%. The following mean changes were recorded after 4 months of therapy: weight decreased by 3.1 kg, fasting glucose by 2.3mmol/l, and HbA1c by 1.8% (p<0.0001 for all). Carotid IMT decreased from 1.55±0.45 mm to 1.36±0.31 mm (p=0.0003). Changes in carotid IMT did not correlate with changes in body weight, fasting glucose, or HbA1c. In conclusion, liraglutide has desirable actions on carotid IMT, a surrogate marker of subclinical atherosclerosis, in type 2 diabetes after only 4 months of therapy. This effect seems to be achieved by mechanisms independent of its effect on glucose metabolism. Further studies are needed to explore the nonglycemic athero-metabolic benefi ts of liraglutide and other agents of the GLP-1 analogues class.

419-PIncreased Incorporation of Antiplasmin into the Fibrin Network: A Possible Mechanism Behind Impaired Fibrinolysis in Patients With Type 1 DiabetesANNA ÅGREN, GUN JÖRNESKOG, PER-ERIC LINS, GRACIELA ELGUE, HÅKAN WALLEN, BJÖRN WIMAN, Stockholm, Sweden

Patients with type 1 diabetes have a tighter fi brin network, which has been associated with impaired fi brinolysis. Antiplasmin is an important fi -

brinolysis inhibitor. We thus investigated the incorporation of antiplasmin into fi brin network in patients with type 1 diabetes.237 age and sex matched patients (107 women) with type 1 diabetes, mean age 44±13 and diabetes duration 23±14 years were investigated. 78 healthy subjects (44 women) with mean age 48±10 years served as controls. We measured the incorpora-tion of antiplasmin into a fi brin clot (by an ELISA method), plasma levels of plasminogen activator inhibitor type 1 (PAI-1), tissue plasminogen activator (tPA)- PAI-1 complex, antiplasmin, plasmin-antiplasmin (PAP) complex, D-dimer and the glycemic control by HbA1c (Mono S; reference level <5.2%).More antiplasmin was incorporated into the fi brin network in patients (1.66± 0.31 μg/mL) vs controls (1.35 ± 0.18 μg/mL) (p<0.01) and no gender differ-ences were found. PAI-1 activity was lower in patients than in controls, median 2.2 (IQR 1.0-5.4 IU/mL) vs 4.3 (IQR 2.0-9.1 IU/mL) (p<0.01), while tPA-PAI-complex, PAP-complex and D-dimer were not signifi cantly different. In females, plasma levels of antiplasmin were lower in patients than in controls (81±11.4 vs 89±16 μg/mL; p<0.01). In patients, no gender differences were found in levels of PAI-1 activity and PAP, while women had higher levels of antiplasmin, D-dimer, lower tPA-PAI-complex (p<0.01, for all) and higher HbA1c (7.2±1.5%) than men (6.8±1.2%) (p<0.02).In conclusion, more antiplas-min is incorporated into fi brin network in patients with type 1 diabetes, sug-gesting a more stable fi brin. This could be one of the mechanisms adding to the risk for cardiovascular disease or to the cause of diabetes angiopathy. The reasons for the reduced levels of PAI-1 in the whole diabetes group and, in addition, the lower plasma levels of antiplasmin in women with type 1 diabetes are unclear.

420-PThe Role of SET7/9 and LSD1 on Macrovascular Complications in Patients With Type 2 DiabetesYONG XU, GUO CHEN, CHEN-LIN GAO, Luzhou, Sichuan, China

Covalent modifi cation of the histone represent a important mechanism by which cells control the structure and function of chromatin. Methylation on different histones has distinct functions. However, the effect of histone methylation on the diabetic nephropathy is still unknown. To explore the role of histone methyltransferase SET7/9 and Lysine-specifi c histone dem-ethylase 1(LSD1) on the diabetic macrovascular diseases, we selected 10 healthy volunteers randomly as Normal control group (NC group), 10 type 2 diabetes patients without macrovascular complication group (DM group) and 10 type 2 diabetes patients with macrovascular complications group (DV group). Peripheral blood sample was drew from each research object. West-ern blot and RT-PCR were used to detect the protein and mRNA expression of SET7/9,LSD1 and infl ammation factor, nuclear factor appa B (NF- B). The results showed that, compared with NC group, the expression of SET7/9 and NF- B was increased in DM group (P<0.05) and further increased in DV group (P<0.05). Compared with NC group ,the expression of LSD1 was decreased in DM group and further decreased in DV group (P<0.05,respectly). In con-clusions, imbalance of histone methyltransferase SET7/9 and demethylase LSD1 may be the important epigenetic mechanism of the occurrence and development of type 2 diabetic macrovascular complications.

Supported by: NNSF of China

421-PMYH9 Gene Polymorphisms are Associated With the Cerebral Blood Flow in Type 2 Diabetes PatientsCHAO LING, CHUNYOU CAI, BAOCHENG CHANG, FENGJIANG WEI, PING YU, WENTAO SHI, LIMING CHEN, WEI-DONG LI, Tianjin, China

Previous studies have shown that intensive glucose control does not af-fect the incidence of stroke in patients with type 2 diabetes mellitus (T2D). In order to decipher genetic components of diabetic cerebrovascular dis-eases, we carried out a quantitative trait association study in T2D patients among candidate gene polymorphisms and transcranial Doppler sonography (TCD) measurements.We studied 337 T2D individuals with diabetes. Cere-bral blood fl ow velocities were evaluated by TCD, and 54 phenotypes were documented, including the peak, minimum, and mean blood fl ow velocities of the bilateral cerebral arteries, vertebral artery, and the basilar artery. Outliers (> 4 SD) were deleted from the data set, and all phenotypes were adjusted by age within sex. We have genotyped 52 single-nucleotide poly-morphisms (SNPs) from 31 candidate genes. Quantitative allelic associations and haplotype analyses were performed with the PLINK program.Two SNPs of gene MYH9 (rs875726, rs735853) were associated with the peak veloc-ity of the right cerebral middle artery (P=0.0044, and 0.0019, respectively). SNPs rs875726, rs2009930, and rs375246 were associated with the mean velocity of the right-anterior and posterior cerebral artery (P=0.0030, 0.0037,

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and 0.0087, respectively). Haplotype analysis results showed that multiple haplotypes of MYH9 were associated with the TCD phenotypes.Our analysis indicates that polymorphisms of MYH9 are associated with blood fl ow veloc-ity of cerebral arteries in T2D patients. MYH9 polymorphisms might account for genetic susceptibility to cerebrovascular disease in diabetes patients.

Supported by: Chinese National Natural Science Foundation Grant 81070576 (W.D.L.)

422-PAssociations of Coronary Artery Calcifi cation and Possible M Value With Incidence of Coronary Heart Disease in Japanese Patients With Type 2 DiabetesTAKAYOSHI SASAI, NORIKO TAKEBE, MITSUTAKA ONO, HARUHITO TANEICHI, JO SATOH, Morioka, Japan

The Coronary artery calcifi cation score (CACS) is a known predictor of coronary heart disease (CHD) in Western countries. In this study, we ob-served a relationship of CACS with incidence of CHD and cerebrovascular disease (CVD) in Japanese patients with type 2 diabetes.Subjects were 589 patients with type 2 diabetes. CACS was measured by multi-detector com-puted tomography. The patients were divided into three groups according to the CACS (Agatston unit [AU]); no CAC (0 AU), mild-moderate CAC (1-400), and extensive CAC (> 400). The period to onset of CHD or CVD from the time of the CACS measurement was retrospectively calculated and analyzed by the Kaplan-Meier method and Cox proportional hazards model.Kaplan-Meier analysis indicated that incidence (per 1,000 person-years) of CHD was 0, 7.6, and 11.6 in the no CAC, the mild-moderate CAC, and the extensive CAC, respectively (Log-rank test: P = 0.045), whereas that of CVD was 0, 7.6, and 11.6 (Log-rank test: P = 0.055). By the Cox proportional hazards model, adjusting for multiple covariates, the CACS is an independent predictor for CHD (hazard ratio [HR] = 1.002 per 1 AU; 95% confi dence interval [CI], 1.000 - 1.003, P < 0.05), whereas no independent predictor was obtained for CVD. In addition, the M value, a marker of blood glucose variability, was a marginally signifi cant predictor for CHD (HR, 1.017; 95% CI, 0.999 - 1.035; p = 0.059). CACS is a signifi cant predictor for CHD in Japanese patients with type 2 diabetes. The M value may also be a predictor.

423-PLeft Ventricle Hypertrophy With Preserved Systolic Dysfunction, a Common Feature of Cardiomyopathy in Type 2 Diabetic Patients—Additional Role of Ischemia and Cardiac Autonomic NeuropathyMINH TUAN NGUYEN, ISABELLE PHAM, EMMANUEL COSSON, ISABELA BANU, SABRINA CHIHEB, PAUL VALENSI, Bondy, France

Diabetic cardiomyopathy is defi ned by structural or functional myocardial alterations related to diabetes after exclusion of hypertension and coronary disease. Aim: to estimate in asymptomatic type 2 diabetic patients the prev-alence of left ventricle hypertrophy (LVH) and systolic dysfunction in those free of silent myocardial ischemia (SMI) and hypertension and examine the role of SMI, silent coronary stenoses (CS) and cardiac autonomic neuropathy (CAN) in myocardial alterations.We included 656 patients, 59.7±8.7 years, HbA1c 8.7±2.1%, with diabetes for 14±8 years and other risk factors but no coronary or heart failure history. They were screened for LVH and systolic dysfunction (hypokinesia, ejection fraction) using transthoracic echocar-diography, SMI (abnormal stress scintigraphy and/or echocardiography), CS (angiography performed in SMI+ patients) and CAN ( 1 abnormal test among deep breathing, lying-to-standing, Valsalva).SMI and CS were detected in 206 and 71 patients, respectively, and CAN in 199 of 282 patients tested. LVH prevalence was high (26%) among patients free of SMI and hypertension, did not differ according to SMI and/or CAN, but was higher in the patients with CS (44% vs 32% in the others; p<0.05). Hypokinesia affected only 3 of the 109 patients free of SMI and hypertension. Hypokinesia was more prevalent in the patients with both SMI and CAN than in those free of SMI and CAN (SMI- CAN-/SMI+ CAN-/SMI- CAN+/SMI+ CAN+: 2/0/8/17%, respectively; p<0.05) with a lower ejection fraction (70±6/69±6/68±8/65±12%; p<0.01). In multivariate analysis, SMI+ CAN+ status was associated with hypokinesia (OR=4.1[1.4-12.2]), independently of hypertension.In conclusion, in high risk type 2 diabetic patients, asymptomatic cardiomyopathy is mostly character-ized by LVH with preserved systolic function; systolic dysfunction must lead to suspect the combined role of SMI and CAN.

424-PAdipose Triglyceride Lipase and Comparative Gene Identifi ca-tion-58 are Downregulated in the Hearts of Diabetic Fatty db/db Mice: A Possible Animal Model for Diabetes-Related Triglyceride Deposit CardiomyovasculopathyTOMOAKI INOUE, KUNIHISA KOBAYASHI, TOYOSHI INOGUCHI, NORIYUKI SONO-DA, EIICHI HIRATA, HISASHI YOKOMIZO, YOHEI MINAMI, RYOICHI TAKAYANAGI, Fukuoka, Japan

Adipose triglyceride lipase (ATGL) was recently identifi ed as a rate limiting triglyceride (TG) lipase and ATGL activity is stimulated by comparative gene identifi cation-58 (CGI-58). Mutations in the human ATGL or CGI-58 gene are associated with neutral lipid storage diseases characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy (TGCV), shows massive TG ac-cumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial TG content is signifi cantly higher in patients with prediabetes or diabetes and is associated with impaired left ventricular diastolic function. Therefore, we investigated roles of ATGL and CGI-58 in the development of myocardial steatosis in the diabetic state or possibility for diabetes-related TGCV.Triglyceride contents in the hearts of diabetic fatty db/db mice are higher than in those of db/+ control mice. His-tological examinations assessed with oil red O staining showed marked lipid deposition within the hearts of db/db mice. Next, we determined expres-sion of genes and proteins that affect triglyceride metabolism and found that ATGL and CGI-58 expression were decreased in the hearts of db/dbmice. Also, we found increased expression of genes that affect triglycer-ide synthesis (SREBP1c, monoacylglycerol acyltransferases and diacylglyc-erol acyltransferases). In addition, we analyzed the expression levels of key modulators of apoptosis and PKC activity. It was found that Bcl-2 levels were lower and phosphorylation of PKC was increased in diabetic hearts. These results suggest that reduced ATGL and CGI-58 expression with increased TG synthesis may induce myocardial steatosis and cardiac apoptosis, leading to cardiomyopathy in the diabetic state.

425-PSerum FGF21 Concentration is Associated With Cardiometabolic Risk Factors and Pericardial Fat Accumulation, Independent of Obe-sity, But Not With Current Coronary Artery DiseaseYENNA LEE, EUN SHIL HONG, HAK C. JANG, SUNG HEE CHOI, EUN KY KIM, MIN KYEONG KIM, SOO LIM, JUNG HEE KIM, SEON MEE KANG, Seoungnam-Si, Gyeonggi-Do, Republic of Korea, Seoul, Republic of Korea

Fibroblast growth factor 21 (FGF21) is an emerging metabolic regulator associated with glucose and lipid metabolism. These associations have been demonstrated in humans, but have been largely confounded by the effects of obesity. We investigated the association between serum FGF21 concentrations and glucose and lipid metabolism, coronary artery disease, and pericardial fat deposition in subjects strictly matched for obesity pa-rameters.We enrolled 189 patients who had undergone cardiac multide-tector coronary computed tomography. We measured various metabolic parameters and serum FGF21 levels within body mass index (BMI)-matched groups. Correlations and linear regressions were analyzed between serum FGF21 levels, pericardial fat volumes, and various cardiometabolic param-eters. Serum FGF21 levels were compared in patients with and without diabetes, metabolic syndrome, and coronary artery disease.Serum FGF21 levels were signifi cantly higher in BMI-matched patients with metabolic syndrome (107.2 ± 83.6 vs. 82.1 ± 67.4 ng/L without metabolic syndrome, P < 0.05) but not those with diabetes (84.32 ± 56.39 vs. 96.30 ± 98.91 ng/L without diabetes, P = 0.300) or coronary artery disease (89.6 ± 65.8 vs. 84.2 ± 83.1 ng/L without coronary artery disease, P = 0.633). Serum FGF21 levels correlated positively with triglycerides, low-density lipoprotein-cholesterol, insulin, HOMA-IR, and pericardial fat volume. They showed an independent association with pericardial fat volume ( = 0.108 ± 0.052, P < 0.05).Serum FGF21 concentrations are signifi cantly associated with lipid profi les, insulin resistance, pericardial fat volume, and metabolic syndrome, independent of obesity, but not with overt coronary artery stenosis or diabetes.

Supported by: National Project for Personalized Genomic Medicine (PGM21), #A111218

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426-PProgression of Vascular Calcifi cation is Increased With Statin Use in the Veterans Affairs Diabetes Trial (VADT)ARAMESH SAREMI, GIDEON BAHN, TOM MORITZ, PETER D. REAVEN, Phoenix, AZ, Hines, IL

The effect of statins on progression of vascular calcifi cation in type 2 diabetes (T2DM) remains uncertain. As part of the RACED sub-study of the VADT, the association between statin use and progression of coronary and abdominal aortic artery calcium (CAC and AAC) was determined in 197 T2DM patients. Progression (average follow-up of 4.6 yrs) was estimated as the difference between square root transformation of the follow-up and base-line volumetric scores (mm3). Information regarding concomitant medication was updated at each visit (14-28 visits).Participants who reported statin use

50% of visits (n= 36) were compared with those who reported statin use > 50% (n=161). Except for a higher prevalence of CVD events history in those with statin use > 50% (41% vs. 19% P<0.01), there were no signifi cant differ-ences between the two groups at baseline. Despite improved lipids at the end of the study, CAC progression was signifi cantly (P <0.001) accelerated in those with more frequent statin use (Figure). Adjustments for age, dura-tion of diabetes, hypertension, CVD events, baseline CAC, race/ethnicity, treatment assignment (intensive vs. standard), time on study and change in BMI, TC/HDL, systolic, diastolic blood pressure, did not change the results. Furthermore, after adjustment for all of the above covariates, every 10% increase in frequency statin use (continuous variable), was associated with a 0.33 ± 0.01 mm3 increase in CAC progression (P =0.04). Exclusion of those with prior or new CVD events did not change results. In conclusion, statin use is associated with accelerated CAC progression in T2DM patients.

Supported by: Carl T. Hayden Medical Research Foundation

427-PHyperglycemic Induced Myocardial Mitochondrial DNA (mtDNA) Damage is Mediated through Dysfunctional Mitochondrial Topoi-somerasesSTEVEN HICKS, BRIAN PITEO, JAMIE MATHEW, DIMTRI LAURENT, MARIA MIT-RY, NAZAR LABINSKYY, JOHN G. EDWARDS, Valhalla, NY

Mitochondrial dysfunction has a signifi cant role in the development and complications of diabetic cardiomyopathy. Less clear are the mechanisms that lead to failure since the mitochondria have defense mechanisms to manage diabetic-induced oxidant stress. We have reported previously in the H9c2 cell line that chronically elevated glucose induced mtDNA damage was linked to dysfunctional mitochondrial topoisomerase activity. The present study extends those observations to include both neonatal cardiomyocytes as well as the type 2 diabetic Goto-Kakizaki (GK) rat. Diabetes signifi cantly decreased cytochrome oxidase activity in GK LV compared to Wistar LV and decrease in ATP production in cardiomyocytes. Mitochondrial dysfunc-tion was associated with signifi cantly increased mtDNA damage without a change in mitochondrial copy number. TTGE analysis of the Cytochrome Oxi-dase Subunit 3 identifi ed several different nucleotide substitutions in the GK LV that altered the primary protein sequence; none were observed in Wistar LV. In isolated mitochondria, diabetes signifi cantly increased mitochon-drial dependent DNA cleavage. Immunoprecipitation with a mitochondrial topoisomerase I antibody partially blocked mitochondrial dependent DNA

cleavage indicating a functional role for this enzyme. Incubation of mito-chondrial extracts with 50 μM H2O2 increased mitochondrial topoisomerase dependent DNA cleavage, implicating ROS as a modifi er of topoisomerase function. Separate from a direct impact of oxidative stress on mtDNA, ROS-induced alteration of mitochondrial topoisomerase function propagated mtDNA damage. These fi ndings indicate a signifi cant role for mitochondrial topoisomerase function in the development and complications of diabetic cardiomyopathy.

Supported by: NIH (HD065551, HL43023)

428-PPreserved Cardiac Structure, Function and Sympathetic Innerva-tion in Subjects With Type 1 Diabetes of Extreme DurationOMAR ASGHAR, CHRISTOPHER MILLER, PARTHIBAN ARUMUGAM, MATTHIAS SCHMITT, IAN ARMSTRONG, RAYAZ A. MALIK, Manchester, United Kingdom

The cardiac phenotype in Medalists (individuals with type 1 diabetes (T1DM) ~50 years) has not been previously described. We characterized cardiac structure, sympathetic innervation and autonomic function in this subgroup.12 Caucasian subjects (62±8 years, M:F 4:8) with T1DM of long duration (48.9±5 years) and no clinical history, ECG features or symptoms of cardiac disease were studied. Ischemia was excluded using vasodilator stress CMR. LV structure, volumes, function and myocardial deformation (standard CMR sequences) and diffuse fi brosis (T1 mapping) were assessed. Cardiac sympathetic innervation and autonomic function were assessed with I-123 MIBG scintigraphy and cardiovascular refl ex tests, respectively.Mean 15 year metabolic profi le indicated suboptimal glycemic control (HbA1c 8.5±1.0%), favorable lipid profi le (total cholesterol 4.7±0.3, HDL 1.8±0.5, LDL 2.2±0.5, triglyceride 1.1±0.4 mmol/l) and mean eGFR of 76±16.6 ml/Kg/min. Mean BP was 146/73±18/10 mmHg. LV mass (83.8±20.2 g), mass index (LVMI) (44.5±7.9g/m2), stroke volume (94.3±18.9 ml) and ejection frac-tion (70.9±6.7%) were normal. Global circumferential LV strain (-20.7±3 vs -20.6±1.0%, p=NS) and diffuse fi brosis (0.31±0.04 vs 0.27±0.01%, p=NS) did not differ signifi cantly from controls. Sympathetic innervation (late HMR) (1.6±0.2, normal >1.6) and cardiac autonomic function (LFA/RFA (1.29±1.57), E:I ratio (1.1±0.1) and 30:15 ratio (1.1±0.1) were normal and LF/HF (1.4±1.6) and valsalva ratio (VR) (1.1±0.1) were only mildly reduced.Patients who have survived ~ 50 yrs of Type 1 diabetes (Medalists) exhibit normal cardiac struc-ture and function without fi brosis and sympathetic denervation with minimal autonomic dysfunction despite suboptimal glycemic and BP control, but fa-vorable lipid profi le. These results extend previous limited data on medalists and microvascular complications to include cardiac disease, supporting the need for further studies.

Supported by: NIHR

429-PComparative Effectiveness of Percutaneous Stenting Versus By-pass Surgery Among Adult Type 2 Diabetes Patients With Periph-eral Arterial Disease: A Nationwide Cohort StudyCHIA-HSUIN CHANG, JOU-WEI LIN, LI-CHIU WU, MEI-SHU LAI, LEE-MING CH-UANG, Taipei, Taiwan

Background: The long-term comparative effectiveness of percutaneouss-tenting and bypass surgery for diabetic patients with peripheral arterydis-ease remained unclear.Methods: A retrospective cohort study using the Tai-wan National Health Insurance claims database was conducted to identify adult patients with type 2 diabetes mellitus and who received peripheral ar-tery bypass surgery (n=5,652) or stenting (n=659) during 2000-2007. Primary outcome was lower extremity amputation, further classifi ed as above-knee (thigh) orbelow-knee (leg, ankle, and foot). Patients were followed from the date of index hospitalization to the earliest of outcome occurrence, death, or December 31 2008. A propensity-score matched cohort was created for com-parison. We estimated adjusted hazard ratios and 95% confi dence intervals (CIs) with Cox proportional hazards models controlling for age, gender, and covariates that were imbalanced after propensity score matching.Results: The incidence rate of lower limb amputation per 1,000 person-years was 94.5 for bypass surgery recipients (186 cases) and 57.8 for stent recipients (49 cases) during an average follow-up of 2 years. A signifi cant decrease in risk was found for patients receiving percutaneous stenting (adjusted haz-ard ratio 0.67; 95% confi dence interval: 0.48-0.92). Risk was signifi cantly reduced for below-knee amputation, but not for above-knee. In contrast, a signifi cantly increased risk for stenting as compared with bypass surgery was observed for patients with chronic renal failure (adjusted hazard ratio 1.97; 95% confi dence interval: 1.00-3.91) for future amputation.Conclusions: Percutaneous stenting was associated with a signifi cantly lowered risk of lower limb amputation for diabetic patients withperipheral artery disease.

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However, bypass surgery may provide better outcome for patients with chronic renal failure.

430-PDiabetics Show Better Collaterals toward the Culprit Vessel at the Time of Their First Acute Coronary EventDARIO PITOCCO, FRANCESCO ZACCARDI, GIAMPAOLO NICCOLI, ANDREA LEO, NICOLA COSENTINO, DOMENICO D’AMARIO, CARLO TRANI, ITALO PORTO, FRANCESCO BURZOTTA, FILIPPO CREA, GIOVANNI GHIRLANDA, Rome, Italy

Diabetes is associated with a more extensive and severe coronary ar-tery disease (CAD); however similar age of presentation of non-diabetics has been described despite a more extensive CAD at the time of a fi rst acute coronary syndrome (ACS), thus suggesting that protective mecha-nisms may act in diabetics. We evaluated angiographic CAD severity and quality of the collateral circulation toward the culprit vessel according to the diabetic status in consecutive patients at their fi rst ACS. One-hundred and sixty-seven consecutive patients undergoing coronary angiography be-cause of non-ST-elevation (NSTE)-ACS, as their fi rst clinical manifestation of CAD, and showing obstructive atherosclerosis were prospectively included. CAD severity was graded according to Bogaty’s stenosis score and extent index, while collateral fi lling was graded according to Rentrop classifi cation toward culprit vessel showing a TIMI fl ow 2. Forty-seven patients were diabetics. Diabetics and non-diabetics were similar for age (69±12vs66±14, p=0.11). Multivessel disease rate was higher in diabetics compared to non-diabetics (68%vs42%, p=0.003). At multivariate analysis both stenosis score and extent index were predicted by diabetes (p=0.001), stenosis score was predicted by male sex (p=0.03) and extent index by lack of statins (p=0.05). Good collaterals (Rentrop 2-3) were observed in 28 over 83 culprit vessel with a TIMI fl ow 2. At multivariate analysis, good collaterals were predicted by diabetes (OR 2.33, 95% CI 1.45-6.54, p=0.002) and inversely by extent index (OR 0.87, 95% CI 0.51-0.96, p=0.03). In conclusion, diabetics show at their very fi rst ACS a more extensive and severe coronary atherosclerosis as compared to non diabetics, despite a similar age of presentation. However, angiographically visible collaterals toward the culprit vessel are better de-veloped in diabetics, suggesting that collaterals may delay onset of a fi rst ACS acting as a protective mechanisms.

431-PEffects of Exenatide vs. Metformin on Endothelial Function in Obese Patients With Pre-DiabetesAARON S. KELLY, RICHARD M. BERGENSTAL, J.M. GONZALEZ-CAMPOY, HAR-OLD KATZ, ALAN J. BANK, Minneapolis, MN, St. Louis Park, MN, Eagan, MN, St. Paul, MN

The effect of glucagon like peptide-1 (GLP-1) receptor agonist treatment on endothelial function in humans has not been described. Therefore, we conducted a study comparing the acute and chronic effects of the GLP-1 receptor agonist exenatide vs. metformin on endothelial function in patients with obesity and pre-diabetes. We performed a randomized, open-label, clinical trial in 50 individuals (mean age 58.5 ± 10.0; 38 females) with abdom-inal obesity and either impaired fasting glucose, elevated HbA1c, or impaired glucose tolerance (IGT) who were randomized (1:1) to receive 3-months of ex-enatide or metformin. Microvascular endothelial function (primary endpoint), assessed by digital reactive hyperemia (reactive hyperemic index: RHI), C-reactive protein (CRP), circulating oxidized LDL (oxLDL), and vascular cell adhesion molecule-1 (VCAM-1) were measured at baseline and 3-months. Seven subjects with IGT participated in a sub-study comparing the effects of acute administration of exenatide and metformin on postprandial endothe-lial function. The groups were similar at baseline for all measured variables. There were no differences for the change in RHI ( exenatide: 0.01 ± 0.68 vs. metformin: -0.17 ± 0.72, P = 0.348), CRP ( exenatide: -0.4 ± 2.2 mg/L vs. metformin: -0.4 ± 2.2 mg/L, P = 0.987), oxLDL ( exenatide: -0.1 ± 41.5 U/L vs. metformin: -16.5 ± 30.4 U/L, P = 0.123), or VCAM-1 ( exenatide: 10.4 ± 83.2 ng/mL vs. metformin: -15.3 ± 101.3 ng/mL, P = 0.336) between exenatide and metformin treatment. Triglycerides were reduced more with exenatide compared to metformin ( exenatide: -25.5 ± 45.7 mg/dL vs. metformin: -2.9 ± 22.8 mg/dL, P = 0.032). In the sub-study, there was no difference in postprandial RHI between exenatide and metformin. Three months of exenatide therapy had similar effects on microvascular endothe-lial function, markers of infl ammation, oxidative stress, and vascular activa-tion, as metformin, in patients with obesity and pre-diabetes.

Supported by: Amylin and Eli Lilly

432-PTreatment of Obstructive Coronary Artery Disease With the Reso-lute Zotarolimus-Eluting Stent in Patients With Diabetes MellitusSCOTT W. LEE, JORGE BELARDI, MARTIN B. LEON, LAURA MAURI, IAN T. MER-EDITH, FRANZ-JOSEF NEUMANN, SHIGERU SAITO, PATRICK W. SERRUYS, PETR WIDIMSKY, STEPHAN WINDECKER, ALAN YEUNG, FRANCINE R. KAUFMAN, SIGMUND SILBER, RESOLUTE CLINICAL INVESTIGATORS, Northridge, CA, Bue-nos Aires, Argentina, New York, NY, Boston, MA, Clayton, Australia, Bad Krozingen, Germany, Kamakura, Japan, Rotterdam, The Netherlands, Prague, Czech Republic, Bern, Switzerland, Palo Alto, CA, Munich, Germany

Current guidelines support the use of drug-eluting stents (DES) for treat-ment of coronary artery disease in patients with diabetes, but no specifi c DES is yet indicated for use in the US in this high-risk population. We evalu-ated 1-year outcomes in patients with diabetes treated with the Resolute stent. A prespecifi ed analysis cohort to obtain the indication for use of the Resolute DES in patients with diabetes in the US included 878 patients with Type 2 diabetes and 1903 patients without diabetes from 5 RESOLUTE trials. Patients were treated for 1 or 2 lesions in separate vessels. Safety outcomes include the composite of cardiac death or myocardial infarction related to the treated vessel (TVMI), and stent thrombosis. Effi cacy is reported as the rate of target lesion revascularization. Target lesion failure, a composite endpoint including cardiac death, TVMI, and target lesion revasculariza-tion, represents stent-related outcomes. Of 878 patients with diabetes, 250 were taking insulin. At baseline, the use of insulin did not result in signifi cant differences between groups except that more women were taking insulin (43.6% vs 29.6%, p<0.001) and had hypertension (91.6% vs 86.0%, p=0.023). Major outcomes at 1 year are in the Table. There were no statistically sig-nifi cant differences in the composite endpoint between patients not taking insulin, and patients without diabetes (5.0% vs 4.9%, p=NS). The Resolute DES was shown to be as safe and effective in non-insulin requiring patients with diabetes as in patients without diabetes. Both groups had similar car-diovascular outcomes at 1 year.

Cardiac Outcomes with the Resolute DESOutcome % (n) Patients

WithoutDiabetes(n=1903)

Patients With Diabetes Not Taking Insulin

(n=628)

Patients With Diabetes

Taking Insulin (n=250)

Cardiac death or target vessel MI 3.2 (59) 2.6 (16) 6.1 (15)Target lesion revascularization 2.0 (38) 2.6 (16) 5.3 (13)Target lesion failure 4.9 (92) 5.0 (31) 10.6 (26)Stent thrombosis 0.3 (6) 0.2 (1) 0.8 (2)

Supported by: Medtronic, Inc.

433-PBlood Pressure Trajectories before Death in Patients With Type 2 DiabetesSANJOY PAUL, KERENAFTALI KLEIN, GIJO THOMAS, KAMLESH KHUNTI, Bris-bane, Australia, Leicester, United Kingdom

The aim of this study is to examine the trajectory of blood pressure (BP) over two yrs before death or censoring and its association with mortality in patients (pts) with T2DM.A cohort of 19966 pts with minimum 3 yrs of diabetes duration (DD) was selected from the UK General Practice Research Database (1990-2008), who had four consecutive 6-monthly BP measures over 2 yrs immediately before death or censoring. In the cohort, 46% were female, mean (SD) age of 68 (11) years and 14% had at least one cardiovas-cular event (CVE) before diagnosis of diabetes. During 6.5 years of median duration of follow-up, 7% pts died, of whom 83% were aged 68 yrs, 53% and 20% pts had CVE respectively in dead and alive groups.During 2 yrs of follow-up, pts’ systolic and diastolic BP trajectories were signifi cantly higher by 5 and 2 mmHg respectively in those who died, compared to those who were alive (Fig 1). These estimates were adjusted for the effects of age, DD, sex, CVE, glucose-lowering and anti-hypertensive medication usages, baseline weight and HbA1c.Compared to pts with systolic BP level 125 - 130 mmHg, pts with BP < 110 and > 140 mmHg had signifi cantly higher mortality risk by 240% (adjusted odds ratio : 2.4, 95% CI: 1.9, 3.1) and 210% (adjusted odds ratio: 2.1, 95% CI: 1.8, 2.5) respectively. Mortality risk was not higher in patients with BP within 130 - 140 mmHg (Fig 1).The population level analy-ses of BP trajectories leading up to death are revealing, and provide clear message in terms of the requirement for intensive BP control and related management policies at primary care level. Our fi ndings also establish the J-shaped relationship between systolic BP and mortality.

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434-PGenetic Variability in AMPK 1 Gene Is Associated With Risk of Coronary Artery Disease in Chinese Type 2 DiabeticsXIAOWEI MA, JIANWEI ZHANG, RUIFEN DENG, SHAN DING, XIAODAN MA, NAN GU, GE BAI, JIANPING LI, XIAOHUI GUO, Beijing, China

The increasing evidence suggested that AMP-activated protein kinase (AMPK) played a critical physiological role in the cardiovascular system. To assess the possible association between the genetic variability in AMPK 1gene and the risk of cardiovascular disease in type 2 diabetics, we selected 5 tag-SNPs (rs3805489, rs249428, rs133671707, rs837103 and rs3805486) at AMPK 1 locus according to CHB database from HapMap R#27 (r2<0.8 and MAF 0.05) and genotyped 400 unrelated Han subjects with type 2 diabetes, 255 individuals with coronary artery diseases(CAD+) as cases and 154 controls (CAD-) by using PCR-RFLP assay. We found that minor al-lele C at rs3805489 was protective from CAD in type 2 diabetics compared to allele A (OR=0.688, 95%CI 0.491-0.963, p=0.035) . Interestingly, when we stratifi ed the data by smoking status, the results showed that smoking status had a synergistic effect with rs3805489 on CAD risk (p = 0.036, for interaction). The carriers of genotype AA at rs3805489 had a higher risk of CAD compared to non-carriers in the subjects who ever smoked (OR=3.069, 95%CI =1.529-6.161, p = 0.002; OR’=2.615,95%CI =1.255-5.453, p’ = 0.010 after adjusted for other known CAD risk factors. The smokers with genotype AA at the SNP had 3 times risk of CAD as high as the non-smokers with genotype AC or CC (OR’=3.312, CI=1.533-7.155, p’=0.002) (Fig.1). Our fi ndings suggest that genetic variability at AMPK 1 locus may be associated, and interact with smoking on the risk of CAD in type 2 diabetes in the Chinese Han population.

Supported by: National 973 Project (2006CB503903, 2006CB503908)

435-PSlower Psychomotor Speed Predicts Faster Gray Matter Volume Loss Within the Prefrontal Cortex Over a Three Year Follow-Up TimeCATERINA ROSANO, CHRISTOPHER RYAN, JUDITH SAXTON, RACHEL MILLER, TIMOTHY HUGHES, ELSA S. STROTMEYER, TREVOR J. ORCHARD, Pittsburgh, PA

Adults with T1D often show psychomotor speed slowing. Smaller left dor-solateral prefrontal cortex (ldPFC) is associated with slower psychomotor speed in older adults independent of other chronic conditions, including dia-betes. We conducted this study to identify potential determinants of rate of change in ldPFC over a 3-year period of time.High-resolution brain magnetic resonance images (MRI, 3-Tesla) and Digit Symbol Substitution Test (DSST) scores were obtained concurrently in 2008 from 9 participants (50+ 5.3 yrs old, 2 men) in the Epidemiology of Diabetes Complications Study, an ongo-ing prospective childhood onset (1950-1980) cohort followed biennially since 1986-88. MRI were repeated in 2011 and changes in gray matter volumes of total brain and of ldPFC were computed. Age (or disease duration), cumula-tive HbA1C since diagnosis, insulin dose, retinopathy, urinary albumin/crea-tinine ratio and number of hypoglycemic episodes were obtained concurrent with the 1st MRI.After adjusting for total brain volume change, slower DSST was signifi cantly associated with greater percent gray matter volume loss in the ldPFC (standardized beta = 4.8%, p=0.03) and explained 32% of the variance (F change: 7.954, p=0.03). Associations between other diabetes-related measures and dLPFC volume changes were all not signifi cant (p>0.2 for all). Slower DSST was not signifi cantly associated with total brain gray matter volume loss (standardized beta = 0.4%, p=0.9).This is the fi rst report of an association between a common manifestation of cognitive dysfunction in T1D middle-aged adults and longitudinal rate of focal volumetric reduc-tions within the frontal lobe. ldPFC may be a brain region particularly vul-nerable to atrophy in T1D because of its non-anastomosing vascularization. Future studies should examine potential applications of DSST as an indicator of focal accelerated brain aging in T1D.

Supported by: R01 DK034818-25 and R01 DK089028-01

436-PThe Association Between G174C Polymorphism on Interleukin-6 Gene With Macrovascular Complications in Subjects With Type 2 Diabetes MellitusSTAVROULA PAPAOIKONOMOU, NIKOLAOS TENTOLOURIS, DIMITRIS TOUSOU-LIS, DIMITRIS PAPADOGIANNIS, ANTIGONI MILIOU, GEORGE HATZIS, NIKOLAOS PAPAGEORGIOU, KOSTAS MAKRILAKIS, STAVROS LIATIS, CHRISTODOULOS STEFANADIS, Athens, Greece

Low grade infl ammation is involved in the pathogenesis of atherosclero-sis. Genes encoding for infl ammatory cytokines such as iterleukin-6 (IL-6) are candidates for predisposing to the risk of atherosclerosis. Subjects with type 2 diabetes mellitus (T2DM) are vulnerable to the development of mac-rovascular disease. The aim of this cross-sectional study was to examine for association between Guanine/Cytosine (G>C) polymorphism at position -174 of the IL6 gene (IL6G174C polymorphism) with the prevalence of macro-vascular complications in subjects with T2DM. A total of 431 subjects with T2DM (mean age 66.5 ±9.96 years, male n=218, female n=213) were exam-ined. IL6G174C polymorphism was detected using polymerase chain reaction and appropriate restriction enzyme. A detailed history and examination was performed for the diagnosis of macrovascular complications. The genotype distribution was 49.1% GG, 26.8% GC and 24.1% CC, with no signifi cant gen-der difference. The prevalence of coronary artery disease (26.7%; 23.6%; 24.2%), peripheral arterial disease (37.6%, 35.5%, 36.4%), stroke (14.9%; 12.7%, 13.1%) or any macrovascular disease (55.9%; 42.7%; 48.5%) was not signifi cantly different (P>0.05) among participants with GG, GC or CC geno-type, respectively. However, participants carrying the C allele (GC and CC) had signifi cantly lower prevalence of any macrovascular complication than GG homozygotes (45.5% vs 55.9% respectively, p=0.034). In addition, this association remained signifi cant by multivariate analysis after adjustment for gender, age, duration of diabetes, body mass index, smoking, hyperten-sion, lipids, HbA1c, and glomerular fi ltration rate, (GC+CC vs GG: odds ra-tio= 0.55, 95% confi dence intervals 0.35-0.87, p= 0.012). In conclusion, in subjects with T2DM expression of the C allele by IL6G174C polymorphism protects from the development of macrovascular disease.

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437-PResistin, Major Cardiovascular Events and All-Cause Mortality in Patients With Type 2 DiabetesCLAUDIA MENZAGHI, SIMONETTA BACCI, LUCIA SALVEMINI, CHRISTINE POW-ERS, GIUSEPPE PALLADINO, ANTONELLA MARUCCI, CONCETTA DE BONIS, DAVIDE MANGIACOTTI, GRAZIA FINI, SABRINA PRUDENTE, SALVATORE DE COSMO, ALESSANDRO DORIA, VINCENZO TRISCHITTA, San Giovanni Rotondo, Italy, Boston, MA, Rome, Italy

High serum resistin has been associated with increased risk of cardio-vascular disease (CVD) in the general population. Only sparse and confl ict-ing results have been reported in patients with type 2 diabetes (T2D) of Asian ancestry. Our aim was to study the role of serum resistin on major cardiovascular events and all-cause mortality in European patients with T2D.To this purpose two prospective studies were carried out. 1) The Gar-gano Heart Study-GHS-prospective design for major cardiovascular events (i.e. cardiovascular death, non fatal myocardial infarction-MI-and non fatal stroke), of 350 patients with T2D and CAD, (follow-up=1,108 person-year). 2) The Gargano Mortality Study (GMS) for all-cause mortality, of 1,028 patients (follow-up=5,659 person-year). Serum resistin levels were available in all the GHS-prospective patients and in 783 participants from the GMS. The corre-lation between serum and mRNA resistin levels in white blood cells (WBC) from 68 patients belonging to the GMS has been also evaluated.Among the GHS-prospective participants, 32 cardiovascular deaths, 3 non fatal MIs and 8 non fatal strokes occurred (annual incidence rate 0.04). Serum resistin pre-dicted major cardiovascular events with age, sex and smoking habit adjusted HR per SD increment of 1.35 (95% CI: 1.14-1.59).Among the GMS participants 150 deaths occurred (annual incidence rate 0.027). Serum resistin predicted all-cause mortality with adjusted HR per SD increment of 1.17 (95% CI: 1.07-1.27). These associations were unaffected by further adjustment for BMI, diabetes duration and current therapy. WBC RETN mRNA and serum resistin were positively correlated (R=0.317, p=0.005).High serum resistin (very likely a consequence of increased resistin mRNA expression) is a strong, indepen-dent risk factor for CVD and all-cause mortality in T2D. This points to resistin as a novel biomarker in improving predictability of CVD and premature death in such patients.

Supported by: EFSD/Pfi zer

438-PA Genome-Wide Association Analysis of Coronary Calcifi cation in the Diabetes Heart StudyAMANDA J. COX, MAGGIE C. NG, JIANZHAO XU, J.J. CARR, BARRY I. FREED-MAN, CARL D. LANGEFELD, DONALD W. BOWDEN, Winston-Salem, NC

Studies of genetic risk factors for cardiovascular disease (CVD) in individu-als with type 2 diabetes mellitus (T2DM) may reveal biological pathways which explain the increased risk for macrovascular complications in T2DM. The aim of the current study was to examine association signals for sub-clinical CVD in analyses restricted to individuals with T2DM.A genome-wide association study (GWAS) for coronary artery calcifi ed plaque (CAC) was performed in the family-based Diabetes Heart Study (DHS). Genotyping was completed using the Affymetrix 5.0 Array. Association analysis was per-formed using a variance components analysis implemented in SOLAR v6.3.4 for all n=1181 European American DHS participants (ALL) and then restricting to the n=989 T2DM affected DHS participants only (T2D). Poorly performing SNPs (CR<0.95; Hardy-Weinberg p-value<1x10-6; MAF<0.01) were excluded from further analyses. SNPs with association p-values <1x10-4 in either ALL or T2D were selected and association signals compared between the two analyses.Overall GWAS fi ndings for ALL included n=43 SNPs with associa-tion p-values <1x10-4 with the top hit on Chr6q15 (rs1144159; p=1.18x10-7;MAP3K7) and a lack of association across the previously well-documented 9p21 region. When comparing association signals between ALL and T2D, n=65 SNPs met the threshold of p<1x10-4 in either ALL or T2D with more than half of the selected SNPs (n=35) showing stronger association signals in the T2D analysis. This included n=7 SNPs from the region 6q15 encompassing the genes CGA and ZNF292. Finally, of those SNPs with stronger associa-tion signals in the T2D analysis, 23/35 SNPs failed to meet the threshold of p<1x10-4 in ALL.These data suggest there is potential for novel GWAS signals for coronary calcifi cation in T2DM. A meta-analysis of vascular calcifi cation signals from T2DM cohorts should further elucidate these relationships and identify variants contributing to risk for vascular calcifi cation in T2DM.

439-PPredictors of Coronary Artery Disease in Type 1 Diabetes (T1D) Dif-fer by Level of Glycemic ControlRACHEL G. MILLER, TREVOR J. ORCHARD, Pittsburgh, PA

Though glycemia has not been a consistent predictor of coronary artery disease (CAD) in T1D, white blood cell count (WBC), hemoglobin (Hgb), and erythropoietin (EPO), have recently emerged as potential risk factors. To ex-plore whether these factors may partially explain why individuals with good glycemic control remain at high risk for CAD, we examined risk factors for CAD incidence by level of HbA1c.Data are from 520 individuals in the Epide-miology of Diabetes Complications (EDC) study of childhood-onset (<17 years old) T1D without baseline CAD, but with measured endogenous EPO level (baseline mean age 27.7, T1D duration 19.6 yrs, 50.2% female). Incident CAD (n=130, 25%) was defi ned as a confi rmed event (CAD death, myocardial in-farction, revascularization/blockage 50%, ischemic ECG, or EDC physician-diagnosed angina) occurring prior to the 18-yr exam. The EDC cohort was divided into 2 groups based on baseline median HbA1c <8.5% and 8.5%. Separate proportional hazards models were fi t by HbA1c level. Hazard ratios (HR) are per standard deviation.There was no difference in CAD incidence between glycemic groups (~25% in both). However, higher ln(EPO) (HR=4.5, 95% CI 1.5, 13.4) and WBC (1.6, 95% CI 1.3, 12.1) were univariately only as-sociated with CAD incidence in those with HbA1c <8.5%, while higher fi -brinogen (1.4, 95% CI 1.2, 1.7) was associated with CAD incidence in HbA1c

8.5%. Standard lipid and blood pressure measures also predicted CAD in both groups. After covariate adjustment, beyond age, only triglycerides re-mained a signifi cant predictor of CAD in those with HbA1c 8.5%, while in HbA1c <8.5%, ln(EPO) (2.8, 95% CI 1.0, 7.8), red blood cell count (RBC) (0.6, 95% CI 0.4, 0.8), WBC (1.4, 95% CI 1.1, 1.9), LDL-c, DBP, and BP medication use also predicted CAD.These data suggest risk factor effects differ by gly-cemic status and emphasize that in the setting of lower HbA1c in T1D, EPO, RBC, WBC, lipids, and blood pressure become particularly important factors to monitor and potentially treat.

440-PMacrovascular Complications in Patients With Type 2 Diabetes are Associated With Abnormal HDL Contents of Oxidized Fatty Acids and HaptoglobinCECILIA MORGANTINI, BEATRICE BOLDRINI, ELENA VENTURI, DAVID MERI-WETHER, YUEN YIN LEE, SIMONA BALDI, ANDREA NATALI, SRINIVASA T. RED-DY, Pisa, Italy, Los Angeles, CA

We have previously shown that in type 2 diabetes (T2D) the anti-infl am-matory and anti-oxidant HDL functions are impaired. Recent studies sug-gest that HDL dysfunction correlates with and may be caused by the content of oxidized fatty acids derived from arachidonic (HETEs) and linoleic acid (HODEs), as well as haptoglobin. In this study, we examined whether HDLs from T2D patients contain elevated levels of oxidized fatty acids and hapto-globin and whether these changes are more severe in T2D with macrovas-cular complications (MVC).HDL contents of HETEs and HODEs were deter-mined by LC-MS/MS and haptoglobin levels were measured by ELISA in 20 non diabetics (ND), in 25 T2D without (DMVC[-]) and 25 with MVC (DMVC[+]). HDL function was evaluated by a cell-free assay using dichlorofl uorescein diacetate.T2D patients and ND were similar with respect to major cardiovas-cular risk factors. HDL content of HETEs, HODEs, and haptoglobin progres-sively increased from ND to DMVC[+] patients.

ND DMVC[-] DMVC[+] p (ANOVA)5-HETE (ng/mgHDL-Chol) 19.7±12.6 42.1±22.6 76.2±74.2 <0.0512-HETE (ng/mgHDL-Chol) 2.5±1.8 8.6±4.9 14.0±11.5 <0.0515-HETE (ng/mgHDL-Chol) 2.1±1.4 5.3±3.3 11.5±13.1 <0.059-HODE(ng/mgHDL-Chol) 4.1±2.4 6.8±4.5 13.3±14.4 <0.0513-HODE (ng/mgHDL-Chol) 4.4±2.1 8.2±4.9 15.1±14.8 <0.05Haptoglobin(ng/ml) 1879±1084 2075±1648 3292±2391 <0.05

HDL anti-oxidant index was signifi cantly impaired only in 5 DMVC[+] patients when compared to ND (1.14±0.64 vs 1.91±0.93, p<0.05).A relative HDL enrich-ment in oxidized fatty acids from arachidonic and linoleic acids and in hapto-globin is observed in T2D patients, particularly in diabetic patients with MVC. A loss of HDL antioxidant function is evident in patients with severe vascular disease. Novel therapeutic agents such as apoA-I mimetic peptides, which bind oxidized fatty acids and reduce atherosclerosis in mouse models of diabe-tes might be an important tool in the prevention of vascular events in T2D.

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442-PPredicting Cardiovascular Outcomes by Intima-Medial Thickness (IMT)SANGMO HONG, MIN-YOUNG CHUN, JEONG TAEK WOO, KWAN WOO LEE, MUN-SUK NAM, SEI HYUN BAIK, YOUNG SEOL KIM, YONGSOO PARK, Seoul, Republic of Korea, Suwon, Republic of Korea, Incheon, Republic of Korea

Type 2 diabetes (T2D) is a major cause of cardiovascular disease. Carotid intima-media thickness (IMT) or presence of plaques in common carotid ar-tery has been shown to have a strong relationship with cardiovascular ac-cidents (CVA). However, only few studies have evaluated the usefulness of IMT or presence of plaques in predicting future development of CVA in Asian T2D patients. Using a total of 1489 patients with T2D (M:F=839:650, mean age: 55.1 yr at baseline) recruited from the Korea National Diabetes Program (KNDP), a prospective natural history study of T2D, we examined the infl u-ence of the progression of IMT on predicting the development of CVA in a Korean T2D cohort. B-mode carotid ultrasonographic measurements (the mean IMT, the maximum IMT and the presence of plaques) were performed and repeated annually for a mean of 3.1 years of follow-up. Development of CVAs was evaluated prospectively for 3.1 years, and anthropometrics and biochemical parameters were also measured annually. During the follow-up, 74 patients (5%) developed CVAs and the annual progression of mean IMT was 0.0580 ± 0.1423mm. Newly development of CVAs was associated with abnormal mean IMT (> 0.08 cm) [OR (95% confi dence interval); 1.29 (1.01-1.66)] and the presence of plaques [OR (95% confi dence interval); 1.34 (1.06-1.68)] at baseline. After adjusting for anthropometrics and biochemical parameters with logistic regression, the presence of plaques were clearly associated with the development of CVAs [OR (95% CI); 1.65 (1.02-1.79)], but the association with abnormal mean IMT decreased due to interaction with age ( =0.09; p<0.001), duration of diabetes ( =0.04; p=0.02), and HDL cho-lesterol ( =-0.02; p=0.06). Although the presence of plaques and elevation

of IMT were all found to be associated with cardiovascular outcomes, only the presence of plaques predicted cardiovascular outcomes independent from conventional cardiovascular risk factors in Asian T2D patients during short-term follow-up.

443-PStress and Response in Association With Coronary Artery Disease (CAD) in Type 1 DiabetesTINA COSTACOU, YUEFANG CHANG, GERALD L. SCHAFER, RHOBERT W. EVANS, TREVOR J. ORCHARD, Pittsburgh, PA

We aimed to assess whether the degree to which an individual is able to respond to oxidative stress modifi es the risk associated with oxidative stress and the CAD development. To do this, we evaluated whether CAD incidence was related to plasma levels of vitamin E ( - and -tocopherol) and creatinine adjusted urinary 15-isoprostane F2t (IsoP) measured at three time points during the 20 year follow up of the Pittsburgh Epidemiology of Diabe-tes Complications (EDC) study of childhood onset type 1 diabetes (n=658, baseline mean age 28 and duration 19 years). EDC participants with three samples available during follow up (n=429) were selected for study. Gener-ally, analyses conducted at each time point showed no consistent associa-tion between serum vitamin E and urinary IsoP levels and CAD, adjusting for diabetes duration. In multivariable mixed model analyses, however, allowing for univariately signifi cant risk factors, -tocopherol was inversely ( =-0.03, p=0.08) and IsoP directly ( =0.02, p=0.03) associated with CAD; no asso-ciation was seen for -tocopherol ( =0.006, p=0.57). Moreover, the ratio of

-tocopherol to IsoP (used as a measure of response to stress) was inversely related to CAD incidence ( =-0.02, p=0.003). All models adjusted for univari-ately signifi cant variables (body mass index, lipid levels, albumin excretion rate (log) and white blood cell count). These data provide some support for the hypothesis that a greater capability ( -tocopherol) to respond to oxida-tive stress (IsoP), relates to CAD incidence.

Supported by: NIDDK (DK082900-01A1 and DK34818)

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445-PSurvival in Diabetic Foot Ulcer Patients After Hyperbaric Oxygen TherapyMAGNUS LÖNDAHL, CHRISTER HAMMARLUND, PER KATZMAN, Lund, Sweden, Helsingborg, Sweden

Increasing evidence support the use of Hyperbaric Oxygen Therapy (HBO) in diabetic foot ulcer healing. However, concerns for long-term risks of HBO treatment have been raised. The aim of the present study was to record and discuss three-year survival data following a randomised, double-blinded, placebo-controlled HBO study in diabetic patients.94 patients were ran-domly assigned to forty 90 minutes long treatment sessions with either HBO (n=49) or hyperbaric air (HBA) (n=45) at 2.5 atmospheres absolute pressure.Nonparametric tests were used for comparison, Kaplan-Meier curves for life-table analysis and Cox proportional hazards models to estimate hazard ratios (HR) and 95% CIs.At inclusion patients were 68.5 (48.3-82.1)(median and 10th and 90th percentiles) years old and 67% had type 2 diabetes. One fi fth of the patients were females. There were no differences in basal char-acteristics between groups.HR for three-year survival for those in the HBO group was 1.78 (95% CI 0.80-3.97, p=0.15).The per-protocol analysis evalu-ated survival in patients receiving more than 35 treatment sessions (HBO n=38, HBA n=37). The HR for three-year survival in this population was 3.04 (95% CI 1.08-8.5, p=0.026). In a third analysis we compared survival be-tween the per-protocol HBO group and those patients < 36 HBO sessions (HBA and drop-outs). HR for three-year survival in this analysis was 3.17 (95% CI 1.19-8.45 p=0.021) in favour of HBO treatment.In terms of long-term effects HBO does not seem to increase mortality. Contrarily, our study indi-cates an increased survival in our selected group of diabetic patients with chronic foot ulcers.

Supported by: Thelma Zoegas Foundation, Swedish Diabetes Foundation, R and D Skane County

446-PPlasma Levels of Circulating miR-191 are Altered in Diabetes As-sociated Impaired Wound HealingSEEMA DANGWAL, BERND STRATMANN, KATRIN SCHWARZ, JOHAN LOREN-ZEN, CLAUS J. SCHOLZ, THOMAS THUM, DIETHELM TSCHÖPE, Hannover, Ger-many, Bad Oeyn hausen, Germany, Würzburg, Germany

MicroRNAs (miRNAs), master regulators of gene expression, are stably present in blood. Here we investigated the levels of circulating plasma miR-NAs in diabetic patients with or without impaired wound healing.MiRNAs profi ling using affymetrix array was performed on pooled RNA from the plasma of 36 diabetic patients with or without impaired wound healing. Re-sults were validated by quantitative real-time PCR in patients with diabetes associated impaired wound healing (n=17) and age- and body mass index-matched disease controls i.e. diabetic patients (n=12) presenting no wounds as well as age-matched healthy controls (n=20).Array data revealed a dif-ferential regulation of 41 miRNAs in diabetic patients with impaired wound healing vs. disease controls. PCR validation confi rmed signifi cantly lower level of miR 191 (p 0.05) in disease control vs. healthy control which was reverted in diabetics with impaired wound healing, whereas another 2 miR-NAs (miR 126 and miR 200b) showed a similar trend. Elevated plasma level of miR 191 was associated with manifestation of myocardial infarction (MI) and coronary artery disease whereas miR 126 with MI, and miR 200b with peripheral artery disease. Diabetics with impaired wound healing showed

higher levels of cholesterol, liver transaminase and BNP vs. disease controls (all p 0.05).Lower plasma levels of miR 191 in diabetics with no wounds are reversed in diabetic subjects with impaired wound healing refl ecting a complex pathology of diabetic wound healing complications. Hence miR 191 may serve as novel diabetic biomarker for the patient subset with no wound healing impairment only.

Correlation analysis of differentially regulated circulating plasma miRNAmiRNA Variables in patients without

impaired wound healing (correlation coeffi cient, p value, n=12)

Variables in patients with impaired wound healing (correlation coeffi cient, p value, n=17)

miR-191 C-peptide (r=0.643, p=0.02) Triglyceride (r=-0.522, p=0.03); C-reactive protein (r=0.500, p=0.04)

miR-126 CRP (r=0.664, p=0.05); Body weight (r=0.664, p=0.018)

LDL (r=-0.503, p=0.04); Lipoprotein(a) (r=-0.514, p=0.04)

miR-200b Cholesterol (r=-0.587, p=0.05); GFR (r=0.572, p=0.05)

Creatinin (r=0.619, p=0.008)

447-PNT-proBNP: A Potential Cardiorenal Biomarker in the Management of DiabetesRIEKO KOMI, IKUE NAKADAIRA, JUN SUZUKI, AIKO OHOKA, ASAMI TANAKA, MASAHIRO TAKAHASHI, KUMIKO HAMANO, Kamakura, Japan, Kawasaki, Japan

Individuals with diabetes and CKD are at particularly high risk for CVD and ESRD. We recently reported that NT-proBNP could be a marker of silent myo-cardial ischemia in type 2 diabetes. CVD accelerates CKD progression and in turn, CKD itself amplifi es CVD risk, known as cardiorenal syndrome.In the present study, we tested whether NT-proBNP could be a biomarker of both CKD and CVD in type 2 diabetes.We consecutively recruited 109 patients (mean age 61.8, HbA1c9.1, Cr0.76 mg/dl) with CKD stage1 and 2. Serum NT-proBNP measurement (Roche) and ultrasonographic echocardiogram were performed and subjects were followed up to 5 years. Primary endpoints were defi ned as 1) renal:30% decrease of eGFR and 2) CVD and all-cause mortality.NT-proBNP was well correlated with left ventricular mass index and eGFR.1) Fourteen patients reached the renal endpoint and had signifi cantly higher baseline NT-proBNP (169 vs. 52 pg/ml, p<0.05). Patients were stratifi ed into 2 groups by NT-proBNP concentrations above and below the optimal cut-off points calculated by ROC analysis. Kaplan Meier analysis demonstrated that signifi cantly higher proportions of subjects with NT-proBNP above 120pg/ml had progressive decline of eGFR (Log rank test: p=0.0054).2) Fourteen pa-tients had new incidences of CVD or death. Mean levels of NT-proBNP were 142 and 44 pg/ml for those with and without new events. Subjects with NT-proBNP level above 153pg/ml had signifi cantly higher risks of CVD and death than those below 153pg/ml (HR 3.68, adjusted for sex and age, p=0.038).Elevations of NT-proBNP predicted not only CVD event or death as well as CKD progression in type 2 diabetes. As CKD is a major risk factor for CVD in diabetes, NT-proBNP might be one of key molecules linking kidney with heart. It is possible to stratify diabetic patients by simple measurement of NT-proBNP, which is reproducible and inexpensive, and enables CVD preven-tion and CKD prevention as well. Optimal cut-off value must be elucidated by further investigation.

448-PChemerin is Associated With the Metabolic Syndrome But is Not Linked to Angiographically Determined Coronary Artery DiseaseCHRISTOPH H. SAELY, AXEL MUENDLEIN, ALEXANDER VONBANK, PHILIPP REIN, KATHRIN GEIGER, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, Phila-delphia, PA

The novel adipocytokine chemerin has been suggested to be linked to in-sulin resistance and to the metabolic syndrome (MetS). Its association with coronary artery disease (CAD) is unclear. We hypothesized that chemerin is associated with both angiographically determined CAD and with the MetS. We measured serum chemerin in 498 patients undergoing coronary angiography for the evaluation of established or suspected stable CAD; the MetS was defi ned according to NCEP-ATPIII criteria; signifi cant CAD was diagnosed when coronary stenoses 50% were present.Chemerin was higher in MetS patients (n=150) than in subjects without the MetS (184±77 vs. 150±62 ng/ml; p<0.001). It did not differ signifi cantly between patients with signifi cant CAD (n=250) and those without signifi cant CAD (p=0.327). When both, MetS and CAD status were considered, chemerin was higher in MetS patients both among those who had signifi cant CAD (182±80 vs. 152±60 ng/ml; p=0.002) and among those who did not have signifi cant CAD (187±73 vs. 148±63 ng/ml; p<0.001); it did not differ signifi cantly between

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patients with signifi cant CAD and subjects without signifi cant CAD among MetS patients (p=0.248) nor among subjects without MetS (p=0.263). Analy-sis of covariance (ANCOVA) showed that from the NCEP-ATPIII metabolic syndrome traits large waist circumference as well as elevated trigylcerides were independent predictors of elevated serum chemerin (F=12.5; p<0.001 and F=8.5; p=0.004).We conclude that chemerin is signifi cantly associated with the MetS but not with angiographically determined CAD. The overall association of chemerin with the MetS is carried by its association with visceral obesity and elevated triglycerides.

Supported by: Research Foundation of the Austrian National Bank

449-PCoronary Artery Disease as a Risk Factor for Developing Type 2 Dia-betes MellitusCHRISTOPH H. SAELY, ALEXANDER VONBANK, PHILIPP REIN, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein

Diabetes mellitus is a major risk factor for coronary artery disease (CAD); whether conversely CAD confers an increased risk for diabetes has not been studied so far.We prospectively recorded incident diabetes over 7.5 years in 506 consecutive non-diabetic Caucasian patients undergoing coronary angiography for the evaluation of stable CAD, covering 3795 patient years. During follow-up, diabetes was newly diagnosed in 107 patients, i.e. in 21.1% of the study population or in 2.8% per year. Patients with signifi cant CAD (n=293) when compared to subjects who did not have signifi cant CAD at the baseline angiography were at a 33% (p = 0.027) increased diabetes risk. However, the relationship between CAD and incident diabetes was at-tenuated and no longer statistically signifi cant after adjustment for potential confounders including metabolic syndrome (MetS) status. The MetS as diag-nosed according to the current consensus defi nition in turn was strongly pre-dictive of diabetes, in particular when the more selective NCEP-ATP-III waist cut-off values were applied for its diagnosis (OR = 2.91 [1.83-4.64]; p <0.001).We conclude that albeit apparently not causally related to diabetes inci-dence, the presence of CAD indicates a strongly increased risk for incident diabetes. Repeated diabetes screening of coronary patients and targeted programs to prevent diabetes in these high-risk patients are warranted.

Supported by: Jubiläumsfonds der Österreichischen National Bank

450-PProgression of Diabetic Retinopathy is Associated With Left Ven-tricular Diastolic Dysfunction in Patients With Type 2 DiabetesSOICHI KURIOKA, Osaka, Japan

Diabetic retinopathy (DR) is anindependent predictor of left ventricular diastolic dysfunction (LVDD) inpatients with diabetes mellitus. However, it is unclear if progression of DR isassociated with LVDD, which is recognized to result in subsequent heart failure.The subjects in this study were 120 con-secutive patients with type 2 diabetesmellitus (T2DM) [72 men (60%); age 66±10 years old (mean±SD); diabetic duration11±9 years; HbA1c 8.0±1.8%]. Subjects with overt heart failure or NYHA class>1, history of coronary artery disease, severe valvulopathy or chronicatrial fi brillation were excluded from the study. All patients underwentclinical evaluation, laboratory tests, and echocardiographic examination. Dopplerechocardiographic indices including peak early diastolic mitral annularvelocity (E’) and early diastolic myocar-dial velocity (E) were obtained in eachpatient. The patients were divided into three groups according to DR stage: nodiabetic retinopathy (n=80), simple retinopathy (n=20), and preproliferative orproliferative retinopathy (n=20). No patients showed systolic impairment ofl eft ventricular function (LVEF >50%), whereas LVDD (E/E’ >8) was detectedin 105 cases (87.5%). E/E’ was correlated with age (r=0.303, p=0.001), sex(r=0.208, p=0.021), DR stage (r=0.241, p=0.007), systolic blood pressure(r=0.199, p=0.031), and se-rum creatinine level (r=0.265, p=0.003). In multipleregression analysis, age ( =0.322,p<0.0001) and DR stage ( =0.266, p=0.002)were independently correlated with E/E’. In this study, almost all asymptomaticpatients with T2DM had LVDD. Progression of DR is associated with LVDD and thismay at least in part explain the increased incidence of heart failure inpatients with diabetes mellitus and DR.

451-PAdiponectin Receptors and Adiponectin Action in Circulating Hu-man CD14+ Monocytes in Patients With Diabetes and Cardiovascu-lar DiseaseLING TIAN, DENNIS STEVERSON, WEI ZHANG, YUCHANG FU, W. TIMOTHY GARVEY, Birmingham, AL

We have previously shown that adiponectin regulates insulin action and foam cell formation via adipoR1 and R2 in cultured cells. To investigate a role

of the adiponectin-adipoR1/2 axis in humans with cardiometabolic disease, we studied adipoR1/2 expression in circulating CD14+ monocytes obtained from insulin sensitive normoglycemic subjects (IS, n=12) vs. T2DM patients with coronary artery disease (DM&CVD, n=13).Flow cytometry found no sig-nifi cant differences in CD14, CD16, CD45 and CD11c expression in circulating mononuclear cells between groups. CD14+ monocytes were then cultured for 8 days followed by adiponectin and oxLDL treatment. Adiponectin increased AdipoR1 and R2 mRNA expression in both IS (p<10E-5, p<0.03, respectively) and DM&CVD (p<10E-5, p<0.001), however, there were no signifi cant inter-group differences. Further analyses revealed gender differences that male IS have a trend for a greater adiponectin-mediated adipoR1 increase than male DM&CVD (p=0.13), while the response was signifi cantly less in female IS vs. female DM&CVD (p=0.01). There were no group differences in cellular cholesterol levels and secretions of MCP-1, IL-6 and IL-1Ra. We did observe that cholesterol accumulation was correlated with adipoR2 levels in total subjects (p=0.0398). Importantly, as we previously reported that adiponec-tin-adipoR1/2 signals via APPL1 in THP-1 macrophages, we found that APPL1 mRNA levels were signifi cantly higher in the IS vs. DM&CVD (p=0.024).In conclusion: 1) circulating monocytes do not exhibit an adiponectin-resistant, pro-infl ammatory, or lipid accumulating status in DM&CVD; 2) there are gender differences in adiponectin’s ability to modulate adipoR1 expression; 3) APPL1 expression is greater in IS. Hence, monocytes may become more pro-infl ammatory only after entering adipose tissue or the vascular wall in cardiometabolic disease, and this may be related to an intrinsic down-regu-lation of APPL1 expression.

Supported by: DK038746 DK083562 P60-DK079626 Merit Review Program of VA

452-POxidative Stress Injury in Type 2 Diabetics Undergoing Coronary Artery Bypass SurgeryASHVINI MENON, EBRAHIM MULLA, S. HUGHES, J. MASCARO, MARTIN STE-VENS, ROBERT BONSER, Birmingham, United Kingdom

Poly (ADP-ribose) polymerase (PARP) is a nuclear enzyme that repairs damaged DNA strands. Increased oxidative stress in T2DM excessively ac-tivates PARP and depletes its substrate resulting in energy defi ciency and cell death. This may relate to a reduced ability of the diabetic myocardium to cope with ischaemia-reperfusion injury. Pre-operative PARP status has not been characterised in humans. We therefore evaluated myocardial PARP activation and plasma protein carbonyls (marker of oxidative injury) in T2DM and non-T2DM patients undergoing CABG.Right atrial biopsies were obtained at initial venous cannulation. Samples were fi xed in 10% formal saline and 4μm paraffi n sections prepared. Sections were incubated with mouse monoclonal anti-PAR (poly-ADP-ribose) antibody. Images were cap-tured using a Ziess Axioshop-plus microscope and analysed using Axiovision 4.4 programme. The percentage of PAR stained nuclei was determined by image analysis using a Scion image programme. Plasma protein carbonyl was measured using the Biocell PC Test kit (Biocell, Auckland, New Zea-land) at 0,6,12,24,48 and 72 hours following CABG.Twenty non-T2DM and 28 T2DM right atrial biopsies have been analysed so far. The median age (78 vs. 73 years), extent of coronary artery disease and median ejection fraction (>50% in both groups) were not different. A signifi cantly higher percentage of PAR positive nuclei was detected in the T2DM group (71 [65.2-76.9] %) compared to the non-T2DM subjects (45.2 [38.6-51.8] %), p<0.001. In both groups, protein carbonyl values varied signifi cantly over time (p=0.02). Pro-tein carbonyl values at 12, 24, 48 hours post CABG were signifi cantly higher in T2DM (p<0.05).Cardiac PARP activation and oxidative stress markers are increased in T2DM. This may promote increased susceptibility to ischaemia-reperfusion injury and may contribute to worse post-CABG outcomes in T2DM. Ongoing studies are in progress to evaluate this relationship.

453-PEffects of Atorvastatin on Endothelial Nitric Oxide Release, Blood Pressure and Nitroxidative Stress Levels in Hypertensive Rats With DiabetesR. PRESTON MASON, ROBERT F. JACOB, J. JOSE CORBALAN, ALEKSANDER CISZEWSKI, TADEUSZ MALINSKI, Boston, MA, Beverly, MA, Athens, OH

Clinical trials have shown that patients with diabetes benefi t from treat-ment with the HMG-CoA reductase inhibitor, atorvastatin, despite having normal LDL levels. Most patients with diabetes also have hypertension, both of which are cardiovascular risk factors associated with endothelial cell (EC) dysfunction and reduced nitric oxide (NO) release. In this study, we tested the hypothesis that atorvastatin reverses EC dysfunction in diabetic, spontaneously hypertensive (SH) rats by enhancing endothelial NO synthase (eNOS) activity. Male SH rats with normal LDL levels were treated with

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streptozotocin (65 mg/kg/BW) to induce diabetes, followed by treatment with atorvastatin at 20 mg/kg/day (or vehicle for control animals). After 5 weeks, glomerular ECs were isolated from renal tissue and assayed for NO and peroxynitrite (ONOO−) release using amperometric nanosensors. Study animals were also examined for changes in fasting glucose and mean blood pressure (BP) levels. As an additional control, we performed similar analy-ses in non-diabetic SH rats. Atorvastatin was found to increase NO release in diabetic SH rats by 98% (63 ± 7 nM to 125 ± 16 nM) while decreasing ONOO- release by 38% (180 ± 12 nM to 112 ± 16 nM) as compared to vehicle alone (p<0.001). The NO/ONOO− ratio, an indicator of normal EC function, increased more than three-fold with atorvastatin treatment. Diabetic SH rats also had elevated fasting glucose (355 ± 38 mg/dL) and mean BP (172 ± 15 mmHg) levels. Atorvastatin reduced mean BP by 21% (to 136 ± 18 mmHg, p<0.001) but had no signifi cant effect on fasting glucose levels. Atorvastatin also increased the NO/ONOO− ratio in non-diabetic SH rats by more than two-fold (p<0.001) and reduced mean BP by 11%. These results suggest that atorvastatin treatment reverses endothelial dysfunction and reduces blood pressure in diabetic SH rats by enhancing eNOS coupling and NO release independently of glucose control.

454-PThe Infl uences of High Glucose and Hypoxia on Hcdc14A, Cyclin Ex-pressions, Cell Proliferation and ApoptosisHOUGUANG ZHOU, LING LIU, YU ZHANG, YANYAN HUANG, ZHUANGLI GUO, RENMING HU, QIANG DONG, Shanghai, China, Nanjing, China, Qingdao, China

Long-term hyperglycemia in T2DM increased glycated hemoglobin, re-duced red blood cell oxygen-carrying capacity, raised blood viscosity, and caused microvascular perfusion defects and chronic hypoxia in tissue cells. But the mechanism of vascular injury and complication induced by high glu-cose and hypoxia(HGH) in T2DM is still not completely clear. The purpose of this study was to observe the infl uences of HGH on human cell division cycle protein 14a(Hcdc14A), the related cycle protein expressions, cell prolifera-tion and apoptosis in human brain vascular endothelial cells(HBVEC). Using different conditions of high glucose and hypoxia stimulated HBVEC. RT-PCR and Western-blot(WB) detected Hcdc14A mRNA and protein expression respectively. CyclinB, cyclinD3, cyclinE and P53 protein expressions were tested with WB. XTT and CCK8 methods were used for the determination of the rate of cell proliferation and survival, fl ow cytometry and caspase3 activity for apoptosis, fl ow cytometry for cell cycle, fl uorescent probe for cytoskeletal protein F-actin spatial structure, and electron microscope for utrastructure. HGH could downregulate Hcdc14A mRNA and protein expres-sions, reduced cyclinB, cyclinD3 and cyclinE protein expressions, while up-regulated P53 protein expression. HGH reduced the cell proliferation and survival rates, increased the apoptosis rate and the caspase-3 activity, signifi cantly lowered the cells in S phase and G2-M phase, infl uenced cy-toskeletal protein F-actin space structure. Electron microscope observation also revealed that apoptosis cells increased. In conclusion, HGH could obvi-ously downregulate the Hcdc14A in HBVEC, infl uence related cyclin expres-sions, block cell cycle, reduce cell proliferation, induce apoptosis, and affect cytoskeletal protein F-actin spatial structure and cell ultrastructure, which representing some key factors in the development of micro- and macrovas-cular dysfunction in T2DM.

Supported by: The National Natural Science Foundation of China (81170322)

455-PInhibition of an Olfactory Receptor Mediates Cell Death in Human Arterial Endothelial CellsJENIE Y. HWANG, CHANG HEE JUNG, WOO JE LEE, JOONG-YEOL PARK, Seoul, Republic of Korea

Olfactory receptors (ORs) are the largest gene family in human genome. Although they are expected to be expressed specifi cally in olfactory tissue, about 10% of ORs have ectopic expression. However, the function of ec-topic ORs is not well explored except their chemotactic function of sperm. The present study explores the expression of one of the representative OR, OR1D2, in a human aortic endothelial cells (HAEC) and assesses the potential functional implication of such ectopic expression.HAECs(BioWhittaker) were cultured in endothelial groth medium-2 supplemented with specifi c growth factors. Using RT-PCR, we demonstrated the existence of OR1D2 in HAEC. Also, we identifi ed that well-known odorant bourgeonal, rosiglitazone and alpha-lipoic acid(aLA) elicit activation of OR1D2 in endothelium. By using siRNA, expression of OR1D2 was down-regulated and apoptosis of human endothelial cells was seen in dose dependent manner. Exposure to siRNA resulted in the activation of members of the MAPK family and inhibition of cell proliferation. Our data give support to the hypothesis that OR signaling

may mediate cell proliferation in HAEC , thus specifi c receptor ligands might be potential candidates for endothelial dysfunction.

456-PThe Estrogen-Related Receptor g (ERRg) Is Involved in the Develop-ment of Vascular Calcifi cationJI-HYUN KIM, YOUNG-KEUN CHOI, CHAE-MYEONG HA, EON-JU JEON, HYUN-AE SEO, JI-YUN JEONG, YEON KYUNG CHOI, KWI-HYUN BAE, SANG-JUN LEE, KEUN-GYU PARK, JUNG-GUK KIM, IN-KYU LEE, Daegu, Republic of Korea

Vascular calcifi cation, which refers to ectopic mineralization in vascular cells occurs frequently in many diseases such as chronic kidney disease, atherosclerosis, and diabetes. Vascular calcifi cation is vastly correlated with high risk of cardiovascular morbidity and mortality, due to arterial stiffness and impaired vascular compliance. Estrogen-related receptor (ERR ), a member of orphan nuclear receptor superfamily is shown to be related to bone formation. However, the role of ERR in vascular calcifi cation has not yet been investigated. This study was undertaken to examine the role of ERR in vascular calcifi cation.ERR expression was upregulated during in-organic phosphate (Pi)-induced calcifi cation of vascular smooth muscle cells (VSMCs), along with increased expression of bone morphogenic protein-2 (BMP2), osteocalcin and alkaline phosphatase. Transient overexpression of ERR stimulated promoter activity of osteogenic genes including BMP2 and osteocalcin. Adenovirus-mediated overexpression of ERR also augmented osteogenic gene expression and induced BMP2 expression and phosphory-lation of Smad1,5,8, consequently, exacerbates Pi-induced calcifi cation of VSMCs. Moreover, inhibition of ERR by a selective inverse agonist attenu-ated both Pi-induced osteogenic gene expression and calcium deposition in cultured VSMCs. Finally, ex vivo aortic tissue culture showed that the ERRinverse agonist signifi cantly reduced Pi-induced calcifi cation in mouse aorta. These results demonstrate that ERR stimulates vascular calcifi cation by up-regulating osteogenic genes. Therefore, our fi ndings suggest that inhibition of ERR may be a potential therapeutic strategy for prevention of vascular calcifi cation.

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457-PComparison of Doppler (D) and Photoplethysmography (PPG) in Measuring the Ankle-Brachial Index (ABI) in People With DiabetesLIVIU DANESCU, CHERYL ROE, LEWIS JOHNSON, Syracuse, NY

American Diabetes Association guidelines recommend measuring ABI in people >50 yrs old with diabetes. It is infrequently done in offi ce practice, in part, because the D technique is time consuming and diffi cult to master. Previous studies have compared use of PPG and D but not specifi cally in dia-betes. We report results from our diabetes clinic comparing the PPG and D methods for measuring ABI. Consecutive patients with diabetes (n=103) had ABI measured by both methods. In 3 individuals we were unable to obtain PPG readings. Mean age was 60 ±12.8 yrs, 57% were female, 79% had type 2 diabetes and the average duration of diabetes was 17 ±12 yrs. Of 200 limbs evaluated, 17 (8.5%) had non-compressible vessels (NC). A comparison of the 2 methods on the remaining 183 limbs yielded a correlation coeffi cient of 0.864 (Figure). Mean ABI was 1.11 ±0.14 for D and 1.12 ±0.14 for PPG. The dif-ference between the means was non-signifi cant. ABI results were classifi ed as discordant if D and PPG varied by more than 0.15 (reported variability of D), placing them in different diagnostic categories [low (<0.9), normal (0.9-1.3) and high (>1.3) / NC groups]. There were only 2 (1%) discordant results. In conclusion, an excellent correlation was found when comparing PPG with the gold standard D technique in the measurement of the ABI in people with diabetes. PPG requires less training and takes less time to perform making it highly suitable for use in the offi ce setting.

458-PReinfarction and Mortality Rate in Patients With Coronary Artery Disease and the Relation to Glucose ToleranceJEANETTE KUHL, GUN JÖRNESKOG, MALIN WEMMINGER, MATTIAS BENGTS-SON, MAJID KALANI, Stockholm, Sweden

We investigated the importance of glucose tolerance for long term car-diac prognosis in patients with coronary artery disease (CAD).1151 consecu-tive patients, 836 men and 315 women, aged 32-80 years, admitted to the coronary care unit at Danderyd University Hospital, Stockholm, for acute coronary syndrome (ACS) from year 2006 to 2008. At discharge, the patients were categorized according to an oral glucose tolerance test (OGTT) as having normal (NGT; n=303 (26%)) or impaired (IGT; n=311 (27%)) glucose tolerance, or diabetes (n=537 (47%)). In 2010, after a mean follow-up time of 3 years, all patients were invited to answer a questionnaire and to give fasting venous blood samples for analysis of metabolic control. Information about reinfarction and mortality were obtained from the Swedish Coronary Angiography and Angioplasty Registry and from the Swedish National Reg-istration.At the follow-up, 36 patients (12%) in the NGT group had had rein-farction compared to 50 patients (16%) in the IGT and 123 patients (23%) in the diabetes group. Mortality was 7.1% (82 patients): 3 patients with NGT (1.0%), 8 with IGT (2.6%), and 71 patients with diabetes (13.2%). 548 (48%) patients (136 women, 412 men) accepted the invitation for a follow-up. At the follow-up, a signifi cant increase in fP-glucose was seen in all groups as compared to baseline values (p<0.05) despite intensifi ed antidiabetic and risk factor medications since discharge from the hospital. In contrast, lipo-protein profi le had improved in all groups as compared to baseline (p<0.001).

Patients with diabetes showed a signifi cant increase in serum creatinine (93.0±4.0 μmol/L vs 97.2±4.2 μmol/L; p<0.05), while no signifi cant changes were seen in patients with NGT or IGT. In conclusion, a majority of patients admitted for ACS have disturbed glucose metabolism including diabetes. Long term cardiac prognosis is poor in patients with CAD and dysglycaemia despite intensifi ed antidiabetic and risk factor medications.

459-PThe Metabolic Syndrome Signifi cantly Affects the Association be-tween Resting Heart Rate and All Cause as Well as Cardiovascular MortalityALEXANDER VONBANK, FABIAN SCHMID, PHILIPP REIN, CHRISTOPH H. SAELY, HEINZ DREXEL, Feldkirch, Austria, Triesen, Liechtenstein, Philadelphia, PA

Epidemiological studies suggest that the resting heart rate (RHR) is an independent predictor of cardiovascular and all cause mortality. However, the power of the RHR to predict cardiovascular events in patients with the metabolic syndrome (MetS) is not known.We prospectively investigated the relationship between RHR and cardiovascular events in 756 consecutive pa-tients undergoing coronary angiography for the evaluation of coronary artery disease (CAD) over a follow-up period of 7.1 ± 0.1 years. The MetS was de-fi ned according to NCEP-ATPIII criteria.In the total study population, both all cause and cardiovascular mortality were increased with an increasing RHR (standardised adjusted HRs 1.03 [1.01-1.04]; p = 0.001 and 1.15 [1.03-1.47]; p = 0.001, respectively). From our patients, 357 (47.2%) had the MetS and 399 did not have the MetS. Among patients without the MetS, a higher baseline RHR indicated a signifi cantly higher risk of total mortality (HR = 1.14 [111 - 1.16], p = 0.001) and cardiovascular mortality (HR = 1.13 [1.12- 1.16], p = 0.001) after multivariate adjustment. However, the RHR did not signifi cantly affect total mortality (p = 0.120) or cardiovascular mortality (p = 0.244) in patients with the MetS. Interaction terms RHRxMetS were signifi cant for both total and cardiovascular mortality (p = 0.027 and p = 0.037, respec-tively), indicating that the respective risks conferred by a high RHR were signifi cantly higher in patients without the MetS than in patients with MetS.We conclude that among angiographically characterized coronary patients, the metabolic syndrome status signifi cantly affects the association of the RHR with total and cardiovascular mortality: RHR is a strong predictor of both total and cardiovascular mortality among subjects without the MetS, but not among MetS patients.


460-POne in Three Patients Referred to The Type 2 Diabetes Clinic, Steno Diabetes Center Suffers from Obstructive Sleep ApneaHEIDI STORGAARD, THOMAS P. ALMDAL, MICHAEL LAUB, LISE TARNOW, Gentofte, Denmark, Copenhagen, Denmark

Several studies suggest that obstructive sleep apnoea (OSA) is associ-ated with cardiovascular disease (CVD), and CVD is common among patients (pts) with type 2 diabetes (T2DM). We therefore aimed to investigate the prevalence of OSA among T2DM pts referred to Steno Diabetes Center.All pts referred to SDC T2DM clinic September 2010-June 2011 were offered to participate in a three-stage screening program for the diagnosis of OSA; 1.The Berlin questionnaire. A “high likelihood of OSA”-score qualifi ed for; 2. An outpatient ApneaLink® monitoring. Pts with a positive test (apnoea-hypopnoea index (AHI) 5/hour) were referred to; 3. Diagnostic polygraphy (with Embletta®) in a sleep clinic.A total of 180 pts (men, 61%; age 59.7 ± 10.5 years, BMI, 31.8 ± 6.7 kg/m2, T2DM duration 8.2 ± 6.3 years, HbA1c, 7.3 ± 1.2%) participated. Amongst these 104 (61%) had a “high likelihood of OSA”-score according to the Berlin questionnaire, and 77 pts (43%) were af-ter ApneaLink® study referred to the sleep clinic. So far, polygraphy has been performed in 49 pts, and 44 have been diagnosed with OSA - corresponding to an estimated prevalence of 34 % of the entire population (n=180).Ap-neaLink® positive (n = 77) pts had higher BMI (34.3 ± 7.4 versus 30.3 ± 5.7 kg/m2, p <0,001 ), higher HbA1c (7.6 ± 1.3 versus 7.2 ± 1.1%, p = 0.008) and lower HDL-chol (1.1 ± 0.3 versus 1.3 ± 0.4 mmol / l, p = 0.003) as compared with those without symptoms (n = 77) or signs of OSA on ApneaLink® (n = 26). The groups were comparable with respect to: blood pressure, albuminuria, p-creat, retinopathy, LDL chol, treatment with OAD, RAS blockade and sta-tins. In a multiple linear regression analysis, AHI increased signifi cantly with higher age, increased BMI and declining HDL-chol. Sex, HbA1c and insulin dose per kg bodyweight were not related to AHI.These preliminary results suggest that more than 30 % of T2DM pts referred to a hospital outpatient clinic have OSA - this may play a role in the high cardiovascular mortality seen in these pts.

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461-PSerum Cystatin C Shows a Greater Association With Peripheral Ar-tery Disease than Albuminuria in Type 2 Diabetes With Normal or Mild Impairment of Renal FunctionJANG-YEL SHIN, MI YOUNG LEE, CHOON HEE CHUNG, Wonju-Si, Gangwon-Do, Republic of Korea

Some studies have shown a link between chronic kidney disease and peripheral artery disease (PAD) in general population. Cystatin C has been suggested to be a sensitive marker for early detection of renal impairment in type 2 diabetes. We examined the association of serum cystatin C with PAD in type 2 diabetes with normal or mild impairment of renal function.We enrolled 272 type 2 diabetic patients [age 57.7±9.7 years; male 58.5%; duration of diabetes 6.3±6.0 years; HbA1c 7.7±1.8%]. Patients were excluded if they had estimated glomerular fi ltration rate (eGFR) < 60 mL/min per 1.73 m2, 24 hour urine albumin (24h-uAlb) 300 mg/day, serum creatinine (Cr) > 1.3 mg/dL, or ankle-brachial index (ABI) > 1.4. eGFR was calculated using the Modifi cation of Diet in Renal Disease equation. PAD was defi ned as an ABI 0.9.Median values (inter-quartile range) of cystatin C, eGFR, and 24h-uAlb were 0.92 (0.80, 1.08) mg/L, 93.7 (82.1, 109.2) mL/min per 1.73 m2, and 18.3 (10.1, 36.0) mg/day. Patients with PAD were 7.4%. Comparing to those without PAD, patients with PAD had more likelihood to be hypertensive and current smoker, higher values of 24h-uAlb, cystatin C, and serum Cr, and lower value of eGFR. In partial correlation after adjusted for age and gender, cystatin C showed positive correlations with waist circumference, smoking, 24h-uAlb, and serum Cr, but was negatively correlated with eGFR. Also, after adjusted for age and gender, PAD was positively correlated with smoking, cystatin C, and 24h-uAlb. Odds ratios (ORs) for PAD after adjusted for age, gender, and smoking, were 1.81 (95% CI, 1.10-2.99) for cystatin C and 1.61 (95% CI, 1.01-2.56) for 24h-uAlb per 1 SD increase. After further adjustment for 24h-uAlb or cystatin C, only the OR for cystatin C remained signifi cant.Our fi nding suggests that serum cystatin C shows a greater positive association with PAD than albuminuria in type 2 diabetes with normal or mild impairment of renal function.

462-PPrevention by Sulforaphane of Diabetic Cardiomyopathy is Associ-ated With Nrf2 Up-Regulation and ActivationYANG BAI, YANG ZHENG, QIANG CHEN, XIAO MIAO, CHI ZHANG, WENPENG CUI, YI TAN, LU CAI, Louisville, KY, Changchun, China, Wenzhou, China

This study was to investigate whether sulforaphane (SFN) as one of NF-E2-related factor 2 (Nrf2) activator can protect diabetic cardiomyopathy. Type 1 diabetes was induced in 8-week-old male FVB mice by multiple intraperitoneal injections of low-dose streptozotocin (STZ). Five days after the last injection of STZ, mice with hyperglycemia (blood glucose levels 250 mg/dl) were defi ned as diabetic. Diabetic and age-matched control mice were subcutaneously given SFN at 0.5 mg/kg for fi ve days of each week for 3 months. At the end of the experiments, cardiac function was assessed using M-mode echocar-diography and cardiac pathological changes, fi brosis, infl ammation and oxi-dative damage were assessed by western blotting and immunohistochemical staining. SFN was found to signifi cantly prevent diabetes-induced cardiac dysfunction, shown by increased left ventricular posterior wall and decreased left ventricular ejection fraction. SFN also signifi cantly prevented diabetes-induced structural disarrangements, examined by hematoxylin-eosin staining, and cardiac remodeling, detected by fi brotic makers of transforming growth factor (TGF)- 1 expression and collagen deposition (Sirius-red staining materi-als). These pathological changes were accompanied by signifi cant increases in oxidative damage, measured by protein nitration (3-nitrotyrosine), lipid per-oxidation (4-hydroxynonenal), infl ammation (plasminogen activator inhibitor-1,PAI-1) in diabetic heats, but not in SFN-treated diabetic hearts. SFN induced signifi cant increases in Nrf2 expression, measured by western blotting and function, refl ected by increase Nrf2 downstream gene NAD(P)H: quinone oxi-doreductase1 (NQO1) expression. These results suggest that diabetes-induced cardiac structure disarrangement and remodeling as well as oxidative dam-age can be signifi cantly prevented by SFN probably via up-regulation of Nrf2 expression and function.

463-P

464-PCoronary Response to an Increased Oxygen Demand is Altered in Asymptomatic Type 2 Diabetic Patients With Coronary Artery Dis-ease: A New Non-Invasive ApproachEMMANUEL COSSON, MINH TUAN NGUYEN, ISABELLE PHAM, ALAIN NITEN-BERG, PAUL VALENSI, Bondy, France

Cold pressure test (CPT) induces an increase of double product ( DP: blood pressure x heart rate after/before, %) and a dilation of epicardial coronary arteries. The aim of the study was to investigate if the attenu-ated dilation during CPT previously shown by coronary angiography in type 2 diabetic patients (T2Ds) without coronary artery disease (CAD) was also observed with a non invasive method (NCT00685984).The fl ow velocity was measured in the left anterior descending artery (aCV) by trans-thoracic echo-doppler (3-7MHz) before and after CPT in 118 T2Ds, 30 overweighed or obese non diabetic subjects (OS), and 25 control subjects 40 years. T2Ds were screened for silent myocardial ischemia (SMI: abnormal stress myocardial scintigraphy and/or echocardiography), and in case of SMI for CAD (coro-nary angiography).Thirty-fi ve T2Ds had SMI, 15 of them CAD. aCV during CPT could be measured in 56% T2Ds, 37% OS and 64% controls. At rest DP (p<0.0001) and aCV (p<0.01) were higher in T2Ds than in OS and controls, and aCV correlated with DP (p<0.05). During CPT, DP (43±34/36±27/32±19% increase) and change in aCV ( aCV: 30±26/21±34/17±10% increase) did not differ signifi cantly in the three groups. aCV and DP correlated in con-trols (r=0.58, p<0.05), OS (r=0.78, p<0.01) and T2Ds without CAD (r=0.56, p<0.0001) but not in T2Ds with CAD. SMI status was associated in T2Ds with aCV (SMI- 21±22, SMI+CAD- 36±28 and SMI+CAD+ 39±26%, p=0.03) despite similar DP; and with gender, BMI, nephropathy, triglycerides and haptoglobin (p=0.02 to 0.05). In multivariate analysis, SMI was only associ-ated with aCV (per 10% increase OR: 1.3[1.1-1.5], p<0.05). To conclude, (i)non invasive study of the coronary reactivity is feasible by trans-thoracic echography-doppler with a lower feasibility in OS, (ii) the coronary response to CPT is increased but unadapted in T2Ds with silent CAD, probably through defects in coronary dilation or microcirculation.

Supported by: Société Francophone de Diabétologie

465-PAlterations in Monocyte Surface Markers in Diabetes ComplicationsDANQING MIN, BELINDA BROOKS, JENCIA WONG, ROBERT SALOMON, WEN-SHENG BAO, BRIAN HARRISBERG, STEPHEN M. TWIGG, DENNIS K. YUE, SUSAN V. MCLENNAN, Sydney, Australia

The monocyte and macrophage cell lineage is central to the infl amma-tory response and alterations in this response are associated with diabe-tes and its complications. Monocytes express many cell surface markers indicative of their infl ammatory and activation status. Whether these mark-ers are affected by diabetes and its complications is not known and was investigated in this study. Blood was obtained from 22 non-diabetic controls and 43 diabetic patients with duration of diabetes >10 years, including 25 without and 18 with diabetic complications. In each subject the percentage of monocytes (CD45+CD14+) expressing pro- or anti-infl ammatory markers (CD16 and CD163 respectively), various activation markers and chemokine receptors were determined by fl ow cytometry. Clinical data were collected and selected plasma cytokines and chemokines were also measured. Dia-betes increased monocyte number (P<0.05) but did not alter the expression of cell surface markers related to activation status or the expression of chemokine receptors. However, the percentage of both pro-infl ammatory CD16+ and anti-infl ammatory CD163+ monocytes were decreased in the group with diabetic complications compared to those without (1.9 and 16.2 fold respectively, each P<0.05). The plasma pro-infl ammatory cytokines in-cluding interleukin-6, interleukin-8, tumour necrosis factor- and interferon-

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gamma also showed the same pattern of decrease in those with complica-tions. These results suggest that monocyte phenotype is altered by diabetic complications status. Both anti- and pro-infl ammatory surface markers are decreased by the presence of complications but a higher anti-infl ammatory profi le is associated with no diabetic complications. These changes may be causally related and if confi rmed in subsequent longitudinal series, could potentially be used to predict susceptibility to diabetic complications. How these fi ndings affect monocyte and macrophage function also warrants fur-ther investigation.

Supported by: Endocrinology and Diabetes Research Foundation, The University of Sydney

466-PA Novel Diagnostic Procedure of Asymptomatic Coronary Artery Disease in Diabetic Patients using Carotid-Aall Intima-Media Thickness as a Surrogate Marker and Coronary Computed Tomog-raphy AngiographyYUICHIRO YOSHIKAWA, HIROKI KAGAYA, TAKAHIRO KAGEYAMA, HIROMI MAEDA, SHIGEKI IMAMURA, KAZUO MISUMI, KEMPEI MATSUOKA, AIZAN HI-RAI, Togane, Japan, Matsudo, Japan, Tokyo, Japan

Diabetes is associated with a marked increase in the risk of coronary artery disease (CAD). Also, diabetic patients without previous myocardial infarction have as high a risk of myocardial infarction as non-diabetic pa-tients with previous myocardial infarction. It is well known that patients with diabetes have often asymptomatic CAD. Carotid-wall intima-media thickness (IMT) is a surrogate marker of atherosclerosis associated with cardiovascular risk factors and with cardiovascular outcomes. The present study was performed in order to establish a diagnostic procedure for as-ymptomatic CAD in diabetic patients using the maximum IMT (maxIMT) of carotid artery as a surrogate marker followed by coronary computed tomog-raphy angiography (CCTA), a new noninvasive diagnostic test for CAD. In the present investigation, 317 diabetic patients and 224 non-diabetic patients with lifestyle-related diseases (hypertension, dyslipidemia) without any epi-sode of chest pain and having maxIMT over 1.5mm were studied. CCTA using a 256 channel MDCT scanner was performed for all of them. Then coronary angiography (CAG) was performed for the patients with positive fi ndings in CCTA. In the present study, 51.4% of diabetic patients and 25.4% of non-diabetic patients have coronary lesions with stenosis more than 25% in the CAG (p <0.0001). Also, 36.3% of diabetic patients and 13.8% of nondiabetic patients have coronary lesions with stenosis more than 75% in the CAG (p <0.0001). False-positive rate of CCTA was 9.1% in diabetic patients, 17.4% in non-diabetic patients (p <0.0001), respectively. Among various parameters, age, HDL-C, eGFR and maxIMT have correlation with the degree of coro-nary lesion. Thus, maxIMT of carotid artery over 1.5mm is a useful surrogate marker of asymptomatic CAD in diabetic patients. The present diagnostic procedure requires further studies of subsequent outcomes.

467-PEffects of GLP-1 Receptor Agonist on Ca2+ Handling of Coronary Smooth Muscle Cells from Metabolic Syndrome Ossabaw Swine With Coronary Artery DiseaseMIKAELA L. MCKENNEY, MOUHAMAD ALLOOSH, KYLE SCHULTZ, NICKI BELL, NAGA CHALASANI, MIKE STUREK, Indianapolis, IN

Chronic effects of the glucagon-like peptide (GLP-1) receptor agonist AC3174 were studied in Ossabaw swine with metabolic syndrome (MetS). MetS was induced by hypercaloric atherogenic diet for 6 months. Subcuta-neous injections of placebo or AC3174 were given twice daily for 6 months after induction of MetS. Consistent with clinical effects, AC3174 attenuated food consumption from 96±1% to 82±2% of allotted amount (p<0.05) and weight gain decreased from 116.2±2.4 kg to 99.5±5.8 kg, respectively. Both groups were glucose intolerant, but placebo pigs had greater intolerance after the 6 month treatment as determined by intravenous glucose tolerance tests. AC3174 stimulated a trend towards an increase in insulin secretion with peak insulin levels at 123±25 vs 67±12 μU/mL (p=0.07). MetS increases coronary artery disease (CAD) and dysfunction of intracellular Ca2+ handling by coronary smooth muscle (CSM). CSM cells were isolated enzymatically from the coronary arteries and Ca2+ was digitally imaged with the fl uores-cent Ca2+ indicator fura-2. Ca2+ levels were measured by a fl uorescence ra-tio of 360/380nm. There was no difference in basal Ca2+ levels. In normal physiological salt solution we released sarcoplasmic reticulum (SR) stores with caffeine, which binds to ryanodine receptors in the SR membrane. AC3174 cells showed a larger peak increase in Ca2+ in response to caffeine,

F360/380=0.26±0.02, compared to F360/380=0.16±0.01. A sustained Ca2+ signal was present during recovery to basal levels in the treated cells,

implying there is greater Ca2+ infl ux after chronic AC3174 treatment. The sus-tained fl uorescence ratio from the treated cells was 0.07±0.01 compared to a lesser signal of 0.04±0.003. It appears chronic AC3174 treatment elicits greater Ca2+ release and infl ux in MetS. Our interpretation is that AC3174 attenuates the decline in CSM Ca2+ handling associated with more severe CAD and dedifferentiation of CSM.

Supported by: NIH (HL062552), Amylin Pharmaceuticals, Inc.

468-PMACE Associated With Pioglitazone: A Meta-Analysis of Random-ized, Controlled TrialsALFONSO PEREZ, ERIC SONG, ANTHONY EDMONDS, Deerfi eld, IL

The benefi t of pioglitazone (PIO) with respect to major adverse cardiovas-cular (CV) events (MACE) has been shown in previous studies. This analy-sis evaluated risk of MACE (primary; composite of CV death and nonfatal myocardial infarction or stroke) and serious CHF (secondary) in patients with type 2 diabetes mellitus (T2DM) receiving PIO (n=12,506) vs placebo or active control (n=10,212) in all 36 randomized controlled trials in the PIO clinical database. Treatment durations were 4 to 42 months. Events were identifi ed by searching preferred terms of adverse events identifi ed through standard safety monitoring; in 4 studies, pre-specifi ed central adjudication was performed. The primary endpoint was time from fi rst dose to fi rst event; hazard ratios (HRs) and 95% CIs for events on PIO vs comparator (COMP) were calculated using a Cox proportional-hazards model stratifi ed by study with treatment as the independent variable. Kaplan-Meier estimates were also produced. The primary analysis showed a statistically signifi cant reduc-tion in risk of MACE with PIO vs COMP (HR, 0.82; Table), and Kaplan-Meier plots showed shorter time to fi rst event with COMP. Secondary analyses as well as sensitivity analyses (also in the Table) showed similar results. The analysis also confi rmed the known CHF risk of pioglitazone (HR for CHF causing hospitalization, 1.41; 95% CI, 1.15-1.74), with no corresponding in-crease in mortality. This analysis supports the CV safety of PIO with respect to standard MACE endpoints; PIO consistently showed favorable CV out-comes compared with placebo and active control in T2DM, independent of background CV risk.

MACE in Pioglitazone Trials: Comparison between Pioglitazone and Active or Placebo ControlAnalysis Study Set N HR (95% CI) for MACE

(PIO vs COMP)Primary analysis All controlled studies 22,718 0.82 (0.71, 0.95)Secondary analyses Double-blind

controlled studies22,131 0.82 (0.71, 0.95)

Controlled studies excluding PROactive

17,480 0.82 (0.62, 1.09)

PROactive study 5238 0.82 (0.70, 0.97)Sensitivity analyses(all controlled studies)

Excluding events >30 days after last dose

22,718 0.83 (0.72, 0.97)

Excluding studies with no MACE

20,569 0.82 (0.71, 0.95)

Supported by: Takeda Global Research and Development Center, Inc.

469-PP53 Silenced Endothelial Progenitor Stem Cells (EPC) Improve Col-lateral Circulation Post Femoral Artery Occlusion in Diabetic MiceSABYASACHI SEN, MARY YOUNG, CYRIL CHOU, BROOKE BENTLEY, JOSEPH JERRY, Springfi eld, MA, Amherst, MA

Literature shows that EPCs contribute to increased collateral vessel for-mation following vaso-occlusion. However diabetes reduces EPC number and reduces collateral vessel formation. We cultured human EPCs and exposed them to 5.5 mM (equivalent to 99mg%) and 20mM (equivalent to 360mg%, HG) glucose, which resulted in signifi cant EPC death within 48hrs. We noted HG exposure was associated with up-regulation of P53 and its downstream genes such as P21, PUMA and Caspase-3. We hypothesized that EPC reduc-tion in hyperglycemia is secondary to up-regulation of pro-apoptotic gene, P53. We obtained mouse peripheral blood derived EPCs from P53 null mice and observed that p53 KO EPCs are more resistant to cell death in HG. P53 null EPCs evolved into mature mouse EC (MEC) and retained endothelial properties such as cobblestone appearance and tube-formation on matrigel. Next, we used Lenti and Adenovirus mediated si-RNA methods to silence P53 (long and short term respectively) in human EPCs and P53 silenced EPc showed better survival in HG. We developed femoral artery occlusion model to mimic peripheral vascular disease in diabetic animals. We used strepto-

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zotocin induced type 1 diabetic or Leptin resistant type 2 diabetic mouse model. We delivered saline, P53 WT and P53 null EPCs intra-muscularly around the femoral occlusion in these mouse models (n=10 in each group) and counted CD31+ve number of capillaries in a 20x microscopic fi eld. High-est number was noted in the group that received P53null EPCs ( 26.3±4.2, 49.6±7.4 and 95.7±9.7 respectively). Summary: Transplantation of P53 null EPCs in hyperglycemic mice with femoral occlusion demonstrate better col-lateral vessel formation post femoral artery occlusion setting compared to WT-EPC transplanted group. Conclusion: This fi nding indicates towards pos-sible therapeutic benefi t in transplanting short-term P53 silenced human EPC to prevent peripheral vascular disease in patients with diabetes.

Supported by: Collaborative Biomedical Research Fund

470-PEffects of Amaryl on Aquaporin-1 Expression in Diabetic RatsYADONG SUN, HAIYING WANG, Changchun, China

Role of infl ammation in the pathogenesis of diabetic cardiomyopathy is concerned recently. Infl ammatory reaction is increased by obesity resulting from excessive intake of nutrient in the early stage of type 2 diabetes. In-fl ammatory cytokines in conjunction with other harming factors of diabetes, by causing oxidative stress and cell death, have a bad effect on the cardio-vascular system generally and locally. There is an urgent need to develop a hypoglycemic agent with protective effects on cardiovascular system. Ama-ryl, an agent of glimepiride, has excellent glycemic controls, and decrease insulin resistance, even in nearly diagnosed type 2 diabetic patients.Studies have shown that infl ammatory cytokines such as tumor necrosis factor- and IFN- have increased the expressing of aquaporin (AQP). AQPs are a series of intrinsic homologous membrane proteins related to transporting water, and it has been confi rmed that a variety of AQPs are expressed in myocardial tissue, including AQP1.After establishment of type 2 diabetes model induced by combined high sugar and high fat diet with low-dose streptozotocin (STZ) intraperitoneal injection, the 25 diabetes rats were randomly divided into three groups: low dose Amaryl (2mg/kg, i.g., once daily), high dose Amaryl (4mg/kg, i.g. once daily), model group (an equal volume of normal saline, i.g., once daily). After 8 weeks, levels of serum cardiac enzyms and HbA1c were assayed, and the expression of AQP1 in myocardial tissue was evaluated by immuno-histochemistry and Western bolt individually. Compared with the model group, the levels of cardiac enzymes and HbA1c were signifi cant de-creased in treatment groups (P<0.01), the expression of AQP1 proteins was increased in low dose group (P<0.05) and signifi cantly increased in high dose group (P<0.01).As relation above, we concluded that Amaryl attenuates the occurrence and development of diabetic cardiomyopathy, and its mechanism may be related to stabling the expression of AQP1 in myocardial tissue.

471-PType 2 Diabetes and the Progression of Visualized Coronary Athero-sclerosis to Clinical Cardiovascular EventsHEINZ DREXEL, ALEXANDER VONBANK, PHILIPP REIN, KURT HUBER, CHRIS-TOPH H. SAELY, Feldkirch, Austria, Triesen, Liechtenstein, Vienna, Austria

We aimed at prospectively evaluating to what extent pre-existing coro-nary artery disease (CAD) accounts for the increased long-term vascular event risk of patients with type 2 diabetes (T2DM).Vascular events were recorded over 8 years in 750 consecutive patients whose baseline CAD state was verifi ed angiographically.The baseline prevalence of CAD (87.8% vs. 80.4%; p=0.029) and of signifi cant coronary stenoses 50% (69.5% vs. 58.4%; p=0.010) as well as the extent of CAD, defi ned as the number of signifi cant coronary stenoses (1.7±1.6 vs. 1.4±1.5; p=0.014) were higher in patients with T2DM (n=164) than in non-diabetic subjects. During follow-up, T2DM and CAD proved to be mutually independent predictors of vas-cular events: T2DM predicted vascular events (n=257) independently from the presence and extent of baseline CAD (hazard ratio (HR) 1.36 [1.03-1.81]; p=0.032); conversely, the presence and extent of baseline CAD predicted vascular events independently from T2DM (HRs 3.29 [1.93-5.64]; p<0.001 and 1.37 [1.23-1.53]; p<0.001, respectively). However, the overall risk in-crease conferred by T2DM was driven by an extremely high 53.3% event rate of patients with both T2DM and signifi cant CAD at baseline; individuals with T2DM but without signifi cant baseline CAD showed a signifi cantly low-er event rate (22.0%; p<0.001).We conclude that T2DM and angiographically visualized coronary atherosclerosis are mutually independent predictors of vascular events. However, the overall risk increase conferred by T2DM is driven by accelerated progression of pre-existing atherosclerosis to clini-cal cardiovascular events, whereas vascular risk is much lower in diabetic patients without pre-existing signifi cant CAD.

Supported by: Jubiläumsfonds der Österreichischen National Bank

472-PCardiovascular Dysfunction in Newly Diagnosed Pre-Diabetic SubjectsJUNPING CHEN, DONGXU FU, MINGYUAN WU, JULIE A. STONER, TIMOTHY J. LYONS, Oklahoma City, OK

Cardiovascular dysfunction (CVD) is responsible for a majority of morbidity and mortality in diabetes, and is thought to commence in the pre-diabetic stage. We hypothesized that early cardiovascular dysfunction is present in pre-diabetes and is associated with infl ammation and accumulation of advanced glycation end products (AGEs). In a cross-sectional study, 131 sub-jects were classifi ed by 2-hour 75-g oral glucose tolerance tests into pre-diabetic (n=73, 12M/61F) and normal glucose tolerant (NGT, n=58, 8M/50F) groups. Cardiovascular function was assessed non-invasively by the pulse wave analysis (PWA). Fasting serum CRP, TNF-alpha, IL-1-alpha, adiponec-tin, leptin, VCAM-1, and ICAM-1 were analyzed by ELISA assays. Skin AGE content was determined non-invasively by an investigational device (Scout, Veralight, Inc.) which scans skin AGE-related autofl uorescence to provide a ‘SCOUT score’. Means were compared between groups using a two sample t-test, correlations were quantifi ed using Pearson’s correlation coeffi cient for unadjusted analyses, and partial correlation coeffi cients were calculated for adjusted analyses. Compared to NGT, newly diagnosed pre-diabetic sub-jects exhibited signifi cantly lower large and small artery elasticity indexes (P=0.002 and 0.03), and higher pulse pressure (p=0.004) and total vascular impedance (p=0.01). In addition, they had signifi cantly higher SCOUT scores (p=0.006); higher serum CRP (p=0.01) and TNF-alpha (p=0.01), and lower adi-ponectin (p=0.01) and IL-1alpha (p=0.06). SCOUT score and serum levels of CRP, leptin, and adiponectin were signifi cantly correlated with certain pa-rameters from PWA with and without adjustment for age, BMI, and gender. Newly diagnosed pre-diabetic subjects exhibited abnormal cardiovascular function and pro-infl ammatory status. The associations of AGEs and the tested pro-infl ammatory markers with cardiovascular dysfunction suggest these factors may be implicated in the early pathogenesis of the disease.

Supported by: Talley Research Award and OCAST Award

473-PMetabolic Predictors of Ischemic Heart Disease and Cerebrovascu-lar Attack in Elderly Diabetic Individuals: The Roles of HDL-Choles-terol and the LDL-C/HDL-C RatioTOSHIO HAYASHI, HIDEKI NOMURA, KOICHIRO INA, HIROSHI WATANABE, KOUTARO YOKOTE, KOUTARO YOKOTE, TAKASHI OHRUI, JAPAN CDM GROUP, Nagoya, Japan, Hamamatsu, Japan, Chiba, Japan, Sendai, Japan

Objectives: Lipids, especially LDL-cholesterol (LDL-C) , are risk factors for ischemic heart disease (IHD) in middle aged diabetic individuals, however it is not wellknown what predicts IHD and cerebrovascular attack (CVA) in older patients.Research Design and Methods: This study was designed as a pro-spective cohort study (Japan Cholesterol and Diabetes Mellitus Study) with 4,014 type2 diabetic patients (1,936 women; 67.4±9.5y.o., n=1261<65,1731 from 65 to 74 and 1016>75y.o.; disease duration 9.6±8.0yrs.). The levels of lip-ids, glucose, and other factors like blood pressure were investigated relative to IHD or CVA by stepwise and multiple logistic regression. Results: 153IHD and 104CVA (7.8 and 5.7/ thousand people · year) occurred over 5.5-year. The risk factors for IHD and CVA in elderly are different from those in younger. Namely, lower HDL-cholesterol (HDL-C) was related to IHD in patients>=75 y.o. (odds ratio (OR):0.953, P<0.02), compared to higher hemoglobinA1C (HbA1C) and LDL-C in subjects<65 y.o. (p<0.05) and LDL-C/HDL-C in patients<74 y.o. (n=2992, P=0.006). HDL-C was also related to CVAs in patients >=75y.o. (OR;0.852, p=0.021). Kaplan-Meier estimator curves showed that IHD was signifi cantly frequent in patients <65 y.o. in highest quartile for L/H ratio while in patients >=75 y.o. in lowest quartile for HDL. CVA was frequent in patient>=75 y.o. in lowest quartiles for HDL-C compared with other groups.Conclusion:IHD and CVA in elderly diabetic patients are preditcted by HDL-C. While, LDL-C and HbA1C are risk for IHD in non-elderly, and LDL-C/HDL-C may represent both effects of LDL-C and HDL-C. These differences in risk by age are important for an individualized strategy to prevent atherosclerotic diseases.

Supported by: Japanese Ministry of Health, Welfare and Labor

474-PSerum Cystatin C is not Associated With Arterial Stiffness in Type 2 Diabetes Patients With Normal Renal FunctionHYEONGKYU PARK, SEOKCHUN YEOM, YEOJOO KIM, DONGWON BYUN, KYOIL SUH, MYUNGHI YOO, Seoul, Republic of Korea

Cardiovascular disease (CVD) is the main cause of mortality in patients with type 2 diabetes (T2DM). Several studies showed that increased ar-terial stiffness is an independent risk factor for CVD. Pulse wave velocity

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(PWV) is known as a marker for large vessel stiffness. Recent studies show that serum cystatin C, a marker of renal function, is associated with PWV and may predict future cardiovascular events, even in subjects with normal renal function. However, there have been few studies, especially in Asian populations, for the relationship between cystatin C and arterial stiffness in patients with T2DM. In this study, we investigated the relationship between serum cystatin C and aortic PWV in T2DM patients with normal renal func-tion. Patients with urinary albumin/creatinine ratio(ACR) higher than 300 ug albumin/mg creatinine, estimated glomerular fi ltration rate less than 60 ml/min or systemic infection were excluded. A total of 124 patients(64 M/60 F, age 60 ± 1 yr, ACR 35 ± 5 ug/mg) were included. Doppler-derived aortic PWV, serum cystatin C, leptin, adiponectin, and resistin were measured. Cystatin C is signifi cantly related to age(r=0.48, P < 0.001), leptin(r=0.24, P <0.05), HDL cholesterol (r= -0.20, P < 0.05), and lipoprotein(a) (r=0.21, P < 0.05). Aor-tic PWV is signifi cantly associated with age (r=0.28, P < 0.01), leptin(r=0.21, P <0.05), and cystatin C(r=0.21, P < 0.05). In multiple regression analysis, however, there is no signifi cant association between aortic PWV and serum cystatin C levels. In summary, serum cystatin C is not associated with arte-rial stiffness in T2DM patients with normal renal function.

475-PReduced Vasodilation and Enhanced Vasoconstriction of Renal Ar-teries in Insulin Resistance Rats Induced by High-Sucrose DietYU GAO, GUANGYAO SONG, HUIJUAN MA, Chengde, China, Shijiazhuang, China

The aim of this study was to investigate the effects of long-term high sucrose diet on relaxation and contraction of the renal arteries in insulin re-sistance (IR) rat. Wistar rats were fed by chow diet(control)and high-sucrose diet (HS) (n=14 in each group) respectively. IR was graded by glucose infu-sion rate (GIR) using hyperinsulinemic euglycemic clamp technique. Blood pressure, body weight (BW), plasma triglycemia (TG), free fatty acid (FFA), insulin (INS), fasting blood glucose (FBG), endothelin-1 (ET-1) and nitric oxide metabolite (NO2-/NO3-) were determined at 12 and 24 weeks. Arterial con-tractility was evaluated by concentration-response curves to 10nM-100uM noradrenaline. High-sucrose diet caused moderate rise in blood pressure at 12 and 24 weeks (P<0.01), accompanied by increase in plasma lipids and de-crease of whole body insulin sensitivity. A reduction in endothelium-depen-dent vasorelaxation of renal arteries in HS rats was observed at 12 and 24 weeks (P<0.01), while the contraction responses of renal arteries responded to cumulative dose of noradrenaline was increased. No signifi cant differ-ence on endothelial-independent max vasorelaxation to sodium nitroprus-side between control and HS groups was observed. This study suggests that high-sucrose diet can lead to dyslipidemia, increased IR and hypertension re-sulted from decreased endothelial-dependent vasorelaxation and increased cumulative dose contraction.Table 1. Body weight and biochemistry in control and HS rats at 12th and 24th

week after high-sucrose diet.

Time Group BW FBG TG INS FFA NO2-/ NO3- ET-1(g) (mmol/L) (mmol/L) (mU/L) (mmol/lL) (umol/L) (pg/mL)

12w Normal chow diet

401.00±22.40 5.28±0.40 0.74±0.23 20.67±2.07 0.76±0. 07 68.33±4.58 212.43±16.58

High-sucrose 408.33±11.69 6.53±0.29 0.82±0.20 23.15±1.61 0.97±0.13* 48.26±5.61 245.28±37.8724w Normal

chow diet450.00±60.39 5.26±0.34 0.95±0.19 20.81±1.79 0.78±0.07 64.79±15.46 231.86±20.33

High-sucrose 480.71±19.88 6.90±0.17 1.38±0.30 36.33±4.76 1.17±0.16 39.33±5.94 274.79±24.53*p <0.05 , p <0.01 vs. control.

476-PAortic Stiffness is Associated With White Matter Integrity in Type 1 Diabetes MellitusLINDA D. VAN SCHINKEL, NATHANJA TJEERDEMA, JEROEN VAN DER GROND, ALBERT DE ROOS, JOHANNES W. SMIT, Leiden, The Netherlands

Diabetes mellitus type 1 (T1DM) is associated with white matter injury. Aortic stiffness may contribute to white matter damage in those patients. Diffusion tensor imaging (DTI) is a MRI marker of early white matter disease.Our purpose was to assess the association between aortic pulse wave ve-locity (PWV), as a marker of aortic stiffness, and white matter brain integrity in patients with T1DM, using MRI techniques.Forty-two T1DM patients (23 men, mean age 44±12 years, mean DM duration 24±13 years) and 17 age and gender matched healthy controls were included. Aortic PWV was as-sessed with a 1.5 Tesla MRI. Brain DTI measurements were performed on a 3 Tesla MRI. Fractional anisotropy (FA) and apparent diffusion coeffi cient (ADC) were calculated for white and gray matter integrity. Multivariable lin-

ear regression analyses including cardiovascular risk factors as covariates were used for statisticsWhite matter FA was increased and white matter ADC was decreased in T1DM compared to the control group (p=0.019 for FA resp. p=0.046 for ADC). No differences in gray matter FA or ADC between T1DM and controls were observed. Multivariable linear regression analyses revealed aortic PWV as an independent predictor for white matter integrity (Beta=-0.920 p=0.001 for FA resp. Beta=0.662 p=0.030 for ADC) in T1DM patients. This effect was independent of age, gender, MAP, BMI, smoking, duration of diabetes and HbA1c levels. Aortic PWV did not predict gray mat-ter integrity.In conclusion: aortic stiffness is an independent predictor of white matter integrity in patients with DM1.

Supported by: The Netherlands Heart Foundation (Project UL 2009-4548)

477-PCardiovascular Event Rates in Adults With Type 2 Diabetes Melli-tus: SHIELD 5-Year PerspectiveSANDRA J. LEWIS, KATHLEEN M. FOX, ELISE HARDY, SUSAN GRANDY, SHIELD STUDY GROUP, Portland, OR, Monkton, MD, Wilmington, DE

Macrovascular complications of type 2 diabetes mellitus (T2DM) include myocardial infarction (MI), stroke, and coronary revascularization. This study ascertained the self-reported incidence rates of cardiovascular disease (CVD) events over 5 years among adults with and without T2DM. Respon-dents to the US Study to Help Improve Early evaluation and management of risk factors Leading to Diabetes (SHIELD) surveys reported at baseline (2004) whether they had ever been told by a healthcare professional they had a heart attack, stroke, heart bypass surgery, angioplasty, or stents. In the subsequent 5 years, any new CVD events (incident event[s] reported subsequent to baseline) reported by T2DM respondents and those without diabetes were captured.There were 2,305 T2DM respondents and 7,207 re-spondents without diabetes who reported no prior history of CVD events at baseline. A greater proportion of T2DM respondents had at least 1 inci-dent CVD event over 5 years, compared with respondents without diabetes (16.7% vs. 10.3%, p <0.0001). In addition, 32.8% of T2DM respondents and 31.4% of respondents without diabetes had multiple CVD events (2 or more) over 5 years (p = 0.69). Among T2DM respondents with a prior history of CVD events (n = 898), 47.7% reported a new CVD event (different from baseline event) over the subsequent 5 years. For those without diabetes but with a prior history of CVD events (n = 1,744), 45.6% reported a new CVD event over 5 years (p = 0.33 in comparison with T2DM group). For those with a prior his-tory of CVD events, 23.6% of T2DM respondents and 21.8% of respondents without diabetes had multiple CVD events over 5 years (p = 0.51). There is a high prevalence of self-reported CVD events in respondents with T2DM. For T2DM respondents without a prior CVD history, there was a signifi cantly higher incidence rate (62% relative increase), compared with those without diabetes and no prior history of CVD events. This study is consistent with previous reports suggesting that T2DM is a CVD risk equivalent.

478-PBlood Glucose Improvement Ameliorates Nocturnal Oxygen De-saturations in Type 2 Diabetic PatientsALBERT LECUBE, ANDREEA CIUDIN, GABRIEL SAMPOL, SILVIA VALLADARES, JORDI MESA, CRISTINA HERNÁNDEZ, RAFAEL SIMÓ, Barcelona, Spain

There is growing evidence suggesting an association between type 2 diabetes and sleep apnea syndrome. However, there are no studies to deter-mine whether blood glucose control could prevent nocturnal arterial oxygen desaturation. For this purpose we designed a case-control between 30 type 2 diabetic patients and 10 non-diabetic subjects closely matched by age, gender, and BMI who where admitted to our Diabetes Unit. Nocturnal oxim-etry was assessed with a pulse oximeter (Model 3100®; Nonin Medical Inc, Plymouth, MN USA) at baseline and 5-days after improving blood glucose control. A desaturation event was considered when the haemoglobin satura-tion level (SaO2) fell bellow 3%, 4%, and 6% from baseline saturation. The total number of desaturations was divided by the hours in bed and oxygen desaturation index per hour (ODI) was obtained. Type 2 diabetic patients showed a higher percentage of ODI-3%, ODI-4%, and ODI-6% in comparison with non-diabetic subjects at baseline. Signifi cant positive correlations be-tween fasting plasma glucose and HbA1C with pulse oximeter parameters were detected. In addition, multiple linear regression analyses showed that HbA1c was independently associated with ODI-3%, 4%, and 6% at baseline. Finally, a signifi cant reduction in HbA1c (10.2 ± 1.9 at baseline vs. 9.9 ± 1.9% at discharge, p<0.001) and fructosamine (320.1 ± 73.6 vs. 304.3 ± 59.6 mg/dl, p = 0.005) were associated with a signifi cant reduction in ODI-3% (39.8 ± 27.5 at baseline vs. 32.5 ± 27.5 events/hour at discharge, p<0.001), ODI-4% (28.5 ± 23.7 vs. 23.5 ± 24.2 events/h, p<0.001), and ODI-6% (15.1 ± 16.8 vs.

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COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS IN DIABETES

12.9 ± 17.3 events/h, p=0.009) in diabetic patients. However, no changes in ODI events were observed in non-diabetic patients after 5-days of fol-low-up. Therefore, glycemic control is related to nocturnal arterial oxygen desaturation in type 2 diabetes. Our results suggest that improvement in nocturnal arterial oxygen desaturation can be added to the benefi cial ef-fects of metabolic optimization.

479-PImpact of CVD History and GXT Abnormalities on Weight Loss and Fitness GainJEFFREY M. CURTIS, TINA KILLEAN, GEORGE A. BRAY, LAWRENCE J. CHESKIN, EDWARD S. HORTON, KAREN C. JOHNSON, JOHN M. JAKICIC, F. XAVIER PI-SUNYER, JUDITH G. REGENSTEINER, PAUL M. RIBISL, LYNNE WAGENKNECHT, MARK A. ESPELAND, Phoenix, AZ, Shiprock, NM, Baton Rouge, LA, Baltimore, MD, Boston, MA, Memphis, TN, Pittsburgh, PA, New York, NY, Aurora, CO, Winston-Salem, NC

Whether a history of cardiovascular disease (CVD) or an abnormal graded exercise test (GXT) infl uences the capacity to lose weight or improve fi tness is unknown.Look AHEAD is a multicenter randomized trial of a behavioral weight loss intervention (ILI) in overweight or obese adults with type 2 dia-betes (T2D), aged 45-76 years. Participants underwent a maximal GXT at baseline and submaximal GXTs one and four years later. This analysis in-cludes only ILI participants with follow-up data (n=2514, 97.8% of ILI group).At study entry 356 participants reported a history of CVD events (including myocardial infarction, stroke, coronary bypass, etc.). Baseline maximal GXTs were deemed abnormal by angina, ST segment changes, ventricular arrhyth-mia (VA), poor heart rate recovery (HRR), or peak workload < 5 METs; 491 had abnormal baseline GXTs. We compared those with and without a history of CVD or abnormal baseline GXT with regard to weight loss and fi tness gains over 4 years of Look AHEAD intervention.Those without a history of CVD events (p=0.02), lost more weight (6.7%) than those with such history (6.0%). Age- and sex-adjusted weight losses, were similar for those with (6.8%) and without (6.5%) abnormal baseline GXT. However, by specifi c GXT abnormal-ity, those with VA (n=6, 12.4 vs. 6.6%, p=0.008) or angina (n=14, 10.3 vs. 6.6%, p=0.01) during exercise, or poor HRR (n=72, 7.9 vs. 6.5%, p=0.03) lost more weight than those without.A history of a CVD event (0.43 vs 0.61 METs, p=0.006) or low peak workload (n=247, 0.25 vs. 0.62 METs, p=0.01) was as-sociated with lower fi tness gains. However, age-, sex-, and baseline fi tness-adjusted fi tness gains were similar for those with and without abnormal GXT (0.46 vs. 0.61 METs, p=0.89), and better for those with than without VA (1.47 vs. 0.58 METs, p=0.04).A history of CVD events or low peak workload on maximal GXT appear to hamper fi tness gains, but VA is associated with better fi tness gains. A history of CVD may hinder weight loss, whereas ab-normalities on the GXT (VA, angina, or abnormal HRR) do not.

480-PAssociations of Fetuin-A Levels With Insulin Resistance and Vascu-lar Complications in Type 2 Diabetic PatientsCHAN-HEE JUNG, BO-YEON KIM, CHUL-HEE KIM, SUNG-KOO KANG, JI-OH MOK, Bucheon, Republic of Korea

Contradictory results have been reported regarding the role of fetuin-A in macroangiopathies. Moreover, there is few data available for associations of fetuin-A with microangiopathies in T2DM. Therefore, we examined the relationship between fetuin-A and macro- and microangiopathies including cardiac autonomic neuropathy(CAN) in Korean T2DM patients. A total of 185 T2DM patients (119 males and 53 females) were recruited and evaluated for diabetic nephropathy, retinopathy, peripheral neuropathy and CAN. Serum fetuin-A levels were assessed by ELISA and the insulin resistance status was assessed by the HOMA-IR. Atherosclerotic burden was assessed by ankle-brachial index(ABI) and brachial-ankle pulse wave velocity(baPWV).The mean levels of serum fetuin-A were 525 ± 162 ug/ml and not differ-ent between males and females. Serum fetuin-A levels showed signifi cant positive correlations with HOMA-IR (r=0.196, p=0.022), baPWV (r=0.165, p=0.036), systolic BP (r=0.165, p=0.036), total cholesterol (r=0.164, p=0.032), triglycerides (r=0.215, p=0.006) and signifi cant negative correlations with ABI (r=-0.183, p=0.019). Serum fetuin-A levels were also negatively corre-lated with serum adiponectin (r=-0.187, p=0.027) and positively correlated with serum TNF-alpha (r=0.204, p=0.016). The mean levels of HOMA-IR were borderline signifi cantly increased progressively across fetuin-A tertiles (p for trend=0.06). The mean levels of serum fetuin-A were not signifi cantly different according to the presence of each microvascular complications. This present study showed that levels of serum fetuin-A are signifi cantly as-sociated with IR and arterial stiffness while there are no associations with microangiopathies in T2DM patients. Future prospective studies with larger

numbers of patients are required to establish a direct relationship between serum fetuin-A levels and the development or severity of diabetic vascular complications.

481-P

COMPLICATIONS—MACROVASCULAR—CELLULAR MECHANISMS OF ATHEROGENESIS

IN DIABETES

Guided Audio Tour: Diabetes and the Arterial Wall—Focus on the Endo-thelium (Posters 482-P to 489-P), see page 13.

& 482-PTargeted Imaging of Myocardial Angiogenesis in Type-1 Diabetes With Novel PET Radiopharmaceutical Demonstrates Impairment of Alpha-v Integrin ActivationMATTHEW R. SCHUELKE, IWONA T. DOBRUCKI, SUZANNE LAPI, LAWRENCE W. DOBRUCKI, Urbana, IL, St. Louis, MO

We have previously assessed temporal changes in peripheral angiogen-esis in mice subjected to type-1 diabetes (DM). Peripheral angiogenesis in DM was signifi cantly reduced as assessed by microSPECT-CT imaging using Tc-99m labeled agent targeted at v integrin. We hypothesize that a novel PET radiopharmaceutical based on cRGD peptide structure can assess the differences in chronic myocardial angiogenesis associated with DM.Surgical ligation of LAD was performed on male non-DM (n=3) and DM Lewis rats at 8 weeks after streptozotocin treatment (n=4, 50 mg/kg i.p., bolus). DM rats demonstrated signifi cant weight loss and fasting glycemia (>400 mg/dL). At two weeks post-ligation, Cu64-cRGDyK (~400uCi) was injected and 60min later in vivo contrast microCT followed by microPET imaging were performed. Blood was collected for measurement of glucose and plasma insulin (assessed with ELISA kit). After imaging, rats were euthanized and organs collected for biodistribution studies. Hearts were excised, cleaned with ice-cold saline and imaged postmortem with a hybrid microPET-CT scanner. After imaging, hearts were cut into 2-mm short axis slices and anterior, septal, posterior, and lateral segments for gamma well counting (GWC) of Cu64 activity.In DM rats, glucose levels were chronically elevated, and insulin levels signifi cantly reduced (p<0.05). Non-invasive microPET-CT imaging with Cu64-cRGDyK and GWC analysis demonstrated a signifi cant (p<0.05) impairment of v integrin activation in all myocardial segments of DM rats.A novel Cu64-cRGDyK agent targeted at activated v integrin pro-

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vided favorable distribution for PET-CT imaging of myocardial angiogenesis which appears to be impaired in the onset of uncontrolled DM. This targeted

v integrin imaging strategy may allow for monitoring of therapeutic inter-ventions directed at stimulation angiogenesis early post-MI.

Supported by: AHA (10SDG4180043)

& 483-PAblation of PDK1 in Vascular Endothelial Cells in Diabetic Condition Deteriorates Vascularity Only in Skeletal Muscles But Not in LiverKAZUHITO TAWARAMOTO, MITSURU HASHIRAMOTO, TOMOHIKO KIMURA, HIDENORI HIRUKAWA, MASASHI SHIMODA, WATARU OGAWA, MASATO KA-SUGA, KOHEI KAKU, Okayama, Japan, Hyogo, Japan, Tokyo, Japan

We previously reported that the vascular endothelial cell-specifi c PDK1 knockout (KO) mice with diabetes deteriorated systemic insulin sensitivity due to lowered vascularization in the skeletal muscle. In contrast, the he-patic insulin sensitivity was not deteriorated, whereby we hypothesized that vascularization in the liver is intact in KO mice compared to the control. KO and control mice (Con) at 6 weeks of age were injected intraperitoneally with streptozotocin (STZ), 150μg/kgBW, or vehicle (Veh). As results, vascu-larization in gastrocnemius muscle (Gastro) and the liver was not different between Veh-treated Con and KO mice (Liver; Veh-Con 0.528±0.145μm2 vs. Veh-KO 0.804±0.141μm2, n.s., Gastro; Veh-Con 0.416±0.049μm2 vs. Veh-KO 0.461±0.045μm2, n.s.). In STZ-injected group, however, vascularization in Gastro was expectedly lowered in KO (Veh-KO 0.461±0.045μm2 vs. STZ-KO 0.323±0.035μm2, p<0.05), but not in Con (Veh-Con 0.416±0.049μm2 vs. STZ-Con 0.453±0.045 μm2, n.s.). On the other hand, vascularization in the liver was not signifi cantly different between STZ-Con and STZ-KO. Protein ex-pression of vascular cell adhesion molecule 1 (VCAM1), located in PI3K signal and regulated by PDK1, was signifi cantly up-regulated by 2.4 times higher in Gastro of STZ-KO than in that of Veh-KO, but in the liver, no signifi cant difference was observed between two groups. Vascular endothelial growth factors (VEGF), VEGF receptor (VEGFR), and hypoxia induced factor 1 (HIF1) gene expressions in both Gastro and liver were not different between STZ-KO and Veh-KO. The present results indicated that ablation of PDK1 in vascu-lar endothelial cells in diabetic state induces the lower vascularization and VCAM1-upregulation in skeletal muscle but not in liver, and the underlying mechanism may be totally different from the reported angiogenic factors, including VEGF and HIF1.

& 484-PGlucagon-Like-Peptide-1 (GLP-1) Activates AMPK and Diminishes Infl ammation in Human Aortic Endothelial CellsNADIA M. KRASNER, JOSE M. CACICEDO, YASUO IDO, NEIL B. RUDERMAN, Boston, MA

GLP-1 therapy is prescribed for type 2 diabetes treatment due to its ef-fects on insulin sensitivity and secretion, glucagon secretion, and food in-take, resulting in improved glycemic control. Recent epidemiological studies also suggest that it may have cardio- and cerebrovascular benefi ts; however, the mechanisms responsible for them are not known. AMP- activated pro-tein kinase (AMPK) has been implicated as a therapeutic target for the pre-vention of cardiovascular disease by virtue of its effects on lipid metabolism, infl ammation and other atherogenic events in vascular cells. In the present study, we examined whether GLP-1 activates AMPK and affects infl amma-tion in human aortic endothelial cells (HAECs). We found that GLP-1 (0.3nM) transiently activates AMPK in HAECs, with phosphorylation of its substrate acetyl-CoA carboxylase (ACC), peaking between 5-15min, and activation of CREB by 20min. GLP-1 also signifi cantly decreased the infl ammatory re-sponse (indicated by increased expression of adhesion factors; VCAM-1, ICAM and TNFa) caused by hyperglycemia (HG, 25mM), lipopolysaccharide (LPS, 1ug/mL) or TNF (10ug/mL). Treatment of HAECs with LPS or TNFalso increased monocyte adhesion and this too was diminished by GLP-1. RT-PCR revealed that HAECs also express dipeptidyl peptidase-4 (DPP4), the primary enzyme responsible for GLP-1 degradation. Incubation with HG increased HAEC DPP4 activity, as well as GLP-1 degradation, potentially ex-plaining the reduced level of GLP-1 in diabetic patients. The results suggest that the benefi cial effects of GLP-1 on atherosclerotic risk could be medi-ated by AMPK activation and the resulting decrease in infl ammation. Further studies are needed to determine if the sustained effect of GLP-1 is attribut-able to AMPK, and if so, whether it works by acute effects on metabolism and/or the regulators of transcriptional activation.

& 485-PCirculating Microparticles From db/db Diabetic Mice Induce Dys-function and Apoptosis to Cerebral Endothelial CellsJI CHEN, CHENG ZHANG, PENG LIU, SHUZHEN CHEN, ZHEN YAO, YANFANG CHEN, Dayton, OH, Sanya, China

Type-2 diabetes is a well known risk factor for cerebral vascular diseases. Our previous study showed that circulating microparticles (MPs) of db/db diabetic mice are high in number and cause dysfunction to endothelial pro-genitor cells. This study was designed to investigate whether circulating MPs from db/db mice have direct effects on cerebrovascular endothelial cell (cECs). Adult (9-12 weeks) male db/db and their age-matched control (db/+) mice were used for the study. Circulating MPs were isolated from db/db (dMPs) and db/+ mice (cMPs). Primary cECs were cultured from db/+ mice. The 3-5 passages of cECs were used for pre-incubation with dMPs, cMPs (5 x 105/ml) or vehicle (culture medium) for 48 hrs. The tube formation ability of cECs was determined using a commercial assay kit. Cell apoptosis was ana-lyzed by fl ow cytometric method. The expression levels of endothelial nitric oxygen synthase (eNOS) and zonula occludens protein-1 (ZO-1) were mea-sured by western blot. Caspase-3 activity was determined by fl uorogenic substrate assay. Results showed: 1). Pre-incubation with dMPs decreased the ability of tube formation (15.8 ± 0.6, 29.6 ± 2.2 and 32.6 ± 3.2 tubes/fi eld, n=5/group, P<0.01, dMPs vs. cMPs or vehicle) and increased the rate of cell apoptosis (20.6 ± 1.2%, 6.2 ± 0.4% and 5.6 ± 0.2%, n=5/group, P<0.01, dMPs vs. cMPs or vehicle) in cECs; 2) Pre-incubation with dMPs down-regulated eNOS and ZO-1 ( eNOS: 0.58 ± 0.08, 1.09 ± 0.08 and 1.00 ± 0.05; ZO-1: 0.62 ± 0.08, 1.08 ± 0.07 and 1.02 ± 0.06 fold of control, n=4/group, P<0.01, dMPs vs. cMPs or vehicle) and up-regulated caspase-3 activity (1.1 ± 0.1 nmol, 0.5 ± 0.04 nmol and 0.4 ± 0.02 nmol, n=4/group, P<0.01, dMPs vs. cMPs or vehicle) in cECs. In conclusion, our data suggest that circulating MPs could be one of the mechanisms which mediate cEC dysfunction and/or injury in diabetes.

& 486-PDerivation of Induced Pluripotent Stem Cells from Patients With Greater Than 50 Years Duration of Type 1 DiabetesSHWETA BHATT, ADRIAN K. TEO, SUSANA SILVA, CHONG WEE LIEW, DAN KAWAMORI, REBECCA WINDMUELLER, HILLARY KEENAN, GEORGE L. KING, AMY J. WAGERS, ROHIT N. KULKARNI, Boston, MA

Type1 diabetes (T1D) is an autoimmune disorder characterized by the loss of insulin-producing -cells. At Joslin, a cohort of patients who survived 50 years of T1D (“medalists”), with or without complications presented a unique opportunity to study mechanisms underlying the disease. The impetus for the study came from observation that serum from several medalists tested positive for C-peptide, and autopsied pancreases from 9 patients showed insulin+ -cells, suggesting unique mechanism(s) protecting these -cells and preventing some Medalists from developing complications. To inves-tigate pathways protective towards the -cells and underlying mechanisms for diabetic complications we obtained skin fi broblasts from medalists or age-matched controls and subjected them to in vitro reprogramming using excisable polycystronic lentivirus expressing OCT4, SOX2, KLF4 and c-MYC to induce pluripotent stem cells (iPSCs). Although reprogramming was nor-mal in both groups, evidenced by positive staining for early (Alkaline phos-phatase), intermediate (OCT4, NANOG, SOX2, SSEA4) and late (Tra1-60 and 1-81) markers of pluripotency, we observed signifi cantly compromised ef-fi ciency in establishing iPSCs from medalists, both with (0.002%) and with-out (0.004%) complications, as compared to age-matched controls (0.02%) [p<0.001; n=3-5]. Intriguingly, medalist iPSCs also exhibited striking morpho-logical differences and reduced proliferation rates (20% of controls, n=5). Further, iPSCs derived from medalists with complications demonstrated impaired spontaneous differentiation potential (10%), unlike the iPSCs from controls (90%) or from medalists without complications (70%) [p<0.001]. These data suggest that unique cellular mechanisms regulate the growth and differentiation of iPSCs from medalists, alterations in which, through genetic manipulation or in vitro disease modeling, may enable the discovery of novel therapeutics to reduce the burden of T1D.

& 487-PHyperglycemia Exacerbates Endothelial Cell Injury during Hypoxia/Reoxygenation via p300-Mediated Persistent Epigenetic Regula-tion of Egr-1 GeneYAQIAN DUAN, BO ZHOU, YUHANG LIU, CHAO DU, HONG SU, Chongqing, China

Hyperglycemia is an independent risk factor of short- and long-term mor-tality in acute myocardial infarction (AMI) patients with and without diabe-tes. However, the mechanisms are not fully understood. We investigated

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whether p300, a transcriptional coactivator with histone acetyl transferase (HAT) activity, mediates glucose-induced upregulation of transcription factor early growth response gene (Egr)-1 and subsequent increase of vasoactive factors during hypoxia/reoxygenation (H/R) in human umbilical vein endothe-lial cells (HUVECs). A H/R injury model was established by exposing HUVECs to 5h of oxygen and glucose deprivation, followed by 24h of reoxygenation in different glucose concentrations. Hyperglycemia (25mM glucose) led to up-regulated p300 expression and increased Egr-1 levels in H/R injured HU-VECs. ChIP assay of the Egr-1 promoter in H/R injured HUVECs showed that hyperglycemia promoted p300 binding and acetylation of histones H3 and H4. Treatment with the p300 activity inhibitor, garcinol, or shRNA targeting p300 caused obvious decreases in p300-Egr-1 promoter binding and Ac-H3 and Ac-H4 levels, ultimately resulting in down-regulation of Egr-1 expres-sion. p300 inhibition also prevented the hyperglycemia-induced increase of two Egr-1-activated genes: intercellular adhesion molecule 1 (ICAM-1) and tissue factor (TF). Furthermore, during 24h of exposure to hyperglycemia, ac-tive recruitment of p300 to the Egr-1 promoter was observed, accompanied by increasing histone acetylation. Remarkably, these epigenetic changes persisted during subsequent incubation at 5.5mM glucose for three days, and were prevented by p300 inhibition.Our data suggest that p300 plays an important role in hyperglycemia-induced endothelial cell injury during H/R. Epigenetic histone modifi cations may be a key mechanism that promotes the progression of vascular damage in AMI patients, even after normoglycemia is achieved.

& 488-PEndothelial Overexpression of PKC 2 Accelerates the Develop-ment of AtherosclerosisQIAN LI, KYOUNGMIN PARK, CHENZHONG LI, CHRISTIAN RASK-MADSEN, AKI-RA MIMA, GEORGE L. KING, Boston, MA

Endothelial dysfunction due to loss of insulin action on the endothelium clearly can contribute to the risk of atherosclerosis. At the molecular level, we have reported that hyperglycemia and elevated fatty acid can activate several protein kinase c (PKC) isoforms and selectively inhibit insulin’s ac-tivation of endothelial nitric oxide synthase (eNOS) via phophatidylinositol 3-kinase (PI3K)/Akt pathway. This study demonstrated the effect of PKC 2activation in the endothelial cells can increase endothelial dysfunction and atherosclerosis in ApoE null mice. PKC 2 isoform was overexpressed spe-cifi cally using endothelial promoter VE-Cadherin and crossed into ApoE null mice (PKC EApoE). Being on high fat diet, PKCBEApoE null mice did not dif-fer from ApoE control in weight, systemic insulin sensitivity, glucose toler-ance, plasma lipid or blood pressure. However, the size and complexity of the atherosclerotic lesions in the aorta increased by 70% as measured by en face fat staining and plaque content of smooth muscle cells and extracellular matrix. Insulin specifi c action on the endothelial cells and femoral artery from the PKC EApoE null mice was impaired by 40% with respect to PI3K/Akt/eNOS phsophorylation (activation) selectively, since the activation of MAPKkinase was not effected. We have reported that angiotensin II (Ang II) can activate PKC and phosphorylate Thr 86 of P85/PI3K to inhibit insulin phosphorylation of eNOS. In PKCBEApoE null mice, the same phophorylai-ton site on P85/PI3K was increased in endothelial cells. Infusion of Ang II increased ET-1 expression in the aorta of PKC EApoE by 1.7 fold more than ApoE null control. Thus, we propose that the acceleration of atherosclerosis in diabetes and insulin resistance is due to loss of endothelial insulin action caused by PKC activation and subsequent enhancement of angiotensin’s atheogenic actions in the vessel wall.

& 489-PEndogenous Secretory Receptor for Advanced Glycation Endprod-ucts (esRAGE) and AGEs-to-esRAGE Ratio as Biomarkers of Athero-sclerosis Risk in Type 2 DiabetesZDENKA TURK, SPOMENKA LJUBIC, JOZO BORAS, Zagreb, Croatia

Endogenous secretory receptor for advanced glycation endproducts (es-RAGE) an isoform of soluble RAGE, acts as decoy for AGEs. We hypothesized ratio of AGE-to-esRAGE could be link to atherosclerosis in diabetes. To examine this issue, we compared serum levels of esRAGE and methylgly-oxal-adduct as well as the AGE-to-esRAGE ratio between type 2 diabetic patients with and without established macrovascular disease.Methylglyox-al-adducts, AGEs and esRAGE were measured in type 2 diabetes (n=130) and healthy controls (n=30). A history of macrovascular events (cardiovascular disease, cerebral vasculopathy or peripheral vascular disease) was recorded in 50 patients.esRAGE levels were lower in type 2 diabetic patients com-pared with control subjects (0.330±0.197 vs. 0.458±0.074 ng/ml, p=0.003). In diabetic population esRAGE correlated positively with MG-adducts, HbA1c,

BMI, and triglyceride, and inversely with CRP, LDL and homocystein. Mul-tiple regression analysis was performed to determine which parameters best predicted esRAGE level. Finally, urinary MG-adduct excretion (p<0.001), AGEs (p<0.001), and AGE/RAGE (p<0.001, inversely) remained to be indepen-dently associated circulatory esRAGE. Multiple stepwise regression model was used to evaluate the relationship between macrovascular disease as a dependent, and metabolic and AGE-parameters as independent variables. Analysis pointed to LDL ( =0.40 P<0.001), HbA1c ( =0.37 P<0.001), AGE/es-RAGE ( =0.31 P=0.007) and homocysteine ( =0.25 P=0.013) as signifi cant independent contributors to diabetic macrovascular disease.High AGE/es-RAGE ratio and esRAGE down-regulation observed in type 2 diabetes were found to be independent predictors of macrovascular disease. A decreased level of esRAGE may therefore be a biomarker of atherosclerosis acting as either a decoy receptor or another mechanism.

490-P

491-PSerum- and Glucocorticoid-Inducible Kinase-1 Increases Telom-erase Activity and Delays the Onset of Endothelial Cell Senes-cenceKATIA BASELLO, FRANCESCA FERRELLI, MARCO FELICE LOMBARDO, DONA-TELLA PASTORE, BARBARA CAPUANI, ANDREA COPPOLA, ROBERTO ARRIGA, SARA CARATELLI, FRANCESCA PACIFICI, GIULIA DONADEL, PAOLO SBRACCIA, MASSIMO FEDERICI, DAVIDE LAURO, Rome, Italy

In Type 2 Diabetes (T2D) precocious endothelial cell senescence increases the risk of developing atherosclerosis; telomeres shortening is a molecular clock of ageing. Telomerase, a RNA-directed DNA polymerase, extends te-lomeres length of eukaryotic chromosomes and human telomerase reverse transcriptase (hTERT) is the catalytic subunit of the telomerase. In this study, it has been examined whether Serum- and Glucocorticoid-inducible Kinase (SGK)-1 can regulate cellular senescence in human umbilical vein endothelial cells (HUVECs). SGK-1 is a serine threonine kinase that promotes cell survival and reduces the intracellular levels of ROS. HUVECs have been infected with SGK-1wt (full length), SGK-1 60 (deleted of the Nh2- 60aa) or the empty vec-tor using a retroviral system. Telomerase activity, detected with TRAP as-say, was found increased in HUVECs infected with SGK-1wt (p<0.001), after eighth populations doubling level (PDL). Protein levels and phosphorylation of hTERT and SGK-1 have been measured using Western Blot. Furthermore, specifi c inhibition of SGK-1 with GSK 650394 completely prevented hTERT phosphorylation at Ser824, with or without insulin stimulation. SGK-1 was differently localized after infection of HUVECs with SGK-1 60 or SGK-1wt constructs in cytoplasmic or nuclear compartments, respectively. Moreover, the senescent status has been determined with SA- -galactosidase in situ staining, with a signifi cant decrease of senescent cells (p< 0.05) in HUVECs infected with SGK-1wt after 14 PDL. These results demonstrate that over-expression of SGK-1 delays the onset of senescence with a telomerase-dependent mechanism. In conclusion, SGK-1 increases telomerase activity

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and reduces the beta-galactosidase activity. SGK-1 may have an anti-aging effect that need further investigation and can be a new molecular target for anti-aging therapy.

Supported by: Rome Foundation, Ministry of University 2009, Di Mario Foundation

492-PRho-Kinase Mediates ER Stress-Induced Endothelial DysfunctionDAIJI KAWANAMI, KEIICHIRO MATOBA, RINA OKADA, MASAMI TSUKAMOTO, JUN KINOSHITA, TOMOKO ITO, SHO ISHIZAWA, YASUSHI KANAZAWA, TAMOT-SU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan

The process of atherosclerosis is affected by interactions among numer-ous biological pathways including endoplasmic reticulum (ER) stress. C/EBP homologous protein (CHOP), which induces cellular apoptosis in response to ER stress, has been reported to promote atherosclerosis. Rho-kinase, an effector of small G protein Rho, has been implicated in the pathogenesis of atherosclerosis. However, its role in ER-stress mediated cellular response remains unknown. In this study, we investigated the role of Rho-kinase in ER stress-mediated endothelial dysfunction. Tunicamycin, an ER-stress inducer, increased Rho-kinase activity in human umbilical vein endothelial cells. Tu-nicamycin also increased CHOP mRNA expression. Interestingly, fasudil, a specifi c Rho-kinase inhibitor, inhibited this induction. From a mechanistic standpoint, we found that fasudil attenuated eukaryotic initiation factor (eIF)2 phosphorylation and subsequent induction of activating transcrip-tion factor (ATF)4 that are key factors to induce CHOP. It is shown that eIF2activates not only ATF4 but also NF- B signaling pathway. Parthenolide, a specifi c NF- B inhibitor, attenuated tunicamycin-mediated vascular cellular adhesion molecule 1 (VCAM-1) expression, suggesting that VCAM-1 induc-tion by tunicamycin is NF- B-dependent. We next used a variety of MAPK in-hibitors to elucidate signaling pathway that is involved in VCAM-1 induction. SB203580, a specifi c p38MAPK inhibitor, attenuated VCAM-1 induction. Furthermore, fasudil inhibited tunicamycin-mediated p38MAPK phosphory-lation, indicating that Rho-kinase mediates VCAM-1 induction via p38MAPK signaling pathway under ER stress. These fi ndings demonstrate that Rho-kinase plays a critical role in tunicamycin-mediated endothelial dysfunction by activating eIF2 and subsequent ATF4 and NF- B/p38MAPK-dependent signaling pathways. We conclude that Rho-kinase is an attractive target against atheroscelrosis by inhibiting ER stress-induced endothelial dysfunc-tion.

493-PRole of p66Shc in Cytokine-Mediated Leukocyte Recruitment by Endothelial CellsMAURA R. ORLANDO, LUIGI LAVIOLA, MARIA A. INCALZA, ANNA LEONARDINI, ANGELO CIGNARELLI, FEDERICA TORTOSA, ROSSELLA LABARBUTA, MARI-ANGELA MELCHIORRE, SABINA MARTEMUCCI, SEBASTIO PERRINI, ANNALISA NATALICCHIO, FRANCESCO GIORGINO, Bari, Italy

Endothelial cells actively participate in vascular infl ammatory events by regulating leukocyte recruitment via the expression of adhesion molecules. The protein p66Shc has been proposed as a mediator of vascular dysfunc-tion and a sensor of oxidative stress, and is upregulated in diabetes. The aim of this work was to investigate the role of p66Shc in regulating leukocyte recruitment by human umbilical vein endothelial cells (HUVEC). Exposure of HUVEC to 50 ng/ml TNF-alpha resulted in increased phosphorylation of p66Shc on Ser36 and intracellular ROS levels, and promoted leukocyte trans-migration through the HUVEC monolayer (p<0.05). TNF-alpha also induced a marked increase in the expression of the adhesion molecule E-Selectin (p<0.05). Adenovirus-mediated overexpression of p66Shc in HUVEC resulted in enhanced p66Shc phosphorylation on Ser36, increased ROS and E-Selec-tin levels, and marked augmentation of leukocyte transmigration (p<0.05), which appeared to be further increased when cells were exposed to TNF-alpha. Conversely, overexpression of a phosphorylation-defective p66Shc protein, in which Ser36 was replaced by Ala, did not augment ROS levels or E-Selectin expression beyond those found in wild-type cells, and minimally infl uenced leukocyte transmigration. Moreover, silencing of p66Shc with two different siRNAs markedly reduced E-Selectin expression, both under basal conditions and following TNF-alpha exposure (p<0.05), and this was associated with decreased leukocyte transmigration (p<0.05). In conclusion, p66Shc acts as a novel signaling intermediate in the TNF-alpha pathway regulating E-Selectin expression and the adhesive properties of endothelial cells, and its action requires phosphorylation of p66Shc on Ser36.

494-PSuppressive Effect of Acute Hyperinsulinemia on Serum Soluble Forms of Adhesion Molecules in Young Healthy SubjectsMONIKA KARCZEWSKA-KUPCZEWSKA, AGNIESZKA ADAMSKA, AGNIESZKA NIKOLAJUK, MAGDALENA ZIELINSKA, NATALIA MATULEWICZ, MARIA GOR-SKA, IRINA KOWALSKA, MAREK STRACZKOWSKI, Bialystok, Poland, Olsztyn, Poland

Markers of endothelial dysfunction, including soluble E-selectin (sE-selectin), soluble intercellular adhesion molecule 1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) are positively related to insu-lin resistance and cardiovascular disease. Atherosclerosis is increased in hyperinsulinemic states, but the direct effect of insulin on atherogenesis remain unclear. The aim of the present study was to investigate the effect of acute hyperinsulinemia on serum sE-selectin, sICAM-1 and sVCAM-1 concentrations in young healthy population. We studied 67 healthy male subjects (mean age, 23.33±2.54; mean body mass index, 25.54±3.79 kg/m2). Serum sE-selectin, sICAM-1 and sVCAM-1 concentrations were measured before and after 2 hour euglycemic hyperinsulinemic clamp. In 20 subjects, clamp was prolonged to 6 hours and at the end additional measurements of serum sE-selectin, sICAM-1 and sVCAM-1 were taken. Hyperinsulinemia de-creased serum sE-selectin, this effect was present in both 2 and 6 hours of the clamp (both p<0.001). The suppressive effect of hyperinsulinemia on se-rum sICAM-1 was also observed in 2 and 6 hours of the clamp (both p<0.001). We also observed decrease in serum sVCAM-1 after 2-hour and 6-hour acute infusion of insulin (both p<0.001). Baseline sE-selectin was positively related to BMI (r=0.37, p=0.02), waist circumference (r=0.42, p<0.001) and triglycer-ides (r=0.32, p=0.008) and negatively to insulin sensitivity (r=-0.29, p=0.017). Our data show that acute hyperinsulinemia decreased soluble forms of ad-hesion molecules, suggesting the antiatherogenic effect of insulin in healthy subjects.

Supported by: Grant UDA-POIG.01.03.01-00-128/08; from the Program Innovative Economy 2007

495-PWillow Bark Extract is a Robust Natural Indirect Antioxidant through the NRF2 ActivationATSUSHI ISHIKADO, YOKO SONO, MOTONOBU MATSUMOTO, AYA OKUNO, STACEY ROBIDA-STUBBS, GEORGE L. KING, T. KEITH BLACKWELL, TAKETOSHI MAKINO, Osaka, Japan, Boston, MA

NRF2 is a redox-sensitive master regulatory transcriptional factor that plays an important role in vascular protection. NRF2 activation may prevent some diabetic vascular complications such as nephropathy. In this study, we have discovered that willow bark extract (WBE), which is listed in Eu-ropean Pharmacopoeia, induces a robust phase II response as indicated by NRF2 activation in human umbilical vein endothelial cells (HUVECs) and Caenorhabditis elegans (C. elegans). WBE (25-400 μg/ml) dose-dependently increased NRF2 target genes HO-1 (200 μg/ml; 20.7 fold), GCLM (3.6 fold), GCS-1 (1.5 fold) or p62 (2.8 fold) mRNA and their protein expressions, and also increased intracellular total glutathione level. WBE (200 μg/ml) in-creased the intranuclear expression (3.2 fold) and DNA binding (2.8 fold) of NRF2. WBE also increased luciferase activity (200 μg/ml; 14 fold) assessed by ARE (antioxidant response element) reporter plasmid dose-dependently. The siRNA of NRF2 signifi cantly reduced the WBE-induced HO-1, GCLM or p62 mRNA and their protein expressions in HUVECs. Pretreatment with WBE (100 μg/ml) prevented the tert-butyl hydroperoxide (tBHP)-induced cell toxicity, while the effect of WBE was canceled by the siRNA of NRF2. WBE-induced mRNA expressions of NRF2 downstream molecules were partially reduced by a specifi c inhibitor of p38, SB203580, but not those of PI3K, PKC and MAPK. We also evaluated in vivo the effects of WBE on NRF2 activation and antioxidative stress activity using C. elegans. WBE (10 mg/ml) signifi -cantly increased GFP expression in C. elegans using a gcs-1 transgene fused with GFP and pretreatment of WBE signifi cantly extended the longevity of C. elegans in case of providing the oxidative stress with tBHP. These re-sults provided the fi rst evidence that WBE increased antioxidative activity through the activation of NRF2 and may contain substances that can protect against the development of diabetic vascular complications and the other oxidative stress-induced diseases.

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496-PInsulin Pensitizer Therapy Improves Atherothrombotic and Infl am-matory BiomarkersROZALINA GRUBINA, BRIAN IRVING, MATTIAS SOOP, MANIVANNAN SRINI-VASAN, LAURA TATPATI, SUMIT BHAGRA, LISA CHOW, AUDREY WEYMILLER, RICKEY CARTER, K. SREEKUMARAN NAIR, Rochester, MN

Insulin resistance, a defi ning feature of diabetes, metabolic syndrome, critical illness, and other chronic diseases, is associated with disproportion-ately high rates of atherothrombotic complications such as cardiovascular disease (CVD). Several pro-thrombotic and pro-infl ammatory biomarkers have been identifi ed as risk factors for CVD and mortality. In a randomized double-blinded placebo controlled study, we demonstrated that targeted pharmacologic improvement of insulin sensitivity (SI) signifi cantly lowers these risk factors. Individuals with impaired fasting glucose or diabetes who never received anti-hyperglycemic therapy (n = 28), were randomized to receive placebo or combination of metformin and pioglitazone (M/P) for 3 months; 25 participants completed the study. SI, measured by the hyper-insulinemic-euglycemic clamp, improved signifi cantly with metformin/pio-glitazone (n = 12), while fasting glucose, insulin, and HbA1c decreased with treatment (all p< 0.001). Although BMI increased slightly with treatment (32.43 ± 5.53 kg/m2 to 32.89 ± 5.49 kg/m2), there were no signifi cant changes in fat free mass or percent body fat. There was also no signifi cant change in resting heart rate and blood pressure. After adjustment for interim changes in fasting glucose, HbA1c, and BMI, M/P resulted in signifi cant improvements in key CVD risk factors compared to placebo (n = 13): 10% increase in HDL cholesterol (p = 0.03), 5.5% decrease in non-HDL cholesterol (p = 0.03), 12% decrease in triglycerides (p = 0.02), 42% decrease in plasminogen-activator inhibitor-1 (p = 0.001), 45% decrease in CRP (p = 0.03), 9% decrease in TNF-(p = 0.008), and 150% increase in adiponectin (p = 0.001). Fibrinogen did not change with treatment. Interventions to improve insulin sensitivity may thus be considered as a therapeutic options to reduce the burden of CVD in insulin resistant states. ClinicalTrials.gov number NCT00443755.

Supported by: NIH RO1-DK41973-A23, KL2-RR024151, CTSA UL1-RR24150

497-PEndothelial Cells Protection by G-CSF Against FFAs-Induced Oxida-tive Stress and Apoptotic Cell Death was Mediated by NF- B Path-way and Cell Cycle Regulatory Protein Hcdc14aHOUGUANG ZHOU, LING LIU, YU ZHANG, YANYAN HUANG, ZHUANGLI GUO, RENMING HU, QIANG DONG, Shanghai, China, Nanjing, China, Qingdao, China

Plasma free fatty acid (FFA) concentrations increase in state of metabolic syndrome and impair endothelial function. The damage of vascular endothe-lial cells is the key event in the pathogenesis of atherosclerosis. The aim of this study was to elucidate whether FFA infl uence human cell division cycle protein 14a(Hcdc14a), oxidative stress, proliferation and apoptosis in human brain vascular endothelial cells(HBVEC), and to clarify whether granulocyte colony stimulating factor(G-CSF) possess ECs protection, and identify its possible signaling pathway. After culturing HBVEC with FFA (50-200 μmol/l, 24-72 h), we examined FFA’s actions on Hcdc14a, cell cycle distribution, as-sociated cell cycle protein expressions, oxidative stress and apoptosis in HBVEC. We investigated whether NF- B mediated FFA-induced injury and apoptosis and G-CSF infl uenced the above effects. After exposure to FFA, Hcdc14a gene and protein expressions in HBVEC were downregulated, P53 protein expression was upregulated, and cyclinD3 downregulated. There were low proliferation, high apoptosis, obviously increased ROS and de-creased NO, eNOS and MMP in HBVEC. FFA also reduced signifi cantly gene/protein expressions of NF- B’s inhibitor, I B . G-CSF(100nM) obviously pre-vented above alterations. In conclusion, high concentration FFA damaged HBVEC through downregulating Hcdc14a, inducing cell cycle arrest, initiat-ing oxidative stress and apoptosis, and activating NF- B pathway, which representing key factors in the development of micro- and macrovascular dysfunction. G-CSF could signifi cantly prevent these effects of FFA on HB-VEC and possess ECs protection.

Supported by: The National Natural Science Foundation of China (81170322)

498-PDifferential Role of miRNA-9 in Fibronectin Production in Organs Affected by Chronic Diabetic ComplicationsBIAO FENG, SHALI CHEN, SUBRATA CHAKRABARTI, London, ON, Canada

MicroRNA alterations play important roles in several biological processes. Mir-9 shows widespread biological effects including tumorigenesis and cel-lular differentiation including dedifferentiation of fi broblasts. Furthermore, it targets fi bronectin (FN), an important extracellular matrix (ECM) protein, which is known to be upregulated in chronic diabetic complications and

plays important roles in cell adhesion, migration, growth and differentia-tion. In this study, we investigated the role of microRNA-9, a FN targeting microRNA, in the endothelial cells and in the organs of diabetic animals.PCR array was used to examine microRNA (miR) expression in endothelial cells (HUVEC) exposed to glucose. miR-9 expression was validated by TaqManTM PCR. Retinal, renal and cardiac tissues from the streptozotocin (STZ)-induced diabetic rats after one month were examined for FN mRNA and protein lev-els. Luciferase assay was used to determine the interaction between FN and miR-9.PCR array showed glucose-induced alterations of 125 miRNAs (out of 381) in HUVECS exposed to 25mM glucose (HG) compared to 5mM glu-cose. Fifty-one miRNAs were upregulated and 74 were downregulated. HG decreased miR-9 expression and increased FN mRNA and protein levels. Lu-ciferase assay showed the binding of FN 3’-UTR with miR-9 but not with the mutated FN 3’-UTR. Diabetic animals showed hyperglycemia, reduced body weight and albuminuria. Interestingly, miR-9 expression was decreased in the hearts of the diabetic animals, but was increased in the retinas and kid-neys. However, FN mRNA expression was increased in the retinas, hearts and kidneys.These studies indicate a novel glucose-induced molecular mechanism of increased ECM protein production in which miR-9 regulates FN expressions in diabetes. However, such effects may vary among vari-ous organs. Identifying such mechanisms may lead to potential RNA based treatment for diabetic complications.

Supported by: Canadian Diabetes Association and Heart and Stroke Foundation of Ontario

499-PToll-Like Receptor 2 Mediates Impairment of Vascular Actions of Insulin in Response to High Fat DietHYUN-JU JANG, SIMONE D. RIDGEWAY, PRATIBHA VASHISTA, DANIEL HWANG, MICHAEL J. QUON, JEONG-A KIM, Birmingham, AL, Davis, CA, Baltimore, MD

Obesity is a chronic pro-infl ammatory state that promotes insulin resis-tance in liver, adipose tissue, and skeletal muscle as well as impairing insulin action in vascular endothelium that contributes to endothelial dysfunction. Vascular insulin resistance promotes atherosclerosis, in part, by facilitat-ing macrophage infi ltration and infl ammatory responses. Toll-like receptors (TLRs) that initiate innate immune signaling are implicated in high fat diet (HFD)-induced impairment of energy metabolism as well as cardiovascular complications. TLR4 helps to mediate vascular insulin resistance through fatty acid stimulated pro-infl ammatory responses including increased pro-duction of cytokines and activation of JNK and NF- B. However, the role of TLR2 in impairment of vascular actions of insulin in response to HFD is un-known. Therefore, we examined the specifi c role of TLR2 in HFD or saturated fatty acid (SFA)-mediated impairment of vascular actions of insulin. siRNA knock-down of TLR2 in primary endothelial cells reduced both SFA-stimulat-ed production of pro-infl ammatory cytokines and splicing of X box protein (XBP)-1. Moreover siRNA knock-down of XBP-1 or inositol requiring enzyme 1 (IRE-1 )reduced production of TNF- from endothelial cells. Thus, SFA stimulates pro-infl ammatory and unfolding protein responses to SFA require TLR2. Moreover, impairment of insulin-stimulated phosphorylation of eNOS and production of NO in response to SFA was opposed by siRNA knock-down of TLR2. Mesenteric arteries isolated from TLR2 knock-out mice were pro-tected HFD-induced impairment of insulin-stimulated vasorelaxation when compared with arteries isolated from wild type mice on HFD. We conclude that TLR2 in vascular endothelium helps mediate HFD-induced pro-infl am-matory and unfolding protein response that may contribute importantly to impairment of vascular actions of insulin, endothelial dysfunction, and atherogenesis.

Supported by: NIH

500-PDiabetes Induces Lung Endoplasmic Reticulum Stress and Cell Death in an Angiotensin II Type 1 Receptor-Dependent MannerJUNLING YANG, FENGLIAN ZHAO, PENG CHEN, LU CAI, Changchun, China, Lou-isville, KY

Diabetes damages multiple organs to cause life-threatening complica-tions. Our previous studies showed that diabetes can induce pulmonary fi brosis, in which angiotensin II (Ang II) played a critical role, but how Ang II initiates lung fi brosis remains unclear. The present study was to investigate whether diabetes induces endoplasmic reticulum stress (ERS) and associ-ated cell death, which has been recognized as an initiator of tissue fi brosis, and whether Ang II involves in this pathogenic effect. FVB mice were induced to type 1 diabetes with multiple low-doses of streptozotocin (STZ, 50 mg/kg daily for 5 days) and hyperglycemic mice at 7 days after the last injection of STZ was defi ned as diabetic. Three months after diabetes onset, lungs

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were collected from these diabetic mice and their age-matched controls. ERS-associated proteins were examined at both protein and mRNA levels with Western blotting and real-time PCR assays. Apoptotic cell death was examined with TUNEL assay and Western blotting assays. Results showed signifi cant increases in protein and mRNA expressions of ERS-related mol-ecules, including PERK, ATF6, and IRE1, in diabetic lungs compared to con-trol lungs. There was also a signifi cant increase in ERS-related cell death, measured by CHOP, JUNK, and caspase-12, in diabetic lungs. Ang II type 1 (AT1) receptor blocker (Losartan) can signifi cantly prevent diabetes-induced lung ERS and associate cell death, as we observed for its inhibition of dia-betic lung fi brosis in previous study. These results suggested that diabetes induced ERS and associated cell death via Ang II/AT1-dependent pathway, which may be the major cause of diabetes-induced lung fi brosis.

Supported by: Natural Science Foundation of Jilin Province in China

501-PHigh Glucose Effects on eNOS and SIRT1 Expression and Chromatin Acetylation in Endothelial CellsALESSANDRO DE MARCO, GLORIA FORMOSO, NATALIA DI PIETRO, ASSUNTA PANDOLFI, VINCENZO DE LAURENZI, MASSIMO FEDERICI, AGOSTINO CONSOLI, Chieti Scalo, Italy, Rome, Italy

The ‘‘ bad metabolic legacy’’ exposing to increased vascular risk diabetic individuals who experienced poor glycemic control in the early stages of the disease might be mediated by hyperglycemia related epigenetic modifi ca-tions in the vascular cells.The NAD+ -dependent protein deacetylase SIRT1 regulates gene expression programs in response to cellular metabolic status by modulating chromatin function via several epigenetic mechanisms: a) di-rect histone deacetylation, b) nuclear enzymes recruitment into chromatin or c) transcription factors and coregulators deacetylation. Since increased eNOS expression and activity have been described among the hypergly-cemia related endothelial cells modifi cations, we set to investigate eNOS, SIRT1 and chromatin modifi cations (Histones H3 and H4 Lysine acetylation) in HUVEC and HAEC cultured in Normal Glucose (NG, 5 mmol/L) and High Glucose (HG, 25 mmol/L) conditions for 48 hours.HG increased both SIRT1 and eNOS protein levels. Thus, to investigate whether SIRT1 might affecte NOS expression, HUVEC and HAEC were transfected so to over-express or to down-regulate SIRT1 expression. SIRT1 overexpression resulted in in-creased eNOS protein levels under both NG or HG conditions, while silencing SIRT1 prevented the increase in eNOS levels observed in HG exposed cells. Furthermore, by Chromatin Immunoprecipitation analysis we observed that HG exposure in HAEC resulted in changes in the acetylation levels of Histone H3 at the eNOS promoter. As compared to NG exposed HAEC, acetylation was less pronounced in the enhancer region (-4178 to -4596 bp) and greater in upstream promoter region (-891 to -794 bp).Taken together, these data indicate that, in endothelial cells, SIRT1 is involved in modulating eNOS ex-pression in response to metabolic stress such as hyperglycemia, potentially by epigenetic mechanisms.

Supported by: EFSD, sanofi -aventis

502-PAntithrombotic Function of Apelin is Preserved During ObesityBRUNO M. FEVE, FRÉDÉRIC ADAM, JOSÉ LOPEZ, CAMILLE VATIER, SABRINA TURPIN, ADELINE MUSCAT, CLAUDE LALOU, ISABELLE CASTAN, PHILIPPE VA-LET, ABDEL-MAJID KHATIB, RÉGIS BOBE, GÉRALDINE SIEGFRIED, Paris, France, Le Kremlin-Bicêtre, France, Toulouse, France

Antithrombotic function of apelin is preserved during obesityObesity and type 2 diabetes are associated with an increased thrombotic risk that is partly related to a disturbed metabolic and adipokine pattern. Apelin is an adipokine with pleiotropic functions on cardiovascular system, and on fl uid and energy homeostasis. Apelin acts through its cognate seven-trans-membrane domain receptor APJ, and is overexpressed in adipose tissue and plasma from obese individuals. We identifi ed a new biological and potent antithrombotic function of apelin in vitro and in vivo. Apelin strongly inhib-its thrombin- or collagen-induced platelet aggregation, while it does not interfere with ADP-induced aggregation or ADP secretion. Likewise, apelin prevents activation of ERK1/2, P38 MAPK, and Akt signaling pathways by thrombin or collagen. Accordingly, intravenous injection of apelin increas-es tail bleeding time and delays chemically-induced vessel occlusion and thrombus stabilization. The use of a mutant form of apelin that is resistant to proteolytic cleavage demonstrates that the peptide maturation is required for its antithrombotic properties. Finally, APJ is overexpressed in platelets from genetic or nutritional murine models of obesity or from obsese humans, and the anti-aggregant effect of apelin is preserved under this condition. Collectively, these fi ndings show a new biological function of apelin that

could be of potential interest to reduce the increased risk of thrombosis in patients with obesity, type 2 diabetes, or metabolic syndrome.

Supported by: INSERM, Universities Paris 6, Paris 7, Paris 11, and UPS of Tou-louse

503-PEvaluation of Dysfunctional High-Density Lipoprotein in Patients With Type 2 DiabetesHIROSHI MURAKAMI, KOKI MATSUMURA, MAKI YAMASHITA, KOTA MATSUKI, JUTARO TANABE, HIROSHI MURAKAMI, JUN MATSUI, NAOKI TAMASAWA, TOSHIHIRO SUDA, Hirosaki, Japan

Study Aim: We simultaneously evaluated several functions of HDL in pa-tients with type 2 diabetes, including the cholesterol effl ux, anti-oxidative and anti-infl ammatory activities.Methods: (1) Subjects (n=30; 16 male and 14 female) were patients with type 2 diabetes admitted to our hospital for diabetic control and education. Clinical characteristics were as follows (data are average values): age, 55 y; HbA1c (JDS), 9.3%; total cholesterol (TC), 180 mg/dl; triglyceride (TG), 132 mg/dl; and HDL-C, 40.7 mg/dl. (2) The HDL fraction of human plasma was separated by PAGE (Lipophor®; Jokoh, Tokyo, Japan) and the sharp HDL band was applied to protein recovery equipment (Phoreto-Yield®; Jokor) with a cut-off MW of 15,000. The HDL fraction was then recovered with elution buffer. (3) Cholesterol effl ux capacity (Effl ux) was determined as reported previously. Values were corrected against those with non-specifi c BSA. (4) In HDL fraction, paraoxonase 1 (PON1) ac-tivity (maker of anti-oxidation capacity), and levels of serum amyloid A (SAA) (marker of infl ammatory capacity) and carboxymethy lysine (CML) (marker of ApoA-I glucoxidation) were determined.Results: (1) HbA1c levels were nega-tively correlated with PON1 activity in the HDL fraction (r=-0.32, P<0.10). However, neither SAA nor CML showed any correlations with HbA1c. (2) Effl ux (%) ranged from 19.1 to 28.9%, showing a positive correlation with PON1 activity (r=0.38, p<0.05), and a weak negative correlation with SAA levels (r=-0.32, p<0.10). No relationship was seen between Effl ux and CML levels.Conclusion: We established a method that is able to rapidly separate the HDL fraction from plasma in patients with type 2 diabetes. We then confi rmed that HDL-mediated Effl ux is reduced by decreases in PON1 activity and increases in SAA levels in the HDL fraction. These fi ndings indicate that diabetic status drives HDL remodeling, leading to dysfunctional HDL, and this may explain the pathogenesis of atherosclerosis in type 2 diabetes.

504-PVascular Smooth Muscle Cell Proliferation by Intermittent Hypoxia is Independent on the Glucose-Induced Insulin SecretionYOJI KYOTANI, HIROYO OTA, ASAKO ITAYA-HIRONAKA, AKIYO YAMAUCHI, SUMIYO SAKURAMOTO-TSUCHIDA, JING ZHAO, HIROSHI KIMURA, MASAYUKI UNO, SHIN TAKASAWA, MASANORI YOSHIZUMI, Kashihara, Japan

Sleep apnea syndrome (SAS), characterized by intermittent hypoxia (IH) during sleep, is a risk factor of cardiovascular diseases as well as type 2 dia-betes. The proliferation of vascular smooth muscle cells (VSMC) is a pivotal factor in cardiovascular diseases. Recently, we found that IH attenuates glu-cose-induced insulin secretion. Therefore, there are two possibilities to in-duce VSMC proliferation by IH: One is IH directly induces VSMC proliferation and the other is IH induces VSMC proliferation via induction of diabetes. In this study, we investigated direct effects of IH on cultured rat aortic smooth muscle cell (RASMC) proliferation. WST-8 assay revealed that IH induced the RASMC proliferation (P<0.0001), but neither normoxia nor sustained hy-poxia did. On the other hand, the decrease of apoptosis was not observed in IH-treated RASMC by TUNEL assay. In order to see what induces the RASMC proliferation, we measured of several mRNAs for growth factor receptors which are concerned with VSMC proliferation using real-time RT-PCR and found that the mRNA level of erbB2 receptor, a member of epidermal growth factor (EGF) receptor, was increased by IH, but not by sustained hypoxia. We next investigated the changes of mRNAs for ligands of EGF family recep-tor and found that epiregulin, amphiregulin and neuregulin-1 mRNAs were increased by IH. We also confi rmed that IH specifi cally increased the level of epiregulin in the conditioned medium and phosphorylation of erbB2 receptor of RASMC at the specifi c residue that mediates intracellular signal for cell proliferation. These results indicate that VSMC proliferation by IH is inde-pendent on the attenuation of glucose-induced insulin secretion by IH, and suggest that the EGF family-erbB2 receptor signal system is involved in the IH-induced VSMC proliferation.

Supported by: Grants-in-Aid from the MEXT, Japan

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505-PDecreased Expression and Activity of Kruppel Like Factor-2 in Monocytes Under HyperglycemiaXINPU CHEN, SRIDEVI DEVARAJ, Houston, TX

Diabetes is now a worldwide epidemic and contributes to increased vas-cular complications. Kruppel-like factors (KLFs) are a subclass of the zinc-fi nger family of transcription factors. KLF2 inhibits proinfl ammatory activa-tion of monocytes. Furthermore, KLF2 expression in circulating monocytes is reduced in patients with chronic infl ammatory conditions such as coronary artery disease. Diabetes is a proinfl ammatory state and we and others have shown increased monocyte activation in diabetic conditions in vitro and in vivo. However, there is a paucity of data on KLF-2 exression and activity in the diabetic milieu and its regulation of monocyte infl ammation. Thus, the aim of this study was to examine the expression and activity of KLF-2 in hyperglycemia and examine its modulation of monocytic infl ammation. THP-1 cells were incubated with mannitol, low glucose (5.5mM glucose) or high glucose (15mM glucose) for 48 hours. KLF-2 expression was assessed by western blotting, mRNA and DNA binding was assessed by EMSA. KLF-2 protein expression, mRNA as well as DNA binding activity was signifi -cantly inhibited in hyperglycemia compared to either low glucose or man-nitol, which was used as hyperosmolar control. This was associated with signifi cantly increased activity of NF-kappaB and p47 phox activation and resulted in increased release of cytokines IL-1 and IL-6 from cells. We have shown previously that Resveratrol, a fl avonoid, found in grapes and red wine, signifi canly reduces infl ammation and monocyte activation under high glucose conditions. Addition of resveratrol (100uM) to cells treated with high glucose signifi cantly reversed HG-suppressed KLF-2 expression and activ-ity and resulted in decreased monocyte infl ammation. Thus, KLF-2 apears to be a negative regulator of monocytic activity in the diabetic milieu and strategies to upregulated KLF-2 may be effective in decreasing infl ammation associated with diabetes and cardiovascular disease.

506-PDiabetes Causes Persistent Insulin Resistance and Impairment of Angiogenic Function of Mesenchymal Stem Cells (MSC) by Protein Kinase C (PKC) ActivationKOJI MIZUTANI, IN-KYUNG JEONG, AKIRA MIMA, QIAN LI, KYOUNGMIN PARK, CHRISTIAN RASK-MADSEN, DAVID J. MOONEY, GEORGE L. KING, Boston, MA, Cambridge, MA

Infusion of bone marrow derived MSC can improve perfusion to isch-emic hindlimbs possibly by increases in angiogenesis which is abnormal in diabetes. To test the hypothesis that the dysfunction of MSC in diabetes is due to loss of insulin action, we implanted MSC isolated from control or STZ induced diabetic mice with or without insulin incorporated into poly-mer scaffolds for local delivery into the ischemic hindlimb of nondiabetic mice. One week after implantation, VEGF mRNA expression in ischemic thigh muscle was signifi cantly increased by 2-fold in control MSC, insulin scaffold, and combination groups vs diabetic MSC with and without insulin. After 4 weeks, insulin alone, control MSC, insulin with control and diabetic MSC groups showed signifi cantly higher perfusion than scaffold alone. How-ever, MSC alone or MSC and insulin had signifi cantly better perfusion than diabetic MSC or diabetic MSC with insulin scaffold groups, respectively. In MSC cultured for 2-3 months from control and diabetic mice, insulin induced tyrosine phosphorylation of its receptors (IR- ) and IRS1 was decreased in diabetic vs control MSC by 71% and 64%, respectively, which were trans-lated into comparable decreases of Akt activation (-51%, p<0.05) although activation of p-Erk1/2 was not affected. mRNA and protein levels of VEGF were reduced in diabetic MSC vs control, (p<0.05). Interestingly, PKC activity and PKC 2 protein expression in the membrane were persistently increased in diabetic vs control MSC. Addition of PKC inhibitor (GFX) or PKC -specifi c inhibitor (RBX) partially reversed the inhibitory effect of diabetes on insulin’s activation of p-Akt by 44% and 25%, and enhanced mRNA expression VEGF by 2.1 and 1.5-fold. Thus, diabetes can cause persistent insulin resistance in MSC via PKC 2 activation, which could be a therapeutic target to improve wound healing in diabetic patients.

507-PPro-Infl ammatory Actions of TNF- in Vascular Endothelial Cells are Reduced by Treatment With the Green Tea Polyphenol EGCG via p38 MAPK-, STAT3-, and COX-2-Dependent MechanismsHUI CHEN, JI-WON LEE, YUNHUA LI, MARIA DE LA LUZ SIERRA, MICHAEL J. QUON, Bethesda, MD, Baltimore, MD

Pro-infl ammatory cytokines including TNF- contribute to the pathophys-iology of diseases impacted by vascular endothelial dysfunction including

diabetes. The green tea polyphenol epigallocatechin gallate (EGCG) exerts multiple benefi cial metabolic and vascular actions. Indeed, EGCG treatment of SHR rats reduces hypertension, and ameliorates endothelial dysfunction and insulin resistance. Additional biological actions of EGCG remain undis-covered. In the present study, we investigated anti-infl ammatory actions of EGCG in vascular endothelial cells. TNF- treatment of human aortic en-dothelial cells (HAEC) stimulated phosphorylation of STAT3 and p38 MAPK (upstream of STAT3) as well as STAT3 transcriptional activity that were all inhibited by EGCG co-treatment (10 μM) or inhibition of p38 MAPK (using siRNA knockdown or SB203580 (p38 MAPK inhibitor)). TNF- treatment also increased COX-2 expression in HAEC that was inhibited by co-treatment with EGCG. These anti-infl ammatory actions of EGCG were abrogated by siRNA knockdown of STAT3. Likewise, TNF- treatment of HAEC stimulated secretion of PGE2 (COX-2 product) that was inhibited by EGCG co-treatment or siRNA knockdown of STAT3. Further, TNF- treatment stimulated IL-6 se-cretion from HAEC that was inhibited by co-treatment with EGCG or siRNA knockdown of COX-2. Finally, IL-6 treatment of HAEC stimulated secretion of TNF- that was inhibitable by co-treatment with EGCG. Thus, TNF- and IL-6 synergistically promote pro-infl ammatory actions through feed-forward mechanisms that are dampened by actions of EGCG to inhibit STAT3 and COX-2 function. Our fi ndings defi ne novel molecular mechanisms for anti-infl ammatory actions of EGCG in vascular endothelium that may be relevant for developing effective therapies targeting diabetes and its cardiovascular complications.

Supported by: Intramural Research Program, NCCAM, NIH

508-PIntermittent High Glucose Reduces Androgen Receptor Expression of HUVECs via PI3K/Akt/mTOR Signaling PathwayLIN LI, XUEYAO YIN, FANG WU, JIAQIANG ZHOU, HONG LI, Hangzhou, China

Glucose fl uctuation plays a more signifi cant role in the pathogenesis of vascular diabetic complications than constant high glucose. Androgen recep-tor (AR) is important in coronary atherosclerosis, and decreasing AR expres-sion correlate with more extensive atherosclerosis. But the regulation of AR gene expression in the fl uctuated glucose remains poorly understood, and the signal transduction pathways correlate with AR has not been defi ned well in the cardiovascular system. This study focuses on the effect of in-termittent high glucose concentrations on AR expression and the PI3K/Akt/mTOR signaling pathway in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated in media containing different glucose concentra-tions: 5.5mmol/l, 30mmol/l, or alternating 5.5 and 30mmol/l glucose twice a day. AR expression in HUVECs was downregulated in constant high glucose condition than that in normal concentration condition. Furthermore, AR was reduced more in HUVECs exposed to intermittent rather than constant high glucose concentration. Constant and intermittent high glucose concentra-tion could induced activation of the PI3K/Akt/mTOR pathway. When we blocked or knocked down PI3K/Akt/mTOR signaling pathway, by LY 294002, rapamycin, Akt siRNA and mTOR siRNA, led to increasing levels of AR mRNA and protein associated with reduced phosphorylation of Akt or mTOR. These results suggest that intermittent high glucose has more prominent effect in reducing AR expression in HUVECs than constant high glucose. The patho-genesis of this effect at least partly related to the enhanced activation of PI3K/Akt/mTOR signaling pathway.

Supported by: S&T Major Project of Zhejiang (2009C03010-4) National Key R&D (2009BAI80B01)

509-PEarly Intensive Insulin Treatment Stimulated Production of Nitric Oxide via Activation of the AMPK/Akt/eNOS Pathway in Aorta of Diabetic RatsDONGHONG FANG, ZHIMIN HUANG, HONGYU GUAN, HAI LI, HAIPENG XIAO, YANBING LI, Guangzhou, China

In this study, we compared the effects of early versus late intensive insu-lin therapy on aortic endothelial function and explored the potential causal mechanisms.High-fat diet and low-dose-STZ induced diabetic rats were ran-domly divided to diabetes group (DM group, no insulin treatment, n=8), early intensive insulin therapy group (EI group, n=9) and late insulin therapy group (LI group, n=7). Non-diabetic SD rats served as controls (NC group, n=8). As soon as diabetes was confi rmed, NPH (6-10 U per day) was used in the EI group to maintain near-normal glycemic control for the entire 16 weeks. In the LI group, near-normal glycemic control was induced after 8 weeks of poor glycemic control. Nitric oxide (NO) productions in blood serum at base-line were similar among the four groups. After completing the treatment, the DM group was markedly declined to 156.62±31.49μmol/L (p<0.05). The

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EI group was higher than the DM group (227.99±10.39, p<0.05), while the LI group was not different from the DM group (147.95±25.27, p=0.54). Western blot revealed that as compared with the NC groups, p-eNOS (active)/ total eNOS was decreased in the DM groups. In the EI group, p-eNOS/t-eNOS was maintained at a near normal level, whereas in the LI group was similar to the DM group. The activities of AMPK, ACC and Akt proteins showed similar trends (Fig. 1).Our results suggested that the decreased production of NO in diabetic rats was counteracted by early, but not late insulin therapy. The aortic endothelial protective function of early intensive insulin treatment was mediated by AMPK/Akt/eNOS Pathway.

510-PEffect of In Vivo Hyperglycemia on Granulocyte and Monocyte Acti-vation Markers in Type II DiabetesPETER HORVATH, STACY R. OLIVER, SHLOMIT RADOM-AIZIK, FRANK P. ZALDI-VAR, PIETRO R. GALASSETTI, Irvine, CA

Atherosclerosis and cardiovascular complications of Type II Diabetes (T2DM) are mediated by chronic infl ammatory processes, including exagger-ated activation of immune cells such as granulocytes (Gc) and Monocytes (Mc). Activation of these cells can be measured by levels of expression of cell surface markers (CD11b, CD66b etc), adhesion molecules which facili-tate anchoring to the endothelium and onset/progression of vascular dam-age. While pro-infl ammatory effects of hyperglycemia on immune cells are established in vitro, Gc and Mc activation during in vivo hyperglycemia, when interacting systemic factors may drastically affect infl ammatory re-sponses, remains unexplored. We therefore performed a 4-h hyperglycemic clamp (240 mg /dl, simulating common post-prandial hyperglycemia) in 7 T2DM subjects, sampling at 0, 30, 90, 120, 180, 240 min. Mean fl uorescence intensity (MFI) of CD11b, CD14, CD16, CD62L, and CD66b was measured by fl ow cytometry. During the fi rst 2 h of hyperglycemia CD11b and CD66b ex-pression in Gc increased signifi cantly. In Mc, the % of CD14+CD16+ cells (abundant in atherosclerotic tissue), and CD14 expression in this cell sub-type, also increased. Our data indicates that in T2DM in vivo hyperglycemia, of magnitude/duration similar to that typically observed postprandially even in relatively well controlled patients, markedly increases Gc and Mc adhe-sion potential and activation state. This effect may be a signifi cant factor in the postprandial component of long-term cardiovascular risk, and further underscores the necessity of avoiding hyperglycemic peaks of even moder-ate elevation.

Supported by: NIH (P01HD048721 and UL1RR031985)

511-PTribble 3 is Induced in Macrophages During Foam Cell Formation and Facilitates Lipid Accumulation in a TLR4 MannerDENNIS STEVERSON, LING TIAN, TIMOTHY GARVEY, Birmingham, AL

The tribbles homolog-3 gene (TRIB3) has beenreported to be upregulated in human atherosclerotic plaque, however, the roleof TRIB3 in foam cell formation has not been studied. Therefore, we transformed THP-1 cells, ahuman monocytic cell line, into macrophage foam cells via treatment with oxidizedlow-density lipoprotein (oxLDL), and found that TRIB3 was also in-duced. In THP-1 cell with stable overexpression ofTRIB3, cholesterol accu-mulation upon oxLDL treatment was increased by 1.2 foldincrease (p<.01) indicating a potential role for TRIB3 in facilitating foamcell formation. oxLDL and other lipidsinteract with Toll-like receptor 4 (TLR4) on the macrophage cell surface, andTLR4 has been implicated in the cellularuptake of lipid and formation of atherogenic plaques. To examine whether TRIB3 induc-tion wasdependent of TLR4, peritoneal macrophages from wild type (WT) and TLR4-/- micewere treated with 100ug/ml of lipopolysaccharide (LPS), a strong activator ofTLR4. After 24 h, WT macrophagesexhibited a 10.1 fold increase in TRIB3 expression over that in untreatedcontrols (p<.01). Further, co-incubatedof WT cells with both 100ug/ml oxLDL and 100ug/ml of LPS led to an augmentationof TRIB3 expression up to 17.4 fold (p<.01). However in macrophages from TLR4 -/- mice, theTRIB3 response was markedly reduced under these conditions; specifi cally, theinduction of TRIB3 was signifi cantly decreased by 5.5 fold following treatment withLPS, and decreased by 5.7 fold following treatment with LPS + oxLDL, whencompared with responses in the WT cells.In conclusion: we have shown for the fi rst time that: 1) TRIB3 isinduced during foam cell formation and can mediate an acceleration in lipi-daccumulation, 2) The induction of TRIB3 in response to oxLDL and LPS is inlarge part dependent upon TLR4. Thus,TRIB3 is mechanistically implicated in foam cell formation and potentiallyrepresents a novel target for preven-tion of atherosclerosis.

512-PMD-2 Plays an Important Role in High Glucose-Enhanced, IFNReceptor/TLR4-Mediated MMP-1 Expression in U937 Mononuclear CellsZHONGYANG LU, YANCHUN LI, DEVADOSS J. SAMUVEL, JUNFEI JIN, XIAOMING ZHANG, MARIA F. LOPES-VIRELLA, YAN HUANG, Charleston, SC

Although it is known that interferon gamma (IFN ) receptor and toll-like receptor (TLR)4 play important roles in diabetic complications, the molecular mechanisms involved in the interaction between IFN receptor and TLR4 under hyperglycemic condition have not been well understood. In this study, we showed that while either IFN or lipopolysaccharide (LPS) stimulated matrix metalloproteinase (MMP)-1, a key proteinase involved in athero-sclerosis, by 2.7- and 16.5-fold (0.54 and 3.3 vs 0.2 ng/ml), respectively, in U937 mononuclear cells, priming of U937 cells with IFN augmented LPS-stimulated MMP-1 expression by 76.5-fold (15.3 vs 0.2 ng/ml). We further demonstrated that high glucose markedly enhanced the priming effect of IFN on LPS-stimulated MMP-1 expression by 984-fold (196.8 vs 0.2 ng/ml).

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To explore the underlying mechanisms, we focused on TLR4, CD14 and MD-2 expression since they are all essential for TLR4 signaling. We found that while IFN stimulated MD-2 expression by 3.2-fold and high glucose alone had no effect, treatment of U937 cells with IFN and high glucose simultane-ously increased MD-2 expression by 5.6-fold. In contrast, high glucose and IFN had no effect on TLR4 and CD14 expression, suggesting that MD-2 may be specifi cally involved in high glucose-enhanced, IFN receptor/TLR4-me-diated MMP-1 expression. To confi rm the critical role of MD-2 in the relation of MMP-1 expression, we showed that neutralizing MD-2 antibody or MD-2 siRNA signifi cantly reduced IFN receptor/TLR4-mediated MMP-1 expres-sion under high glucose condition. Surprisingly, results showed that neutral-izing MD-2 antibody or siRNA also signifi cantly reduced MMP-1 expression induced by IFN alone, suggesting that MD-2 is involved in not only TLR4 signaling, but also IFN receptor signaling. Finally, we demonstrated that IFN and high glucose upregulated MD-2 expression via mitogen-activated protein kinase (MAPK) pathways and AP-1 transcription factor.

Supported by: Merit Review Grant from Department of Veterans Affairs and NIH (RO1 DE016353)

513-PAngiopoietin-Like 4 is Elevated in Type 2 Diabetes But is Not Asso-ciated With Angiographically Determined Coronary Artery DiseaseHEINZ DREXEL, KATHRIN GEIGER, ALEXANDER VONBANK, PHILIPP REIN, AXEL MUENDLEIN, CHRISTOPH H. SAELY, Feldkirch, Austria, Philadelphia, PA, Triesen, Liechtenstein

Angiopoietin-like 4 (ANGPTL4, fasting-induced adipose factor), a protein inhibitor of lipoprotein lipase, is synthesized and secreted during fasting in adipose tissue and the liver. Its associations with metabolic syndrome traits are uncertain, and it is not known whether it is associated with type 2 diabetes (T2DM) or coronary artery disease (CAD).We therefore measured serum ANGPTL4 in 493 patients undergoing coronary angiography for the evaluation of established or suspected stable CAD; signifi cant CAD was diagnosed when coronary stenoses 50% were present.ANGPTL4 was signifi cantly positively correlated with age (r=0.177; p <0.001) and fasting glucose (r=0.112; p=0.013) but was not correlated with waist circumference, triglycerides, HDL cholesterol, systolic blood pressure or diastolic blood pressure. ANGPTL4 was signifi cantly higher in patients with T2DM (n=115) than in non-diabetic subjects (28±32 vs. 25±38; p=0.032); however, it was not signifi cantly different between patients with signifi cant CAD (n=246) and individuals without signifi cant CAD (p=0.112).We conclude that ANGPTL4 is positively correlated with fasting glucose and elevated in T2DM but is not signifi cantly associated with angiographically determined CAD.



Guided Audio Tour: Experimental Models of Diabetic Kidney Disease (Posters 514-P to 521-P), see page 15.

& 514-PThe Role of Thioredoxin Interacting Protein (TXNIP) in the Develop-ment of Diabetic NephropathyANU SHAH, LING XIA, HOWARD GOLDBERG, KEN W. LEE, CATHARINE WHITE-SIDE, SUSAN E. QUAGGIN, I. GEORGE FANTUS, Toronto, ON, Canada

Thioredoxin-interacting protein (TXNIP) is an endogenous inhibitor of thioredoxin (Trx), a ubiquitous thiol oxidoreductase that regulates cellu-lar redox status. TXNIP is upregulated by high glucose (HG) and promotes oxidative stress. We have found that Hcb-19 mice (lack TXNIP) are partially protected from streptozotocin-induced diabetes and diabetic nephropathy (DN). Since HG and reactive oxygen species (ROS) are key mediators of the microvascular complications of diabetes, we investigated the potential role of TXNIP in the pathogenesis of DN by utilizing primary mouse mesangial cells (MC) culture from the 2 strains and then exposing MC to HG. C3H MC exposed to HG (25 mM) for 3h showed a signifi cant increase in total cell ROS determined by DCF (2.29 fold, p<0.001) as well as MitoSox (mitochondrial ROS, 3.54 fold, p<0.001) fl uorescence, while Hcb-19 MC had no response. Trx activity was decreased by HG only in C3H MC (i.e. 69% decrease). Of interest lucigenin based NADPH oxidase assay revealed activation by HG only in C3H (2.21±0.28 fold, p<0.05) but not in Hcb19 MC. Recently, the mitochondria-localized NADPH oxidase isoform, Nox4 has been implicated in HG-mediated ROS generation. Nox4 protein level was increased by HG in C3H total cell lysates (1.7±0.19 fold, p<0.05) and isolated mitochondrial frac-

tions (2.14±0.26 fold, p<0.05), but not in Hcb19 MC. To verify our fi ndings, we transfected Hcb19 MC with TXNIP adenovirus and C3H with siRNA against TXNIP, and then repeated the aforementioned experiments. Overexpression of TXNIP in Hcb19 MC in normal glucose (5.5 mM) augmented cellular ROS formation, NADPH oxidase activity, NOX4 levels, and collagen IV (fi brosis marker) expression. In contrast, TXNIP siRNA blocked all these effects of HG in C3H MC. These data indicate that TXNIP is a critical component of the HG-ROS signaling pathway, apparently required for the induction of the mitochondrial NADPH oxidase isoform Nox4. Thus, TXNIP may be a promis-ing target to prevent the complications of diabetes.

Supported by: NSERC-CGS M, Ontario Graduate Scholarship, and BBDC Gradu-ate Awards

& 515-PHigh Glucose Induces Platelet Derived Growth Factor C (PDGF-C) Expression in Glomerular Mesangial Cells via a Glucose Respon-sive Transcription Tactor, Carbohydrate Response Element Binding Protein (ChREBP)HIROYA KITSUNAI, YUICHI MAKINO, HIDEMITSU SAKAGAMI, KATSUTOSHI MI-ZUMOTO, TSUYOSHI YANAGIMACHI, YASUTAKA TAKEDA, YUKIHIRO FUJITA, ATSUKO ABIKO, YUMI TAKIYAMA, MASAKAZU HANEDA, Asahikawa, Japan

High glucose evokes a variety of gene expression in mesangial cells (MC) that alter cellular functions responsible for the development of dia-betic glomerulopathy. We have recently shown that, in MC exposed to high glucose, a glucose responsive transcription factor carbohydrate response element binding protein (ChREBP) activates HIF-1 and downstream gene expression leading to an extracellular matrix expansion in diabetic glomeruli. Besides such observation, however, a role of ChREBP in gene regulation in MC under diabetic circumstances has been scarcely documented. To provide more insight into glucose-responsive gene regulation via ChREBP in MC, we performed genome wide analysis by chromatin immunoprecipitation with anti-ChREBP antibody followed by DNA microarray assays using cultured hu-man MC and identifi ed platelet derived growth factor C (PDGF-C) as a direct target of ChREBP. Consistently, sequence analysis found the ChREBP binding site at 1.6 kbp upstream of transcriptional start site for human PDGF-C gene. In quantitative analyses, high glucose enhanced PDGF-C mRNA expression in both human and mouse cultured MC. Similarly, PDGF-C protein levels in MC increased in a glucose concentration-dependent manner. PDGF-C is known for its mitogenic and fi brogenic action in various disease condtions such as cardiac and liver fi brosis, renal interstitial fi brosis in non-diabetic chronic kidney diaseases. In support of these issues, knockdown of PDGF-C in mouse MC abrogated high glucose-mediated increase in mRNA levels of fi brogenic genes including Collagen 4a-1 and Collagen 6a-1. Taken together, high glucose-mediated induction of PDGF-C via ChREBP could contribute to the development of the mitogenic and fi brotic lesion in diabetic nephropathy and might provide a novel platform for therapeutic intervention of the com-plications in diabetic patients.

& 516-PLeptin-Dependent Effects of CP-900691, a Highly Selective and Po-tent PPAR- Agonist on Diabetic Nephropathy in the BTBR ob/ob MouseTOMASZ WIETECHA, KELLY L. HUDKINS, EDWARD J. FOX, TRI Q. NGUYEN, LAW-RENCE A. LOEB, CHARLES E. ALPERS, BARDIA ASKARI, Seattle, WA

We have recently characterized a new model of type II diabetic neph-ropathy (DN), the BTBR ob/ob leptin-defi cient mouse. Current therapies for DN (control of hyperglycemia, blood pressure and inhibition of the renin-angiotensin system) while effective, are not completely effi cacious. Studies suggest that peroxisome proliferator-activated receptor (PPAR) agonists are promising agents in the treatment of DN. Piperidine-based PPAR- agonists are novel agents that improve lipid, glycemic, and infl ammatory indicators in mammalian models of diabetes and obesity. The purpose of this study was to determine whether CP-900691 (CP), a member of this class of agents, could prevent the development of DN in BTBR ob/ob mice. 4 week-old female BTBR ob/ob mice and wild-type controls received either reglar chow or one containing CP (3mg/kg/day) for 16 weeks. CP treatment reduced body weight in BTBR (28.9 ± 2 vs 19.7 ± 1.5 g, untreated vs treated, respectively, p<0.001) and BTBR ob/ob (57.8 ± 5.2 vs 51 ± 6 g, untreated vs treated, respectively, p<0.001). Treatment also reduced fasting blood glucose in BTBR ob/ob (373 ± 48.1 vs 182 ± 10.9 mg/dL, untreated vs treated repectively, p<0.001), plasma trig-lycerides (124 ± 11.9 vs 47 ± 9.2 mg/dL, untreated vs treated respectively, p<0.01), free fatty acids (1.01 ± 0.2 vs 0.43 ± 0.06, untreated vs treated, p<0.01) and interleukin 6 (35.5 ±14.8 vs 8.4 ± 3.3 pg/ml, untreated vs treated, p<0.01). CP improved insulin resistance (P<0.001) but not plasma insulin levels in the

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BTBR ob/ob. Despite these positive effects, CP did not prevent the develop-ment of DN in the BTBR ob/ob, having no effect on glomerular hypertrophy, mesangial matrix expansion and renal macrophage accumulation. CP also had differential effects on the activation of fatty acid oxidation genes in the liver and kidney. These results suggest that normal plasma leptin levels are integral for PPAR- agonist effi cacy in the treatment of the renal complica-tions of type II diabetes.

Supported by: MMPC MICROMouse Program (U24-DK76126)

& 517-PCCR2 Inhibition Improves Renal Function in Diabetic BKS db/db MiceTIMOTHY J. SULLIVAN, ZHENHUA MIAO, BIN N. ZHAO, ROBERT D. BERAHOVICH, JAY P. POWERS, LINDA ERTL, JUAN C. JAEN, THOMAS J. SCHALL, Mountain View, CA

Diabetic Nephropathy (DN) is a major complication of uncontrolled diabe-tes. The chemokine receptor CCR2 has been implicated in the renal recruit-ment of blood monocytes in response to hypertension and hyperglycemia. We have assessed the therapeutic benefi t of CCR2 antagonism in a model of diabetic nephropathy.CCX872 was dosed daily to extremely diabetic and proteinuric 28-week old male db/db mice for 13 weeks. Assessment of 24-hr excretion of albumin (UAER) and creatinine was performed every 3 weeks. At the end of study, GFR was measured via inulin-FITC clearance. Renal histol-ogy included assessment of infl ammatory cell infi ltration, glomerular size, podocyte density, and extent of mesangial matrix.At study initiation, mice were extremely diabetic (fasting plasma glucose 500-600 mg/dL) and pro-teinuric (UAER 700-800 μg/day). Treatment with a CCR2 antagonist signifi -cantly improved multiple renal parameters in obese, diabetic mice, including albuminuria and serum markers of renal function. CCX872 treatment also improved histologic measures of renal health including leukocyte infi ltra-tion and podocyte density. There were no treatment-related effects on body weight throughout the study and the improvements in renal parameters were seen independently of any glycemic improvements. Profound improve-ments of albuminuria, renal function and renal infl ammation/fi brosis were seen following pharmacological intervention with a small-molecule CCR2 antagonist in a mouse model of severe diabetic nephropathy. These results support the clinical investigation of CCR2 antagonists, such as CCX140-B, for the treatment of diabetic nephropathy.

& 518-PRho-Kinase Inhibition Prevents the Progression of Diabetic Neph-ropathy via Downregulation of Hypoxia-Inducible Factor 1KEIICHIRO MATOBA, DAIJI KAWANAMI, RINA OKADA, MASAMI TSUKAMOTO, JUN KINOSHITA, TOMOKO ITO, SHO ISHIZAWA, YASUSHI KANAZAWA, TA-MOTSU YOKOTA, NORIYUKI MURAI, SENYA MATSUFUJI, JUNKO TAKAHASHI-FUJIGASAKI, KAZUNORI UTSUNOMIYA, Tokyo, Japan

The small GTPase Rho and its effector, Rho-kinase (Rho-associated coiled-coil containing protein kinase, ROCK), are implicated in the pathogenesis of diabetic nephropathy. Recent studies have shown that hypoxia-inducible fac-tor 1 (HIF-1 ) is a potent inducer of renal fi brosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1 in the development of renal dysfunction have not been defi ned. In the present study, we assessed whether Rho-kinase blockade attenuates HIF-1 expression and subsequent fi brotic response using type 2 diabetic mice and cultured mesangial cells (MES-13). Eight-week-old db/db mice were administered Rho-kinase inhibi-tor fasudil (100 mg/kg/day) for 24 weeks through the drinking water. Urinary albumin excretion, mesangial matrix expansion and the expression of fi brotic mediators were attenuated by Rho-kinase inhibition. From a mechanistic standpoint, we observed that HIF-1 accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also atten-

uated by fasudil in the renal cortex. Under hypoxic conditions (1% oxygen), Rho/Rho-kinase signaling was activated in mesangial cells. Rho-kinase in-hibitor decreased the levels of HIF-1 protein and its target genes compared with untreated cells, whereas HIF-1 mRNA levels were not altered. Fur-thermore, proteasome inhibitor MG132 prevented the effect of Rho-kinase blockade on HIF-1 expression. These observations were also confi rmed by hypoxia mimetic deferoxamine. Our data indicated that the Rho/Rho-kinase pathway might be involved in the prevention of proteasome-dependent HIF-1 degradation under low oxygen conditions. The present study shows an unrecognized mechanism for the renoprotective properties of Rho-kinase inhibition and suggests that Rho-kinase could be regarded as a potential therapeutic target for the treatment of diabetic nephropathy.

& 519-PHyperglycemia-Induced SHP-1 Expression Cause Insulin Resis-tance in PodocytesNICOLAS DRAPEAU, FARAH LIZOTTE, PEDRO M. GERALDES, Sherbrooke, QC, Canada

Renal podocytes apoptosis induced by hyperglycemia and insulin re-sistance is an early event leading to diabetic nephropathy. A recent study reported that insulin is critical for podocyte survival. However, how hyper-glycemia induces insulin resistance in podocytes is not fully understood. Recent studies demonstrated that SHP-1, a protein tyrosine phosphatase, is elevated in renal cortex of type 1 diabetic mice (Akita). The aim of this study is to evaluate the role of SHP-1 actions on hyperglycemia-induced insulin resistance leading to podocyte dysfunction. We exposed cultured podocytes to 5.6 mmol/L (NG) or 25 mmol/L (HG) of glucose concentration up to 3-4 days with or without insulin (10-50 nmol/L). SHP-1 mRNA and protein levels were evaluated by qPCR, and western blot. Effects of HG and SHP-1 on insulin signaling pathway (Akt and ERK) were assessed using adenovirus of SHP-1 dominant negative and wild-type. Glomerular fi ltration rate (GFR), urine albumin/creatinine ratio (uACR), foot process effacement, mRNA and protein expression of SHP-1 and insulin signaling pathway were performed on 9 months non-diabetic (NDM) and diabetic Akita (C57BL/6J-Ins2Akita)mice. Podocytes exposed to HG expressed high mRNA and protein levels of SHP-1. Moreover, HG concentrations decreased insulin-induced Akt and ERK phosphorylation by 33% and 77%, respectively. Overexpression of SHP-1 dominant negative in podocytes prevented HG effects and restored insulin actions. Renal pathology was confi rmed in Akita mice by elevated GFR (53%), uACR (4-fold) and foot process effacement as compared to NDM mice. Insulin injection increased Akt and ERK phosphorylation in renal cortex of NDM mice but were unchanged in renal Akita mice. This renal insulin resistance correlates with elevated SHP-1 mRNA (glomeruli) and protein (cortex) levels of Akita compared to NDM mice. In conclusion, our results showed that hyperglycemia and diabetes increase SHP-1 expression causing insulin resistance, podocyte loss and diabetic nephropathy.

Supported by: Kidney Foundation of Canada, JDRF

& 520-PThe Effects of Diabetes and Angiotensin on the Loss of Glucagon-Like Peptide-1’s Protective Actions in Diabetic Endothelial Glom-erulopathyAKIRA MIMA, QIAN LI, KOJI MIZUTANI, KYOUNGMIN PARK, CHRISTIAN RASK-MADSEN, GEORGE L. KING, Boston, MA

The fi nding of Glucagon-like peptide-1 (GLP-1) receptors on renal glomeruli suggests that they may have protective actions in glomerular functions. We studied the expression and signaling of GLP-1R on mice glomerular endothe-lial cells and made the novel fi nding that GLP-1 can activate protein kinase A to phosphorylate c-Raf at serine259 to inhibit angiotensin II (Ang II) induced phosphorylation of serine338 of c-Raf and ERK1/2 and increased the expres-sion of PAI-1, NF- B, TNF- , IL-6 and CD68. When rat glomerular endothelial cells were exposed to phorbol 12-myristate13-acetate (PMA; 100nM), PKCis activated and decreased GLP-1R expression by 46±4% and its actions. Dia-betic mice also exhibited decreased GLP-1R expression by 35±9% and loss of GLP-1’s protective action and increase Ang II’s actions on phosphor-c-Raf (Ser338), phosphor-Erk1/2 and infl ammatory cytokine productions (PAI-1; by 1.3 ± 0.1-fold,NF- B; by 1.4 ± 0.3-fold, TNF- ; by 1.4 ± 0.2-fold, IL-6; by 1.5 ± 0.5-fold, CD68; 6.1 ± 2.8-fold). Mice overexpressing PKC 2 specifi es to the endothelial cells by VE-cadherin (EC-PKC 2Tg) exhibited decreased GLP-1R expression by 45±19% and increased phosphor-c-Raf(Ser338) leading to en-hanced effects of Ang II. Diabetic EC-PKC 2Tg mice exhibited greater loss of endothelial GLP-1R expression by 51±3% and exendin-4 protective actions and exhibited more albuminuria (by 1.5 ± 0.5-fold) and mesangial expansion (by 1.4 ± 0.4-fold) than diabetic controls.Thus, GLP-1 has renal protective

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actions on the glomerular endothelial cells which are mediated by inhibit-ing Ang II action at phosphor-c-Raf(Ser338) via phosphor-c-Raf(Ser259). This action of GLP-1 is diminished in diabetes by PKC activation via downregula-tion of GLP-1R and the enhancement of Ang II active in diabetes.

Supported by: DERC (P30DK036836)

& 521-PHyperuricemia Induce Renal Injury by Down-Regulating CD2-Associated ProteinWEI REN, JIANJIN GUO, DONGMING SU, WEIPING JIA, Shanghai, China, Nan-jing, China

The increasing incidence of the link between hyperuricemia and the metabolic syndrome has been noted. Mild hyperuricemia has been shown to induce hypertension, cardiovascular disease, and renal fi brosis. The CD2 associated protein (CD2AP) is characterized as an adaptor protein that impli-cated in podocyte homeostasis, signal transduction, dynamic actin remodel-ing and also membrane traffi cking. CD2AP knockout mice exhibit defects in podocyte foot processes, accompanied by mesangial cell hyperplasia and extracellular matrix deposition, suggesting that CD2AP plays an essential role in cell-to-cell union. In this study, we examined the effect of hyperuri-cemia on the expression of CD2AP in renal injury. Hyperuricemia has been developed in rat models by blocking uricase enzyme with oxonic acid and low-salt diet. Serum creatinine (Cr), uric acid (UA) and urinary album in excre-tion rate (UAER) were measured. The concentration of CD2AP in renal cortex was analyzed by immunohistochemistry and Western blotting. All statistical analyses were performed using SPSS 13.0. Compared with the control group, the plasma uric acid level (0.9 ± 0.1 vs. 2.2 ± 0.5 mg/dL, P < 0.01), Cr (0.45 ± 0.1 vs. 1.5 ± 0.5 mg/dL, P < 0.01) and UAER(0.44 ± 0.1 vs. 1.12 ± 0.4 mg/24h, P < 0.05) were markedly increased in hyperuricemia groups, and UARE was positively correlated with plasma UA level (r = 0.72; P < 0.01). The expression of CD2AP in kidneys was decreased signifi cantly in hyperuricemia groups (P < 0.01). The plasma UA level plays an important role in the development of nephropathy.The mechanisms maybe related to uric acid level decreased the expression of CD2AP resulting in the dysfunction of podocyte.

Supported by: National Basic Research Program of China (2011CB504000)

Guided Audio Tour: Clinical Studies of Diabetic Kidney Disease (Posters 522-P to 529-P), see page 15.

& 522-PReduced White Blood Cell Telomere Length in Type 1 Diabetes (T1D) Patients, is Consistent With Accelerated Aging, but Lacks Relation-ship With Vascular ComplicationsANDRZEJ S. JANUSZEWSKI, SURYA SUTANTO, CONNIE KARSCHIMKUS, DAVID N. O’NEAL, SUSAN V. MCLENNAN, STEPHEN M. TWIGG, ANTHONY KEECH, ALI-CIA J. JENKINS, Melbourne, Australia, Sydney, Australia, Footscray, Australia

Telomeres regulate cell ageing and death. Shorter telomeres are associated with vascular risk factors, infl ammation and oxidative stress, and are associ-ated with and predictive of vascular disease. T1D is commonly regarded as a condition of accelerated aging.We measured telomere length in T1D and non-diabetic (CON) subjects, to determine its association with age, renal and vascular status, infl ammation and glycemia. T1D subjects; n=144, aged (mean±SD) 38±14 yrs; 83F / 61M 54 with microvascular complications (CX+). 118 CON subjects: 36±14 yrs, 64F / 54M were studied. telomere length was measured in whole blood using quantitative PCR and expressed as T/S ratio in arbitrary units (AU).Age- and gender-adjusted mean T/S in CON vs. T1DM was 1.65±0.04 vs. 1.51±0.04 AU respectively; p=0.01. T/S was similar in T1DCX[-] 1.45±0.06 AU vs. T1DCX[+]1.52±0.05 AU respectively; p=NS. T/S correlated with age: CON (r=-0.21; p=0.02); T1D (r=-0.33; p=0.00005) and T1D duration (r=-0.21; p=0.01). For each decade T/S was less in CON by 0.07 AU and by 0.11 AU in T1D; p=0.01.In CON T/S correlated with CRP (r=-0.23; p-0.02), small artery elasticity (r=0.27; p=0.004), systemic vascular resistance (r=-0.22; p=0.02) and GFR (r=0.27; p=0.004). In T1D T/S was not correlated signifi cantly with HbA1c, renal or vascular function, lipids or CRP. In multiple regression analysis of the combined group T1D (p=0.02), age (p<0.0001) and CRP (p=0.04) were signifi -cant determinants of T/S.Age-related telomere shortening was greater in T1D than in non-diabetic subjects suggesting accelerated aging. Telomere length correlated with T1D duration, but did not differ by complication status nor cor-relate with vascular health or risk factors. In non-diabetic subjects telomere length correlated with infl ammation, renal function and vascular health but not with glycemia or lipids. Age, diabetes and CRP levels were independent determinants of telomere length.

& 523-PPatterns of Urinary Albumin Excretion Rate (AER) in Normoalbu-minuric Normotensive (NANT) Patients (pts) With Type 1 Diabetes (T1D)AMEER A. KHOWAJA, MICHAEL MAUER, M. LUIZA CARAMORI, Minneapolis, MN

We previously evaluated the patterns of AER in normoalbuminuric (NA) T1D pts in the diabetic nephropathy (DN) Natural History Study (NHS). AER levels in these pts followed one of 4 patterns (Table). We analyzed the data from the Renin Angiotensin System Study (RASS) to explore the re-lationships between RAS blockers and patterns of AER in NANT T1D pts.RASS was a multicenter, randomized, double blinded, placebo controlled trial comparing enalapril and losartan to placebo on progression of DN over 5 years. AER was measured at baseline, quarterly and 2 months af-ter drug washout. Patterns were classifi ed per NHS. AER at exit was cat-egorized as < or 20 μg/min. Chi-square and analysis of variance (ANOVA) were used for analysis. There were no statistically signifi cant differences in the distribution of AER patterns among groups (Table). There was also no group difference in the proportion of pts with AER < or 20 μg/min at exit (p=0.1). At exit, AER values were higher in the losartan [4.80 (0.90-264.30) μg/min] than in the placebo [4.30 (0.40-23.90); p=0.007] group. There was no within group differences in AER levels at exit and washout. AER levels increased from baseline to washout among pts randomized to placebo (p=0.01) or losartan (p= 0.003), while they did not change among pts randomized to enalapril. However, there were no group differences in AER values at washout (p=0.139). Our results did not show a treatment ef-fect on AER patterns over 5 years in NANT T1D. Pts randomized to losartan showed the greatest increase in AER levels from randomization till exit.

& 524-PInsulin Dose is Associated With Susceptibility to Overt Nephropa-thy in Type 1 DiabetesTREVOR J. ORCHARD, TINA COSTACOU, DEMETRIUS ELLIS, Pittsburgh, PA

Kidney disease increases mortality risk in type 1 diabetes, but in its ab-sence mortality risk is similar to that of the general population. We assessed the association between modifi able risk factors and susceptibility to overt nephropathy (ON). We used 18 year follow up data from the Pittsburgh EDC study, a prospective investigation of childhood onset type 1 diabetes (n=658, baseline mean age, 28 and duration, 19 years). ON (albumin excretion rate >200 μg/min in multiple timed urine specimen) susceptibility was defi ned as developing ON within 20 years of diabetes onset and at least four years before developing either of the other two advanced microvascular complica-tions (proliferative retinopathy or confi rmed distal symmetric polyneuropa-thy). ON resistance was defi ned as being ON free after 20 years of diabetes or developing ON within 20 years of diabetes duration but at least four years after developing both other microvascular complications. Thus, although susceptible to microvascular disease in general, these groupings assured specifi c susceptibility or resistance to ON. Out of 419 that could be so cat-egorized, 26 (6.2%) were ON susceptible and 393 ON resistant. Of the 393 resistant, 259 were assessed at similar diabetes duration (<20 yrs) to sus-ceptible individuals. Compared to resistant, ON susceptible individuals were more likely to be female (p=0.05), with higher insulin dose per body weight, systolic and diastolic blood pressure (all p-values <0.05). In multivariable lo-gistic regression models, female gender (OR=3.74, 95% CI: 1.33-10.49), insu-

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lin dose per body weight (OR=24.92, 95% CI: 3.62-171.56) and systolic blood pressure (OR=1.07, 95% CI: 1.03-1.11) were independently associated with ON susceptibility. The association with insulin dose merits further study as it is unlikely to solely refl ect insulin resistance or glycemic control, for neither a previously validated equation to estimate glucose disposal rate (eGDR, p=0.36) nor HbA1c (p=0.93) differed by susceptibility group.

Supported by: NIH (DK34818)

& 525-PDevelopment of Risk Prediction Models for Chronic Kidney Disease in Type 2 Diabetes Using Genetic and Clinical VariablesGUOZHI JIANG, ERIC S. LAU, YING WANG, ANDREA O. LUK, CLAUDIA H. TAM, JANICE S. HO, VINCENT K. LAM, HEUNG MAN LEE, MAGGIE C. NG, XIAODAN FAN, WING YEE SO, JULIANA C. CHAN, RONALD C. MA, Hong Kong, China

This hypothesis-generating study examined whether T2D or obesity-related genes can predict development of chronic kidney disease (CKD). Thirty-six single nucleotide polymorphisms (SNPs) associated with T2D, hyperglycaemia or obesity, identifi ed through GWAS, were genotyped in 5371 T2D patients without CKD at baseline from the Hong Kong Diabetes Registry, of which 1287 (24.0%) developed CKD during a median follow-up period of 8 (IQR 4.8-11.0) years. A feature selection method based on Akaike Information Criterion (AIC) was used to select an informative variable sub-set, and Cox proportional hazards regression was used to develop models and detect association effects. Predictive performance was evaluated and comparisons using C statistics and time-dependent area under the curve (AUC) of the ROC curves.Top eighteen variables were selected as predictors of CKD in T2D, including ACR, age, HbA1c, presence of neuropathy, as well as 6 genetic variants. The AUC was 0.80, with tendency to decrease with increasing follow-up time (from 0.87 to 0.80 between 0 and 10 years). The c statistics based on variable subsets were 0.857(inter-quartile range [IQR]: 0.853-0.861) and 0.853(IQR: 0.846-0.858) on training and test data sets re-spectively. Removing genetic variants from the variable subsets resulted in lowering of the c statistics to 0.854(IQR: 0.851-0.858) and 0.851(IQR: 0.845-0.855) correspondingly. Three SNPs were independently associated with CKD after adjusting for top clinical variables, with respective hazard ratios (95% confi dence interval [CI]): 0.85 (0.78-0.94, P=0.001), 1.19 (1.05-1.35, P=0.005) and 1.17 (1.03-1.32, P=0.015). Analysis of joint effect of these 3 SNPs showed additive effect with increasing number of risk alleles (Ptrend = 4.6×10-5). By using the feature selection method based on AIC, we success-fully identifi ed an informative variable subset from our dataset containing genetic and clinical variables.

Supported by: Innovation and Technology Fund (ITS/487/09FP)

& 526-PPrevalence of Stage 5 Chronic Kidney Disease by Cause in a Nation-wide Study of People With DiabetesINEZ BRADY, ON BEHALF OF THE SCOTTISH RENAL REGISTRY AND THE SCOTTISH DIABETES RESEARCH NETWORK, EPIDEMIOLOGY GROUP, Dundee, United Kingdom

Our aims were to examine the prevalence of stage 5 chronic kidney disease (CKD5) and use of renal replacement therapy (RRT) in the Scottish population with diabetes, to examine the primary renal diagnosis (PRD) among those on RRT and to test whether survival following RRT varies by PRD.We linked records from the national database of all those registered with diabetes in 2006 -2008 (SCI-DC) to the Scottish Renal Registry that captures all RRT recipients in Scotland. CKD5 by end 2008 was defi ned as 2 recent MDRD derived eGFR readings 15/ml/min/1.73m2 or being in receipt of RRT. Logistic and Cox regression were used for analyses.The point prevalence of CKD5 in 2008 was 1.6% of 19,847 people with type 1 diabetes (T1D) compared with 0.6% of 168,176 people with type 2 diabetes (T2D) (Odds ratio [OR] = 3.98, p<0.0001) with the difference explained by duration (OR on adjustment for duration = 0.95). 83% of CKD5 T1DM patients and 61% of T2D CKD5 patients were receiving RRT. Diabetic nephropathy (DN) was the PRD assigned by the renal physician in 88% of those with T1D and 56% with T2D (p<0.0001). Other common PRDs in T2D were multisystem diseases (16%), interstitial nephropathies (7%) and primary glomerulonephritis (5%). In those with T2D the following were associated with a PRD of DN; longer duration of diabetes (OR = 1.08, p=0.0001), higher HbA1C (OR = 1.31, p=0.01), absence of cardio-vascular disease pre-RRT (OR = 0.50, p=0.02) and presence of retinopathy pre-RRT (OR= 4.82, p=0.005). Three years from start of RRT 50% (CI 45% - 57%) of those with DN were alive with survival rates being similar for pri-mary glomerulonephritis 54% (CI 36% - 81%), interstitial nephropathies 41% (CI 27% - 62%) and multisystem diseases 39% (CI 29% - 53%).This provides contemporary data on the prevalence of CKD5 in diabetes and illustrates the

heterogeneity of causes of CKD among those with T2D. However mortality rates after starting RRT were not signifi cantly different in diabetic vs. non-diabetic kidney disease among those with T2D in this study.

Supported by: Scottish Government and Wellcome Trust Scottish Health Infor-matics Programme

& 527-PRenal Structural Changes in Patients With T2DM, Impaired Renal Function and ± AlbuminuriaELIF I. EKINCI, ALISON SKENE, KAREY CHEONG, DAVID POWER, SIANNA PA-NAGIOTOPOULOS, KAREN MCNEIL, SOPHIE CLARKE, SCOTT BAKER, PAULA FIORETTO, GEORGE JERUMS, RICHARD MACISAAC, Melbourne, Australia, Van-couver, BC, Canada, Padua, Italy

The structural basis of nonalbuminuric renal insuffi ciency in T2DM re-mains to be elucidated. We compared renal biopsy fi ndings in outpatients with T2DM, eGFR<60ml/min/1.73m2 and normo-(N), micro-(μ) or macro-(M) albuminuria attending a tertiary hospital. A minimum of 6 glomeruli was ex-amined by light microscopy (LM) which included immuno-fl uorescence/his-tochemistry. In subjects with N or μ, eGFR was supplemented by iGFR (DTPA). All biopsies were assessed by light (LM) and electron (EM) microscopy and the fi ndings categorised according to Fioretto classifi cation1- Category 1(C1): Normal/near normal, Category 2 (C2): Typical diabetic nephropathy (DN) & Category 3 (C3): Atypical (disproportionately severe interstitial/tubular/vascular damage with no/mild diabetic glomerular changes). There was a predominance of typical DN (C2) in all stages of albuminuria. Subjects with M all had C2. In subjects with N, 7/8 had varying degrees of arteriosclero-sis. Histological evidence of DN, manifesting as either glomerulopathy (C2) or as interstitial/vascular damage (C3) is still likely to be found in patients with T2DM and non albuminuric renal insuffi ciency (table). This suggests that age, BP and vascular disease contribute to changes in renal structure and function. In μ subjects the present results differ from previous data1 in 34 subjects with T2DM, μalbuminuria and GFR of 101 ml/min/1.73m2. In that study, the biopsies were classifi ed as 35% C1, 30% C2 and 35% C3. This raises the possibility that GFR in subjects in C1 and C3 does not decline as fast as in C2, but studies of GFR trajectory would be needed to confi rm this.

Fioretto Classifi cation (1. P Fioretto et al., Diabetologia 39, 1569 (1996)

N(n = 8)

μ (n = 6)

M (n = 17)

C1 1 (12.5%) 0 (0%) 0 (0%)C2 4 (50%) 5 (83%) 17 (100%)C3 3 (37.5%) 1 (17%) 0 (0%)

Supported by: NHMRC

& 528-PIGF-II Associated Genes May Prevent Progression of Diabetic NephropathyRAM P. NARAYANAN, SIMON G. ANDERSON, BO FU, ANTHONY PAYTON, JOHN P. NEW, ADRIAN H. HEALD, WILLIAM E. OLLIER, JOHN M. GIBSON, Salford, United Kingdom, Manchester, United Kingdom

IGF-II is expressed in the kidney with increased expression in some re-nal neoplasms. Interactions of IGF-II in diabetic nephropathy are unknown despite its structural and functional similarities with insulin. We studied single nucleotide polymorphisms (SNPs) of the IGF2 gene, its closely coupled regulatory gene H19 and the IGF-II receptor gene IGF2R with respect to lon-gitudinal trends in glomerular fi ltration rate (eGFR)528 Caucasian individu-als with type 2 diabetes were studied. Phenotypic data were derived from linked primary care and hospital records. Genotyping of HapMap selected haplotype tagging SNPs was performed on Sequenom iPlex . Quality assur-ance using SVS7 (Golden Helix) was performed. Mixed effects regression was used to analyse longitudinal trends over 8 years in eGFR in STATA 10SE corrected gene-wise for multiple testing.Eight IGF2, eleven H19 and twelve IGF2R SNPs were selected . 7 SNPs were highly associated with longitu-dinal trends in eGFR adjusted for gender, age, diabetes duration and ACE inhibitor use. eGFR was calculated from serum creatinine using the Modifi -cation of Diet in Renal Disease (MDRD) formula.Minor alleles of three IGF2 SNPs were associated with preserved eGFR - rs734351 ( 0.53,[95% CI 0.35 to 0.71], p<0.001); rs10770098( 0.33,[95% CI 0.13 to 0.53], p=0.001) and rs1003483 ( 0.32,[95% CI 0.12 to 0.52], p=0.001). Also associated with good renal function were rs6578973 ( 0.29, [95% CI 0.09 to 0.49], p=0.003) near the H19 gene as well as the IGF2R SNP rs609207 ( =0.28, [95% CI 0.10 to 0.46], p=0.002).Minor alleles of two SNPs favoured a decline in eGFR- IGF2 SNP rs2000993 ( -0.49, [95% CI -0.66 to -0.31], p<0.001), and IGF2R SNP rs761389 ( -0.57, [95% CI -0.85 to -0.28], p<0.001).All associations were

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sustained after Bonferroni correction.These novel associations of IGF2 and related genes with longitudinal renal function suggest a protective role for IGF-II in preventing progression of diabetic nephropathy with implications for cellular action in the kidney.

& 529-PRelationship Between Abdominal Obesity and Diabetic Nephropa-thy According to the Distribution of Fat Tissue in Korean Type 2 Dia-betic PatientsMIN YOUNG CHUNG, JIN OOK CHUNG, DONG HYEOK CHO, DONG JIN CHUNG, Gwangju, Republic of Korea

Waist circumference correlates to both visceral and subcutaneous fat tissue. Recently, abdominal obesity assessed by waist circumference has been implicated in the pathogenesis of diabetic nephropathy. However, the impacts of visceral and subcutaneous fat tissue on cardiometabolic disease have been suggested to be different from each other. In addition, the im-pact of perirenal fat tissue on diabetic nephropathy has not been studied. The aim of this study was to evaluate the difference between visceral, sub-cutaneous, and perirenal fat tissue in relation to diabetic nephropathy in Korean type 2 diabetic patients. Two hundred and twenty type 2 diabetic patients (M: 113, F: 107; mean age: 60.9 ± 11.6 years) were recruited. All subjects were assessed for the metabolic profi les, urinary albumin excre-tion rate (UAE) and estimated glomerular fi ltration rate (eGFR). The visceral, subcutaneous and perirenal fat tissue were measured by ultrasonography. In the univariate correlation analysis, visceral fat tissue was positively as-sociated with UAE. Also, there was a signifi cant positive association be-tween subcutaneous fat tissue and eGFR. However, perirenal fat tissue was not signifi cantly associated with UAE or eGFR. In a multivariate analysis, increased visceral fat tissue was an independent positive determinant for macroalbuminuria (>300 μg/mg creatinine) after adjustment of age, gender, diabetes duration, glycated hemoglobin, systolic blood pressure, and use of angiotensin-converting enzyme inhibitor and angiotensin-receptor blocker, while increased subcutaneous fat tissue showed an independent inverse as-sociation for reduced eGFR (< 60 mL/min/1.73 m2) in multivariate analysis. This study suggests that abdominal fat tissue is associated with the differ-ent natural course of diabetic nephropathy according to body fat distribution in Korean type 2 diabetic patients.

530-PObstructive Sleep Apnea is Independently Associated With Dia-betic Nephropathy in Patients With Type 2 DiabetesABD A. TAHRANI, ASAD ALI, SAFIA BEGUM, KIRAN DUBB, SHANAZ MUGHAL, BIJU JOSE, MILAN PIYA, ANTHONY BARNETT, MARTIN STEVENS, Birmingham, United Kingdom, Coventry, United Kingdom

Diabetic nephropathy (DN) causes signifi cant morbidity and mortality. Understanding DN pathogenesis is essential to develop new therapies. Ob-structive sleep apnea (OSA) is prevalent in patients with type 2 diabetes (T2D). Since OSA and DN may share common oxidative stress and infl amma-tory mechanisms, we hypothesized that OSA is associated with DN.Adults with T2D were recruited randomly from the diabetes clinic of a UK-based hospital. Patients with known respiratory disorders (including OSA) and end-stage renal disease were excluded. DN was defi ned by eGFR of <60 ml/min/1.73m2 or the presence of albuminuria after exclusion of other causes. OSA was assessed using home-based portable multi-channel respiratory monitoring (Alice PDX, Philips Respironics, USA). OSA diagnosed when the apnoea-hypopnea index was 5 events/hour (OSA+).T2D patients (n=234) were included, 64.5% were OSA+ and 39.7% and 36.2% had DN and albu-minuria respectively. OSA+ patients were older, more obese and had higher BP compared to those without OSA. DN (29.7% vs. 55.9%, p<0.001) and albuminuria (24.3% vs. 43.2%, p=0.007) prevalence were higher in OSA+ pa-tients. OSA+ patients had lower eGFR (92.9 ± 25.2 vs. 82.4 ± 26.4, p=0.006).OSA+ remained independently associated with DN (OR 2.251, 95%CI 1.069-4.741, p=0.033) after adjusting for age, gender, ethnicity, BP, HbA1c, total cholesterol, triglycerides, HDL, diabetes duration, smoking, alcohol, obesity, and the use of anti-platelet, anti-diabetes, lipid-lowering and anti-hyper-tensive treatments. In addition, nadir nocturnal oxygen saturations were an independent predictor of DN (OR 0.957, 95% CI 0.920-0.995, p=0.027) when it replaced OSA in the model.In conclusion, we describe a novel association between OSA and DN and nocturnal hypoxia in patients with T2D. Ongoing studies are exploring the mechanisms involved. The ability of OSA treatment to impact DN warrants determination.

Supported by: NIHR (UK)

531-PTriglycerides, NMR Lipoprotein Subclasses and Albuminuria in Men With Type 2 Diabetes: A Veterans Affairs Diabetes Trial Sub-StudyALICIA JENKINS, DERRICK G. KAUFMAN, JULIE STONER, RICK L. KLEIN, MARIA LOPES-VIRELLA, TIMOTHY J. LYONS, VA DIABETES TRIAL INVESTIGATORS, Okla-homa City, OK, Hines, IL, Charleston, SC

In diabetes, nephropathy can induce dyslipidemia which in turn may accel-erate renal decline. Nuclear magnetic resonance (NMR) lipoproteinanalysis can provide detail regarding the lipoprotein species implicated. NMR (Lipo-Science Inc, Raleigh, NC) was performed on a sub-set of 331 VADT men (age 60 (9) y (mean (SD)): diabetes duration 11 (7) y; BMI 32.6 (5.1) kg/m2; HbA1c 7.7 (1.5)%), of whom 91% had hypertension, and 88% were on lipid drugs. Fast-ing samples were obtained within 6 months of the 2-year post-randomiza-tion visit. In cross-sectional analysis, using linear regression of the associa-tion between (natural log-transformed) urine albumin:creatinine ratio (ACR) (dependent variable) and (standardized natural log-transformed) lipoprotein measures, adjusted for total LDL particle concentration but unadjusted for other covariates, fasting triglyceride was the only conventional lipid asso-ciated with ACR regression coeffi cient =0.23, p=0.036). Triglycerides re-mained associated with ACR when adjusted for age, diabetes duration, race, VADT randomization status (hence HbA1c), hypertension, lipid drugs, BMI and WHR ( =0.22, p=0.049). NMR lipoproteins that were inversely related to ACR in adjusted cross-sectional analyses were: LDL particle size; particle concentrations of large LDL and total and medium HDL concentrations (all p<0.05). In longitudinal follow-up (1-5y, mean 2.3y), triglycerides were as-sociated with average ACR over time ( =0.34, p=0.019, fully adjusted), and the following NMR lipoprotein subclasses were inversely associated with average ACR over time: LDL particle size and medium HDL concentration (both p<0.05, fully adjusted). No lipoprotein measure predicted decline in ACR over time. In conclusion, in cross-sectional and longitudinal analyses, triglycerides, and NMR measures of LDL and HDL were associated with ACR during the VADT intervention period, but none predicted change in ACR over an average of 2.3 y.

532-PEstimated Glomerular Filtration Rate and Albuminuria: True Predic-tors of Cardiovascular Events in Obese Patients With Type 2 Dia-betes?MICHAEL RESL, GREISA VILA, RICHARD PACHER, ANTON LUGER, MARTIN HÜL-SMANN, STEPHANIE NEUHOLD, HELMUT BRATH, RUDOLF PRAGER, MARTIN CLODI, Vienna, Austria

The widely used MDRD formula underestimates kidney function in obese diabetic patients. Therefore, we aimed to evaluate the impact of this cal-culation bias on the predictive value of eGFR for cardiovascular events in a typical cohort of diabetic patients.In general 988 patients were analysed. Cox Regression models including the variables HbA1c, age, duration of dia-betes, eGFR and urinary albumin to creatinine ratio (UACR) were run. At fi rst the whole collective was analysed, in a second step the cohort was split into 4 different groups according to BMI and eGFR (Group 1, 475 Pts : eGFR > 60 ml/min; BMI < 30 kg/m2, Group 2, 274 Pts : eGFR > 60 ml/min; BMI >30 kg/m2,Group 3 110 Pts, : eGFR < 60 ml/min; BMI > 30 kg/m2 and Group 4, 129 Pts. : eGFR < 60 ml/min; BMI < 30 kg/m2). The endpoint was defi ned as unplanned cardiovascular hospitalization.Patients (571 male, 417 female) were 61 ± 22 years of age, mean duration of diabetes was 14.3 ± 12.3 years. After a me-dian follow up of 29 months 95 (9.6%) patients reached the defi ned endpoint. The fi rst model, including all patients showed that UACR (HR 1.001, p <0.001) and eGFR (HR 0.957, p<0.001) were signifi cant predictors of the composite endpoint. In obese patients eGFR completely lost its predictive value for cardiovascular events.Noteworthy the prevalence of normoalbuminuria in patients with an eGFR below 60 ml/min was 59.4%. In obese patients eGFR is not predictive for cardiovascular events.

533-PAssociations of Serum Pigment Epithelium Derived Factor Levels With Renal Dysfunction, Body Habitus and Dyslipidemia in Type 2 Diabetes PatientsALICIA JENKINS, DONG-XU FU, JULIE STONER, DERRICK KAUFMAN, SARAH X. ZHANG, RICK KLEIN, MARIA LOPES-VIRELLA, JIAN-XING MA, TIMOTHY J. LY-ONS, VA DIABETES TRIAL INVESTIGATORS, Oklahoma City, OK, Hines, IL, Charles-ton, SC

The serpin Pigment Epithelium Derived Factor (PEDF) has anti-angiogenic, anti-infl ammatory, and anti-oxidant effects, and in cell culture and animal models, is protective against microvascular damage. We determined if se-rum PEDF in Type 2 diabetes patients was related to vascular risk factors

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and renal function. Serum PEDF was quantifi ed by ELISA in a sub-study of 254 male Veterans Affairs Diabetes Trial (VADT) subjects, sampled within 6 months of the 2-year post-randomization visit (age: 60 (9) y (mean (SD); diabe-tes duration: 11 (7) years; BMI: 32.6 (4.8) kg/m2; HbA1c: 7.7 (1.4)%), of whom 88% were hypertensive and 85% were taking lipid-lowering drugs. Univari-ate, cross-sectional associationsbetween PEDF and risk factors were quan-tifi ed by Spearman’s rank correlation coeffi cients. Mixed model regression analyses were used to evaluate longitudinal associations between PEDF and renal function (dependent variable), with and without adjustment for age, ethnicity,diabetes duration, hypertension, body habitus, VADT randomiza-tion, and lipid drugs. Cross-sectional study: Among 17 variables tested, PEDF (ng/ml) correlated with body habitus (BMI: =0.26, p<0.0001; waist-to-hip ratio: =0.20, p=0.0018), renal function (serum creatinine: =0.29, p<0.0001; estimated GFR (mL/min/1.73 m2): =-0.27, p<0.0001), and lipids (fasting triglycerides (mg/dl): =0.35, p<0.0001; HDL-cholesterol(mg/dl): =-0.33, p<0.0001). PEDF correlated inversely with CrCl (Cockcroft-Gault) and GFR (regression coeffi cient =-2.13, p<0.0001 and =-1.95, p<0.0001), but not with log-transformed urine ACR (p=0.11)after adjustment for confounders. Longitudinal study: PEDF was not associated with longitudinal renal function changes. In conclusion, serum PEDF in VADT Type 2 diabetes patients was associated with adiposity, dyslipidemia and with renal dysfunction, but was not associated with subsequent changes in renal function.

534-PObesity Modifi es the Association between Leptin Levels and Kidney Function Decline in Patients With Type 2 DiabetesKO HANAI, TETSUYA BABAZONO, YASUKO UCHIGATA, Tokyo, Japan

We have recently demonstrated that both low and high serum leptin levels are risk factors for kidney function decline in patients with type 2 diabetes (T2D). Obesity has been shown to be a modifi er of the association between leptin levels and cardiovascular events. We tested the hypothesis that obesity modifi es the relationship between serum leptin levels and kidney function decline in patients with T2D.This was a longitudinal observational cohort study of 411 Japanese adult patients with T2D (mean [± SD] age: 58 ± 13 years, men: 61.1%). Patients were classifi ed into 2 groups by sex-specifi c median of serum leptin levels. Obesity was defi ned as body mass index 25 kg/m2. Outcome measurement was the annual rate of decline in estimated glomerular fi ltration rate (eGFR).During the median follow-up period of 4.8 years (range: 2.0 - 8.2), the mean rate of change in eGFR was -1.5 ± 3.7 mL/min/1.73 m2/year. A signifi cant interaction between serum leptin levels (low or high) and obesity (present or absent) on the rate of change in eGFR was detected (P interaction =0.01). In the non-obese group, the adjusted rate of change in eGFR (mean ± SE) in patients with low leptin was steeper than that in patients with high leptin (-1.9 ± 0.3 and -0.6 ± 0.5 mL/min/1.73 m2/year, p =0.04, ANCOVA). In the obese group, the adjusted rate of change in eGFR in patients with high leptin was not signifi cantly different with that in pa-tients with low leptin (-1.5 ± 0.3 and -0.8 ± 0.5 mL/min/1.73 m2/year, p =0.25). When leptin levels were treated as a continuous variable, logarithmically transformed leptin levels were positively associated with the rate of change in eGFR, independent of other variables in non-obese patients (standard-ized estimate = 0.22, p <0.01). In contrast, in obese patients, leptin levels were negatively associated with the rate of change in eGFR (standardized estimate = -0.24, p =0.03).The effects of leptin levels on the kidney function decline may depend on the presence of obesity in patients with T2D.

535-PThe Prevalence of Albuminuria According to Status of Autoimmu-nity and Insulin Sensitivity among Youth With DiabetesAMY K. MOTTL, ABIGAIL LAUER, RALPH B. D’AGOSTINO, JR., MICHAEL MAUER, DAVID MAAHS, LAWRENCE M. DOLAN, LISA K. GILLIAM, JEAN M. LAWRENCE, MARYAM AFKARIAN, KRISTI REYNOLDS, SANTICA M. MARCOVINA, GIUSEPPI-NA IMPERATORE, DANA DABELEA, ELIZABETH MAYER-DAVIS, FOR THE SEARCH FOR DIABETES IN YOUTH STUDY GROUP, Chapel Hill, NC, Winston-Salem, NC, Minneapolis, MN, Aurora, CO, Cincinnati, OH, Seattle, WA, Pasadena, CA, Atlanta, GA

It is unknown how autoimmunity and insulin resistance affect the natural history of diabetic kidney disease. To address this issue, we evaluated the effect of these characteristics on the risk of albuminuria in children with dia-betes.The SEARCH study is an observational study of children with type 1 or 2 diabetes who are followed longitudinally. Diabetes autoantibodies (DAA), insulin sensitivity (using a validated equation based on euglycemic hyper-insulinemic clamp data) and urinary albumin:creatinine ratio (UACR) from a single, random urine specimen were measured. Data from the most recent visit were used for all analyses. Participants were grouped into four etiologic

subclasses of diabetes: 1) DAA+/insulin sensitive (IS) (n=1833); 2) DAA+/in-sulin resistant (IR) (n=1462); 3)DAA-/IS (n=682) and 4) DAA-/IR (n=978). Mul-tivariate regression analyses adjusted for race/ethnicity, sociodemographics and diabetes duration, to assess the relationship between these subclasses of diabetes and albuminuria (UACR 30ug/mg). Interaction with race/eth-nicity, mean arterial pressure (MAP), hemoglobin A1C and body mass index was explored.Participants included in the analysis (n=4,955) had a mean age of 13.7 yrs (SD=4.4) and mean diabetes duration 49 mos (SD=39). Prevalence of albuminuria was 8.2% DAA+/IS, 9.8% DAA+/IR, 7% DAA-/IS and 17% DAA-/IR group. With DAA+/IS as the reference group, the adjusted odds ratio (OR) of albuminuria was signifi cant only in the DAA-/IR group (OR=1.6; 95% CI 1.2, 2.1). Only MAP modifi ed this association, wherein the higher the MAP, the greater the relative difference in prevalence of albuminuria in the DAA-/IR vs DAA+/IS groups (interaction term p-value=0.0003).In youth with diabetes of relatively short duration, , there is a signifi cantly increased prevalence of albuminuria in those with IR and DAA- but not in those with IR and DAA+, compared to those with DAA and IS. This distinction may be mediated by differences in MAP.

Supported by: The CDC with support from the NIDDK

536-PEstimating Early Glomerular Filtration Rate (GFR) Decline in Dia-betes by the Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) EquationRICHARD J. MACISAAC, KAREY CHEONG, ELIF EKINCI, SHILPA VERMA, EROSHA PREMARATNE, ZHONG X. LU, KEN A. SIKARIS, GEORGE JERUMS, Melbourne, Australia, Heidelberg, Australia, Collingwood, Australia

Evaluating GFR using the modifi cation of diet in renal disease (MDRD) equation underestimates reference GFR levels >60 ml/min/1.73m2, whereas an estimated GFR (eGFR) derived from the CKD-EPI equation is reported to be less biased. However, the accuracy of the CKD-EPI equation in people with diabetes remains to be defi ned. We therefore assessed the performance of an eGFR calculated from the CKD-EPI or MDRD equations compared with reference GFR measurements in subjects with diabetes. The reference GFR was measured using 99mTc-DTPA plasma clearance (iGFR) and creatinine was measured by an enzymatic method. In a cross-sectional study, bias (eGFR - iGFR) was compared for the CKD-EPI and MDRD equations for subjects with iGFR measurements > 60 ml/min/1.73m2. In a longitudinal study of subjects with an early decline in GFR i.e., initial iGFR >90 ml/min/1.73m2 and iGFR

3.3 ml/min/1.73 m2 per year, iGFR (based on initial and fi nal values) was compared with eGFR by the CKD-EPI and MDRD equations over a mean of 9 years. In the cross-sectional study, iGFR for the whole group was 80 ± 2.2 ml/min/1.73m2 (n=199, 75% type 2 DM). For subjects with an iGFR >90 ml/min/1.73m2 (iGFR: 112 ± 2.0, n=76), both equations signifi cantly underesti-mated iGFR to a similar extent: bias for CKD-EPI -12 ± 1.4 ml/min/1.73m2 (p < 0.001) and for MDRD -11 ± 2.1 ml/min/1.73m2 (p < 0.001). By contrast, for sub-jects with an iGFR between 60 and 90 ml/min/1.73m2 (iGFR 77 ± 1.2, n=59), there was a non-signifi cant trend for both equations to overestimate iGFR. In the longitudinal study (n=30, 66% type 1 DM), initial and fi nal iGFR values were 102.8 ± 6 and 54.6 ± 6.0 ml/min/1.73m2, respectively. Mean GFR (ml/min/1.73m2 per year) was 6.2 by iGFR compared with 3.1 by MDRD and 3.3 by CKD-EPI (both p < 0.05 vs iGFR). In conclusion, both the CKD-EPI and MDRD equations underestimate reference GFR values >90 ml/min/1.73m2 as well as an early decline in GFR to a similar extent.

537-PMicrovascular Disease in Type 1 Diabetes (T1D): The Predictive Power of A1c Variability (A1cV)STEPHEN A. DELURGIO, PRIYA S. RAMAN, DAVID D. WILLIAMS, MARK A. CLE-MENTS, Kansas City, MO

A1cV has been shown to correlate with increased risk of microvascular disease (MD) for those with T1D; whether A1cV is useful in predicting MD is unknown. This study explores the utility of the standard deviation (SD) of A1c’s in predicting level II nephropathy among T1D patients using Cox Propor-tional Hazard models. The DCCT study cohort (n=1439, 333 events) was split: 2/3 (n=965, 222 events) for model development and 1/3 (n=474,111 events) to test models in risk predictions. All models (M, S, B, F) include age, sex, T1D duration, and treatment group as covariates. Model M included only the Mean (X 2=141, p<0.000); S, the SD alone (X2=120, p<=0.000); B, Both mean and SD (X 2=147, p<0.000). F is Model B fi tted to the Full cohort and serves as a benchmark for accurate predictions (X2=201, p<=0.000) for the 474 pa-tients withheld when developing models M, S, and B. Using SPSS 19, risk predictions with Model B were more accurate than with M. Correlations of the risks of F with those predicted with M, S, and B were rFM=.952, rFS =.913,

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and rFB=.986, (p< 0.0000001 for all). Statistical difference tests of r’s yield t-values of 8.59 for rFB-rFM, 14.30 for rFB-rFS, and 9.59 for rFM-rFS (p<0.00001 for all), indicating signifi cant differences between rFB, rFM, and rFS. Kendall’s W tests confi rmed that there are differences in the 474 predicted risks of all models at p<0.000001. Using both the mean and SD as predictors of level II nephropathy better discriminates between those with medium and low hazards compared to using only the mean (hazard curves, Figure 1). Further analyses using retinopathy data will be presented.

538-PHigh Glucose Induces ROS Production by NADPH Oxidases via a Src-Dependent MechanismKEN W. LEE, LING XIA, HOWARD GOLDBERG, ANU SHAH, CATHARINE WHITE-SIDE, I. GEORGE FANTUS, Toronto, ON, Canada

The pathogenesis of diabetic nephropathy (DN), a leading cause of end stage renal disease, remains incompletely understood. The increased gen-eration of reactive oxygen species (ROS) and consequent oxidative stress is a mediator of the pathologic changes caused by chronic exposure to high glucose (HG). Src has been reported to be both upstream and downstream of ROS. To determine its role in DN, cultured primary rat mesangial cells (MCs) were exposed to either normal glucose (NG 5mmol/L) or HG (25 mmol/L) and total cell ROS measured using 2’,7’-dichlorofl uorescein (DCF) and dihy-droethidium (DHE), fl uorescence. NADPH oxidase activity was assayed with lucigenin. Src activity was blocked by the inhibitors, Dasatinib (DS) and AZD, as well as by transfection with Src-specifi c siRNA. After 3 h, HG signifi cantly induced cell ROS and NADPH oxidase activity that were markedly decreased by DS, AZD, and Src siRNA. In contrast, mitoSOX loaded cells showed that HG-induced mitochondria-specifi c ROS were unaffected. Thus, we examined the role of Nox2 and Nox4. Nox2-specifi c siRNA abrogated ROS production in short-term HG (1-3 h), while Nox4-specifi c siRNA blocked ROS production only in long-term HG (24-48 h). Consistent with these results, HG induced Nox4 protein showed a time-dependent signifi cant increase at 24-48 h. Treatment with DS, AZD, and Src-specifi c siRNA blocked Nox4 induction. Ex-posure to hydrogen peroxide in the setting of NG and in the presence of Src inhibitors, augmented Nox4 protein, while the antioxidant Tempol blocked HG induction of Nox4. These data indicate that a Src-dependent rapid ac-tivation of Nox2 by HG generates a ROS signal to upregulate Nox4, that is responsible for long term increased ROS which mediates the pathological effects of chronic hyperglycemia.

539-PPrognostic Value of Resting Heart Rate on Renal Outcomes in Type 2 Diabetic Patients: A Competing Risk Analysis in a Prospective CohortSAMY HADJADJ, AURELIE MIOT, STEPHANIE RAGOT, WALA HAMMI, PIERRE-JEAN SAULNIER, PHILIPPE SOSNER, XAVIER PIGUEL, RICHARD MARECHAUD, RONAN ROUSSEL, Poitiers, France

Epidemiological studies and clinical trials suggest that resting heart rate (RHR) was an independent predictor of cardiovascular (CV) risk and all-cause mortality. However no data evaluated the relationship between RHR and re-nal complications in type 2 diabetes (T2D) patients.Method: We performed a single-centre, prospective analysis in 1088 T2D patients. RHR was deter-mined by ECG. The study outcome was time to a renal composite criterion (renal replacement therapy or chronic doubling of baseline serum creatinine) adjusted for all-cause death as a competing event.Results: During median follow-up of 4.2 years, 62 patients (6%) experienced the study outcome. At

baseline, a history of CV and of renal disease was noted in 336 (31%) and 367 (34%) patients, respectively. An interaction was found between RHR and history of CV (but not renal) disease at baseline for the relation between RHR and study outcome (p interaction =0.03). In those patients with CV disease at baseline (CVD+), those developing a renal outcome had a higher RHR than non-affected patients: 77 ± 13 versus 66 ± 12 bpm (p <0.0001), while no such effect was noticed in those patients non-CVD+.In CVD+, RHR was associ-ated with renal risk when adjusted for all-cause death as a competing event (p <0.0001). In multivariate analysis in CVD+, predictors of the renal outcome were RHR (SHR=1.04; p=0.001) and renal disease at baseline. In non-CVD+, no relation was found between RHR and the incidence of CV and/or renal events.Conclusion: In the real-life setting, RHR constitutes an easy, quick, effi cient and cost-effective factor that would permit identifi cation of dia-betic patients with an increased risk for severe renal complications.

540-PImpact of High Glucose on the Histone Ubiquitylation in Rat Glom-erular Mesangial CellsYONG XU, CHENLIN GAO, GUO CHEN, Luzhou, Sichuan, China

The modifi cation of histones by ubiquitylation is a prominent epigenetic mark that features in a variety of chromatin-based events such as histone methylation, gene silencing, and repair of DNA damage.The bulk of histone ubiquitylation occurs on chromatin by the addition of a single ubiquitin mol-ecule via an isopeptide linkage to a specifi c lysine residue of histones H2A and H2B. Ubiquitylation on different histones has distinct functions. Howev-er, he effect of histone ubiquitylation on the diabetic nephropathy is unclear. Recently, we cultured HBZY-1 rat glomerular mesangial cells(GMCs) and divided them into 6 groups : normal glucose group( 5.6mmol/L),high glucose group(10,20,30 mmol/L),mannitol group was used as high osmosis control and proteasome inhibitor group (30mmol/Lglucose pretreated with MG132 as a proteasome specifi c inhibitor).The ubiquitylation of histones was mea-sured by Western blot and indirect immunfl uorescence laser scanning con-focal microscope respectively. The results showed that the ubiquitylation of H2A in GMCs in normal group was weak, whereas the ubiquitylation of H2A in GMCs in high glucose group was stronger than that in normal group (P<0.05) in a concentration -dependent manner. After pretreated with pro-teasome inhibitor MG132, the ubiquitylation of H2A in GMCs was decreased obviously compared with 20,30mmol/L glucose group (all P<0.05).But for the ubiquitylation of H2B,we found the opposite changes in high glucose groups (P<0.05).These data support the hypothesis that high glucose affects the ubiquitylation of histone in GMCs and imply that the ubiquitylation of his-tone may be take part in the pathogenesis of diabetic nephropathy.

541-PThe 9p21 Coronary Artery Disease Locus and Kidney Dysfunction in Patients With Type 2 Diabetes MellitusSALVATORE DE COSMO, SABRINA PRUDENTE, OLGA LAMACCHIA, HETAL SHAH, CHRISITINE MENDONCA, DANIELA LUCCHESI, LAURA PUCCI, LUANA MERCURI, DIEGO BAILETTI, GIUSEPPE PENNO, MAURO CIGNARELLI, ALESSANDRO DORIA, VINCENZO TRISCHITTA, San Giovanni Rotondo, Foggia, Italy, Boston, MA, Pisa, Italy, Rome, Italy

Cardiovascular disease and kidney dysfunction share a common patho-genic soil, raising the hypothesis that they also share, at least in part, a common genetic background. We investigated whether rs2383206 at the chromosome 9p21 locus, a single nucleotide polymorphism (SNP) which is strongly associated with coronary artery disease (CAD) both in the general population and in diabetic patients, is also associated with low estimated glomerular fi ltration rate (eGFR) or increased urinary albumin excretion (UAE) in patients with type 2 diabetes mellitus (T2DM). Four independent samples (three from center-south Italy, one from Boston, MA) of White patients with T2DM, for a total of 3,167 individuals, were studied. Low eGFR (<60 ml/min/1.73 m2) was calculated from serum creatinine according to the MDRD Study equation. Increased UAE was defi ned as an albumin-creatinine ratio (ACR) > 2.5 in men or > 3.5 mg/mmol in women. All study participants were typed for SNP rs2383206 by means of TaqMan assay. No association was found between rs2383206 and low eGFR or increased UAE in each individual sample. Similarly, in the pooled analysis, no association was found between s2383206 and low eGFR (594 cases and 2,573 controls; additive OR=1.07, 95% CI=0.94-1.22, p=0.31) or increased UAE (1,009 cases and 2,121 controls; additive OR=1.00, 95% CI=0.90-1.12, p=0.95). No interaction was observed between rs2383206 and HbA1c (i.e., below or above the median value of the pooled sample=7.7%) in modulating eGFR or UAE (p for SNP-by-A1c inter-action=0.42 and 0.90, respectively).In conclusion, variability at the chromo-some 9p21 locus is unlikely to play a role in modulating the risk of developing

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kidney dysfunction in patients with T2DM. Whether other genetic markers are shared between CAD and kidney dysfunction in diabetic patients will have to be investigated by means of further collaborative studies.

542-PAdolescent Type 1 Diabetes Cardio-Renal Intervention Trial: Screen-ing ResultsM. LOREDANA MARCOVECCHIO, ON BEHALF OF THE ADDIT STUDY GROUP, Cam-bridge, United Kingdom

To describe baseline markers of cardiovascular and renal disease in the AdDIT screening population.Adolescents with type 1 diabetes (T1D) are be-ing screened with 2 sets of 3 consecutive early morning urines for measure-ment of albumin-creatinine ratio (ACR). Tertiles of ACR are calculated using an algorithm adjusting for age, gender and duration: the upper tertile identi-fi es the high-risk, the lower and middle tertiles the low-risk group. Blood samples are being collected for centralized measurement of cardiovascular and renal markers and standardized cardiovascular assessments are being performed.2515 adolescents (54% males, mean (±SD) age:13.1±1.7yr; median [IQR] T1D duration 4.8 [2.4-7.7]yr) have been assigned to ACR tertiles: 919 (36.5%) upper, 829 (33%) middle,767 (30.5%) lower. ACR progressively in-creases from the lower to the upper tertile (0.51 [0.42-0.62], 0.70 [0.58-0.85], 1.26 [0.91-1.82] mg/mmol, p<0.001); mean HbA1c is 8.4±1.4%, with small differences across ACR tertiles (8.3±1.3%, 8.4±1.3%, 8.5±1.6%, p=0.03).Renal markers (creatinine and symmetric dimethylarginine), available from 171 high- and 76 low-risk participants, are signifi cantly lower in the high-risk group (48.5 [42.9-57.9] vs 53.9 [46.2-62.7] μmol/l, p=0.002; 398.52±55.53 vs 424.43±60.37 nmol/l, p=0.001, respectively) indicating hyperfi ltration. The prevalence of abnormal lipid profi les is higher in the high-risk group (total cholesterol 5.2mmol/l: 19.9 vs 13.2%) but this is not signifi cant. The cardio-vascular assessments (n 270 high-risk, n 121 low-risk) indicate a higher prev-alence of high diastolic blood pressure (20.2 vs 8.3%, p=0.003), and greater pulse wave velocity (5.00 [4.45-5.50] vs 4.70 [4.40-5.00] m/s, p=0.001) in the high-risk group, indicating increased arterial stiffness.These preliminary data indicate early abnormalities in renal and cardiovascular function in ado-lescents with T1D and ACR in the upper part of the normal range, suggesting a higher risk of developing complications.

Supported by: JDRF, Diabetes UK, British Heart Foundation

543-PPodocyte Excretion in the Urine of Patients With Diabetic Neph-ropathy as a Marker of Podocyte InjuryMASAO TOYODA, EITARO TANAKA, NAOYUKI YAMAMOTO, MASAAKI MI-YAUCHI, MORITSUGU KIMURA, TOMOYA UMEZONO, MITSUNORI YAGAME, MASANORI HARA, DAISUKE SUZUKI, MASAFUMI FUKAGAWA, Isehara, Japan, Niigata, Japan

According to various reports, detection of podocytes in the urine indicates severe injury to these cells in the glomeruli. Therefore, investigation of uri-nary podocyte excretion in relation to the development and progression of diabetic nephropathy (DN) is thought to be important. Cilnidipine is an oral N/L-type calcium channel blocker that has been reported to inhibit sympa-thetic activity and has a stronger renoprotective effect than L-type calcium channel blockers.To investigate the relationship between the stage of DN and the urinary excretion of podocytes, the number of podocytes in the urine wvas measured in patients with type 2 diabetes. Furthermore, we studied the infl uence of N/L-type calcium channel blocker treatment on albuminuria and podocyturia.One hundred patients with type 2 diabetes, ranging from normoalbuminuria to end-stage renal failure, were enrolled in this study. We evaluated the correlations between the number of urinary podocytes and clinical parameters. Thirteen patients with podocyturia and urinary albumin excretion (UAE) >300 mg/g Cr despite treatment with angiotensin-converting enzyme inhibitor (ACE-I) or angiotensin II receptor blocker (ARB) were given additional treatment with the N/L-type calcium channel blocker cilnidipine.A signifi cant increase in the number of urinary podocytes was seen along with the progression of albuminuria, and there was a signifi cant positive correla-tion between the number of podocytes excreted in the urine and UAE. Treat-ment with cilnidipine for 3 months did not reduce the mean UAE. However, there was a signifi cant reduction in the number of urinary podocytes com-pared with the baseline value.Measurement of the urinary podocyte count might be useful for detecting podocyte injury and for assessing the reno-protective effect of treatment. Our results also suggest that cilnidipine may have an additional podocyte protective effect in patients with DN who are already receiving recommended renoprotective treatment.

544-PThe Expression and Role of Notch Signal Related Molecules in Me-sangial Cells Induced by High GlucoseYONG XU, LIU LI, GAO CHENLIN, Luzhou, Sichuan, China

The Notch signaling pathway is a widespread and highly conserved signal-ing pathways. Recent studies have confi rmed a member of the Notch family involved in podocyte injury,tubular differentiation and renal fi brosis. Gamma secretase is the key proteases of cut Notch receptor and form reactive Notch intracellular dormin (NICD), so it is also the core link of regulating Notch sig-naling pathways . This study observe the changes of Notch signal related protein molecules (Notch1, Jagged1, Hes1) and fi brosis molecules(Fibronectin and TGFbeta) in the rat glomerular mesangial cells (GMC) stimulated by high glucose or gamma - secretion inhibitors (DAPT) and explore the mechanism of Notch signaling pathway in diabetic nephropathy. We divided cultured rat GMC into 4 groups:the normal control group (5.5mmol/L glucose), high glucose group (30mmol/L glucose),high glucose + DAPT group and mannitol group (high osmosis control). Western-blot,RT- PCR techniques and immu-nofl uorescence confocal microscopy were used to detect the expression of Notch1, Jagged1, Hes1, Fibronectin (FN), TGFbeta protein and mRNA expres-sion.We found the protein and mRNA expression of Notch1, Jagged1, Hes1 in normal cells were weak. After high glucose treated, the protein expression was enhanced signifi cantly ( P < 0.01 ) . Pretreated DAPT to high glucose, the protein and mRNA expression of Notch1, Jagged1, Hes1 were decreased (P < 0.01 ). The expression of FN and TGF beta were shown the same changes. Our study indicate that high glucose is involved in the development of dia-betic glomerular fi brosis via Notch pathway activation.

Supported by: NNSF of China

545-PGlycemia Infl uences Cystatin C in Youth With Diabetes: SEARCH for Diabetes in Youth StudyROOPA KANAKATTI SHANKAR, DAVID MAAHS, LAWRENCE M. DOLAN, AN-DREA ANDERSON, GIUSEPPINA IMPERATORE, DANA DABELEA, KRISTI REYN-OLDS, IRL HIRSCH, ELIZABETH MAYER-DAVIS, SANTICA M. MARCOVINA, RALPH B. D’AGOSTINO, JR., MICHAEL MAUER, AMY K. MOTTL, Cincinnati, OH, Denver, CO, Winston-Salem, NC, Atlanta, GA, Pasadena, CA, Seattle, WA, Chapel Hill, NC, Minneapolis, MN

There has been increasing interest in serum cystatin C as a potential marker of glomerular fi ltration rate (GFR) in children. The distribution and factors infl uencing cystatin C levels in pediatric patients with diabetes are largely unknown.The SEARCH for Diabetes in Youth Study is a large, mul-ticenter cohort study of youth with type 1 and 2 diabetes identifi ed soon after diagnosis and followed longitudinally. Cystatin C was measured in 952 participants (825 with Type 1 and 127 with Type 2 diabetes) with a mean duration of diabetes of 31 +/- 9.6 months. We used step-wise regression models to explore the infl uence of diabetes duration, fasting plasma glu-cose, glycated hemoglobin (A1c), systolic and diastolic blood pressure (BP) and body mass index (BMI), adjusting for known cystatin C determinants such as age, gender, and race/ethnicity. Given the small sample size of type 2 diabetes and differences in comorbidities by diabetes type, analyses were run separately for Type 1 and Type 2 diabetes.In youth with type 1 diabetes, adjusted cystatin C levels were negatively associated with glucose con-centration (p<0.001, = -0.00023) and A1c (p<0.001, = -0.01126). In youth with type 2 diabetes, adjusted cystatin C was negatively associated with glucose concentration (p<0.001, = -0.00069) but not signifi cantly with A1c, and it was positively associated with BMI Z-score (p=0.005, = 0.05224). We found no statistically signifi cant association with diastolic or systolic BP. Hyperglycemia is associated with lower cystatin C levels in both Type 1 and Type 2 diabetes of short duration, likely refl ecting acute hyperfi ltration. The effects of acute vs. chronic hyperglycemia on cystatin C may differ by type of diabetes and needs to be further explored. In children with Type 2 diabetes who have a greater prevalence of obesity, studies exploring the relationship of cystatin C to BMI are also needed.


546-PSleep-Disordered Breathing as a Modifi able Risk Factor for Micro-vascular Complications in Japanese Type 2 Diabetes MellitusSHINYA FURUKAWA, TERUKI MIYAKE, SHINYA YAMAMOTO, TETSUJI NIIYA, TERUHISA UEDA, TERU KUMAGI, TAKENORI SAKAI, HIROAKI MIYAOKA, SUSUMU SAKURAI, ISAO SAITO, BUNZO MATSUURA, TAKESHI TANIGAWA, MORIKAZU ONJI, Toon, Japan, Matsuyama, Japan, Yawatahama, Japan

Background: The associations between sleep-disordered breathing (SDB) and type 2 diabetes mellitus (T2DM) have been reported in cross-sectional

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study. Although SDB and cardiovascular disease are closely related, the as-sociation between SDB and microvascular complications remains unclear. We hypothesized SDB may be a potential and treatable risk factor for mi-crovascular complications. Aim: To estimate the prevalence of SDB among Japanese T2DM and to examine the association between SDB and micro-vascular complications. Method: We conducted a cross-sectional study of 690 Japanese T2DM aged from 20 to 85 years old. All participants com-pleted self-administered questionnaires on the duration of diabetes, ethanol intake per day, numbers of cigarettes smoked per day, use of medications (antihypertension and antihyperlipidemia) and the score on the Epworth Sleepiness Scale. Urinary albumin-creatinine ratio (UACR) was calculated using fi rst morning urine sample, and microalbuminuria was defi ned by UACR of 30 mg/gcr or greater. A pulse-oximeter was used to assess SDB.Results: The prevalence of SDB, defi ned by 3% oxygen desaturation index (ODI) of 5 events/hr or more, was 45% among Japanese T2DM. There were positive associations between 3% ODI and body mass index (BMI), hypertension, hy-perlipidemia and microalbuminuria (P<0.0005 in all). Multivariate analysis identifi ed BMI (P=0.0001), age (P=0.002), hyperlipidemia (P=0.028), hyper-tension (P=0.030) and microalbuminuria (P=0.032) as associated factors for SDB. Finally, hypertension (P=0.0001) and SDB (P=0.0115) are independently associated with microalbuminuria after adjusting age, sex, BMI and dura-tion of T2DM. Conclusion: We identifi ed the prevalence of SDB in Japanese T2DM, and SDB was independently associated with microalbuminuria. SDB may be a potential and treatable risk factor to prevent microvascular com-plications in T2DM.

Supported by: Grants-in-Aid for Young Scientist (B) (No. 217090583)

547-PZinc Defi ciency Exacerbates Diabetes-Induced Renal Oxidative Damage, Infl ammation and Fibrosis via Down-Regulation of Nrf2 ExpressionWENPENG CUI, BING LI, YI TAN, YANG BAI, QIANG CHEN, XIAO MIAO, LINING MIAO, LU CAI, Louisville, KY, ChangChun, China, Jilin, China

Zinc (Zn) defi ciency often occurs in patients with diabetes; therefore, here the effect of Zn defi ciency on diabetic renal damage was investigated. Type 1 diabetes was induced in FVB mice with multiple low doses of strepto-zotocin. Once hyperglycemia was established, diabetic and age-matched control mice were treated with and without Zn chelator, N, N, N’, N’-tetrakis (2-pyridylemethyl) ethylenediamine (TPEN) at 5 mg/kg daily for 4 months. Renal oxidative damage, infl ammation and fi brosis were examined by his-topathological observation, Naphthol AS-D Chloroacetate esterase assay, immunofl uorescent staining, and Western blotting assay. Mechanistic study was done with HK-11 renal tubular cells in vitro. Chronic TPEN treatment sig-nifi cantly decreased renal Zn levels in both diabetic and control mice. Com-pared to groups of diabetes and TPEN alone, Diabetes/TPEN group showed a signifi cant decrease in nuclear factor-erythroid 2-related factor 2 (Nrf2) expression along with signifi cant increases in renal oxidative damage (pro-tein nitration and lipid peroxidation), infl ammation [infi ltrated infl ammatory cells and infl ammatory mediators (PAI-1 and ICAM-1)], and fi brosis (collagen accumulation in glomerulus mesangial area and up-regulation of profi brotic mediator CTGF). Treatment of HK 11 cells with TPEN signifi cantly removed intracellular Zn with a signifi cant increase in CTGF expression, which de-creased Nrf2 expression and nuclear distribution and also prevented sul-foraphane-induced Nrf2 expression and function. These PTEN effects could be signifi cantly attenuated by Zn supplementation with signifi cant increase in Nrf2 expression and function. These results indicated that Zn is required in part for Nrf2 expression and function; Zn defi ciency signifi cantly enhanced diabetes-induced renal oxidative damage, infl ammation and structural re-modeling, via down-regulation of Nrf2 expression and function.

548-PInteraction of Angiotensinogen and Atrial Natriuretic Peptide Un-der Hyperglycemia in the KidneySHYI J. SHIN, CHAO S. LO, CHAO H. CHEN, KUN D. LIN, Kaohsiung, Taiwan

Hyperglycemia can activate intrarenal angiotensinogen(ANG) and atrial natriuretic peptide (ANP) synthesis. We recently reported that renal ANP can attenuate high glucose-activated TGF- 1, collagen I and NF- B ex-pression through auto/paracrine action in renal tubular cells (J Cell Physiol 219:776-86, 2009). We aimed to investigate the interaction between ANG and ANP in the kidneys under hyperglycemia. In this study, we found high glucose signifi cantly increased ANG mRNA and ANGII immunoreactivity at 12, 24, and 24 hr and ANP mRNA and ANP immunoreactivity at 24 and 48 hr in NRK-52E cells. The increase of ANP mRNA and immunoreactivity stimu-

lated by high glucose was attenuated by the addition of losartan (10-6 M Los) and PD123319 (10-6 M PD) in cultured NRK-52E cells and by the administra-tion of angiotensin converting enzyme inhibitor in STZ-induced diabetic rats. ANP mRNA and immunoreactivity increased 1.75 and 2.45-fold at 1 and 2 hr by adding 10-6 M ANGII in NRK-52E cells. Urine ANP excretion from bilateral ureter was signifi cantly increased at 1 and 2 hr after perfusion of 500ng/kg/hr angiotensin II through left renal artery of normal rats, but no change during perfusion with 0.9% NaCl, 3% NaCl and 50 % glucose. In NRK-52E cells transfected with ANG siRNA, high glucose-stimulated increase of ANP mNRA and immunoreactivity was signifi cantly decreased as compared to control siRNA. Similarly, the transfection of ANP siRNA aggravated the stimulation of ANG, TGF- 1 mRNA and immunoreactivity by high glucose in NRK-52E cells. In conclusion, high glucose-activated ANG can increase ANP synthesis that then attenuates ANG, and TGF- 1 synthesis.

549-PAssociation between TGF-B1, IGF-1 and HLA and Diabetic Neph-ropathy in T1DM Pediatric Patients from BrazilKARLA S. SOUZA, MARCELA A. URURAHY, YONARA M. OLIVEIRA, MELINA B. LOUREIRO, HEGLAYNE P. SILVA, FRANCISCO P. FREIRE-NETO, GUSTAVO H. OL-IVEIRA, FABRICIO M. SANTOS, RICARDO F. ARRAIS, ROSARIO D. HIRATA, MARIO H. HIRATA, EDUARDO A. DONADI, MARIA G. ALMEIDA, ADRIANA A. REZENDE, Natal, Brazil, São Paulo, Brazil, Ribeirão Preto, Brazil

Diabetic nephropathy (DN) is the leading cause of renal failure. TGF-B1 and IGF-1 are crucial mediators in the pathogenesis of DN and likewise HLA region has a possible infl uence. Thus, the objective of present study was to investigate the association between TGF-B1 (rs1800469), IGF-1 (rs35767) and HLA (HLA-DRB1,-DQA1, and -DQB1) polymorphisms with DN in type 1 diabetic children and adolescents assisted at a pediatric hospital (HOSPED/UFRN) in Natal-RN/Brazil. This study included 101 type 1 diabetic patients (T1DM group) and 106 normoglycemic subjects (NG group) aged between 6 and 20 years. Metabolic control was evaluated by glucose and glycated he-moglobin. Albumin to creatinine ratio (ACR) was calculated. Polymorphisms was determined by allelic discrimination technique in real time PCR using TaqMan® pre-designed SNP assays from Applied Biosystems. TGF-B1 and IGF-1 mRNA expression were also evaluated by real time PCR. HLA gene alleles were examined by the polymerase chain reaction (PCR-SSO reverse). Glucose and glycated hemoglobin were signifi cantly increased in T1DM group compared to NG, indicating a poor metabolic control of these patients. Enhanced ACR was observed in T1DM group when compared to NG, suggest-ing an initial stage renal injury. Seven T1DM patients had microalbuminuria and presented the following alleles: DRB1*04:05 (p=0,009; OR=0,009) and DQA1*03:02 (p=0,009; OR=0,009) and genotypes: DRB1*03:01 DRB1*0405 (p=0,041; OR=0,070) and DQA1*03:02 (p=0,041; OR=0,070), but no signifi cant association was seen. TGF-B1 (p=0.015) and IGF-1 (p=0.002) polymorphisms were signifi cantly associated with ACR in those patients, which may have generated a signifi cant increase in TGF-B1 (p=0.001) and IGF-1 (p=0.029) mRNA expression in T1DM patients when compared to NG, which can lead to renal damage. These results suggest that genes TGF-B1 and IGF-1 can infl uence the genetic susceptibility to microalbuminuria in patients with T1DM.

Supported by: CNPq/Brazil

550-PAging, Kidney Function, and Treatment for Type 2 Diabetes: The RIACE StudyANNA SOLINI, GIUSEPPE PENNO, GIACOMO ZOPPINI, CECILIA FONDELLI, GIAM-PAOLO ZERBINI, EMANUELA ORSI, ROBERTO TREVISAN, MONICA VEDOVATO, MARCO G. BARONI, MAURO CIGNARELLI, FRANCESCO GIORGINO, RAFFAELLA BUZZETTI, ELE FERRANNINI, GIUSEPPE PUGLIESE, Pisa, Italy, Verona, Italy, Siena, Italy, Milan, Italy, Bergamo, Italy, Padova, Italy, Cagliari, Italy, Foggia, Italy, Bari, Italy, Rome, Italy

Use of some glucose-lowering agents in type 2 diabetes raises concern in the elderly or in pts with renal impairment. Few studies have described the prevalence of these treatments in real-life situations. We used data from the Renal Insuffi ciency And Cardiovascular Events (RIACE) Italian Multi-center Study. The cohort consists of 15,773 type 2 diabetes pts attending 19 diabetes clinics in 2007-2008. Pts were divided into age quartiles (quartile median [IQR]: 53 [8], 63 [4], 69 [3] and 77 [6] yrs); treatment was coded as no pharmacological therapy (Diet), metformin (Met), sulphonylureas (SU), glinides (Gli), thiazolidinediones (TZD), or insulin (Ins). Glomerular fi ltration rate (eGFR) was estimated by the CKD-EPI equation, identifying four classes of eGFR: 1 (>90); 2 (60-89); 3 (30-59); 4 (<30) ml/min/1.73m2. Across age quartiles, BMI declined (from 29.9±5.8 to 27.8±4.5 kg/m2, p<0.0001), while

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pulse blood pressure increased (from 53±13 to 63±17 mmHg, p<0.0001). HbA1c, LDL-cholesterol, and triglycerides decreased slightly if signifi cantly (all p<0.01) across age groups, while HDL-cholesterol increased (from 47±13 to 51±14 mg/dl, p<0.0001). eGFR declined progressively (from 96±18 to 67±19 ml/min/1.73m2, p<0.0001) - at a time-linear rate of 1.1 ml/min/1.73m2 equally in men and women. Likewise, micro- and macro-albuminuria increased with age quartile (from 18.3 to 27.0% and from 3.9 to 6%, p<0.0001). Between the lowest and highest age quartile, diet declined from 17 to 11%, Met and TZD fell (from 59 to 47% and from 5 to 2%), while SU and Gli rose from 26 to 41% and from 8 to 11% (all p<0.0001): Ins use was stable (24 to 26%). In CKD-EPI 4 class (n=304, age 74±9 yrs, eGFR 23±6 ml/min/1.73m2, 73% micro- or macro-albuminuric), Gli and SU usage was 16 and 18%, respectively, and Met was 14%. Even in CKD-EPI 3 (n=2411, age=73±9 yrs, eGFR=49±8 ml/min/1.73m2), Met and/or SU was 76%. In real-life situation, use of agents that are not recommended in older pts with severe renal impairment is still prevalent.

Supported by: Research Foundation of the Italian Society of Diabetology

551-PTaurine Ameliorates the Progression of Diabetic Nephropathy in Type 2 Diabetic Rat ModelEUN SOO LEE, YEON SIK CHOI, MI YOUNG LEE, EUN YOUNG LEE, CHOON HEE CHUNG, WonJu, Republic of Korea, Soonchunhyang, Republic of Korea

Diabetic nephropathy is a major cause of end stage renal disease in the diabetic complication. Recent studies suggested that high glucose increased reactive oxygen species which lead to tissue damage through the expression of infl ammatory factors.Taurine is a 2-aminoethanesulfonic acid which is found in the most animal tissues. According to previous studies, taurine have several functions such as osmoregulation, bile acid conjugation, cell mem-brane stabilization, cholesterol excretion and cell volume regulation. And it prevents hyperglycemia induced insulin resistance through the reduction of oxidative stress.Thus, we investigated whether taurine may reduced blood glucose, oxidative stress and infl ammation or not.Experimental animals were divided into four groups; normal group (LETO, n=10), DM group (OLETF, n=10), taurine treated group (OLETF, n=10) and losartan treated group (OLETF, n=10). We treated them for 20 weeks from 26 to 45 weeks of age, and measured blood glucose, urine ACR, MCP-1 levels, GBM thickness, slit pore numbers and glomerular volume.As a result, body weight was decreased in DM group compared with control group, which was recovered similar to that of control group in taurine and losartan treated groups. And 24hrs urinary albumin and ACR were signifi cantly reduced in taurine treated group compared with DM group. Moreover, we found taurine decreased vascular endothelial growth factor (VEGF) protein and mRNA as well as, monocyte chemotacticprotein-1 (MCP-1) mRNA expression in the kidney tissue. It also decreased glomerular basement membrane thickness and glomerular volume, and increased open slit pore numbers between podocyte and foot processes. But there were no signifi cant difference in IPGTT data between taurine treated and DM groups.In conclusion, these results suggest that taurine ameliorates the progression of diabetic nephropathy. We need more studies to search the mechanisms why taurrine protect the renal injury in type 2 diabetic rat model.

552-PProteinuria and Chronic Kidney Disease Predict 10-year Cancer-Related and All-Cause Mortalities in Type 2 Diabetic PatientsTSE-YA YU, HUNG-YUAN LI, YI-DER JIANG, TIEN-JYUN CHANG, JUNG-NAN WEI, LEE-MING CHUANG, Tainan, Taiwan, Taipei, Taiwan

Proteinuria has been found to be associated with cancer mortality in gen-eral population. However, this relationship remains unknown in type 2 dia-betes. We conducted a cohort study to determine if proteinuria, associated with diabetic nephropathy, predicts cancer-related mortality in subjects with type 2 diabetes. Between July 1996 and June 2003, we enrolled 646 subjects with type 2 diabetes at the National Taiwan University Hospital. A spot urine sample was collected to determine the presence of proteinuria at enrollment. The vital status of all subjects was ascertained by linking their data with computerized death certifi cates in Taiwan. The medium follow-up period was 10.4 years. Subjects with proteinuria had a hazard ratio (HR) of 2.83 (95% CI 1.87-4.29) for all-cause mortality and 2.02 (95% CI 1.03-4.00) for cancer-related mortality when adjusted for age, gender, smoking, his-tory of cardiovascular disease, obesity, hypertension, use of angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers, hemoglobin A1c, diabetes duration, total cholesterol, use of statins, abnormal ankle-bra-chial index, and estimated glomerular fi ltration rate. The presence of protei-nuria signifi cantly improved the predictive ability of cancer-related mortality (increase in concordance statistics = 0.03 and increase in area under the ROC curve = 0.02). Similarly, the presence of chronic kidney disease (CKD)

was associated with increased all-cause and cancer-related mortality (HR 2.12 [95% CI 1.46-3.09], and 1.94 [95% CI 1.04-3.51], respectively). In conclu-sion, proteinuria and CKD can predict 10-year all-cause mortality and cancer-related mortality independently in individuals with type 2 diabetes, over and above the established risk factors associated with type 2 diabetes.

553-PLow-Grade Albuminuria and the Role of Urine Storage in Type 2 DiabetesNADAN GREGORIC, DRAZENKA P. BARLOVIC, JELKA ZALETEL, Ljubljana, Slovenia

Microalbuminuria is associated with an increased risk for development of kidney and vascular disease in type 2 diabetes. Screening for microalbu-minuria is therefore an important tool in preventive care. Albuminuria deter-mined from overnight urine collections is one of the most direct measures of urinary albumin excretion. However, one of its drawbacks is the requirement of urine storage at low temperatures. Therefore, the aim of our study was to assess whether a more convenient storage of urine samples at ambient temperature affects the reliability of results.In patients with type 2 diabetes included in DEMAND (delapril and manidipine for nephroprotection in dia-betes) study albuminuria was determined by radioimmunoassay with series of three consecutive timed overnight samples on two different occasions. The samples were stored at ambient temperatures. We calculated a differ-ence in albuminuria measurement between the fresh sample and samples stored for 1 or 2 days, respectively. A paired-sample T-test was used to test for a statistical signifi cance between the two groups.From 68 patients with type 2 diabetes (age 67+/-8,5 years; glycated hemoglobin 8,4+/-1,3 %; se-rum creatinine 76+/-13,3 μmol/L; blood pressure 147/81+/-15/8 mmHg; body mass index 30,5+/-5,1 kg/m2; mean+/-SD) 15 were microalbuminuric and 53 normoalbuminuric. The absolute differences in albuminuria among timed overnight urine samples that were stored for 1 or 2 days compared to fresh sample on two different occasions were not statistically signifi cant (4,3+/-7,4 vs. 4,8+/-7,8 μg/min and 5,5+/-8,4 vs. 4,3+/-5,8 μg/min, respectively, all p values >0,05).Urine storage at ambient temperature for 2 days does not affect reliability of albuminuria values in patients with type 2 diabetes and low-grade albuminuria.

554-PPredicting Decline of Renal Function among People With Stage 3 Chronic Kidney Disease and Type 2 DiabetesHELEN C. LOOKER, HARSHAL DESHMUKH, ANDREW D. MORRIS, COLIN N. PALM-ER, HELEN M. COLHOUN, Dundee, United Kingdom, Kircaldy, United Kingdom

The power of trials of drugs for improving renal function in diabetes rely on progression rates in entrants who are typically at stage 3 of chronic kid-ney disease (CKD). Further stratifi cation for rapid progressors among such entrants would help to improve trial design. Therefore we have used longi-tudinal estimated glomerular fi ltration rate (eGFR) data from a contemporary cohort of people with type 2 diabetes (T2D) and stage 3 CKD to identify risk factors which predict a reduction of 50% of baseline eGFR within a 5 year period.Patients were identifi ed from the GoDARTS study which recruited 6500 people with T2D in Tayside, Scotland, between 1 October 1997 and 1 July 2006. EGFR and covariate data were obtained by data linkage to the national clinical diabetes database (SCI-DC) which contains data on ~99% of people with diagnosed diabetes in Scotland. We selected those with stage 3 CKD on enrolment into GoDARTS (eGFR = 30-59 mls/min/1.73m2) and at least one subsequent eGFR measure. Cases were defi ned as people who under-went a 50% reduction in eGFR during follow-up and controls as people with a reduction of eGFR <10% over a maximum follow up of 5 yearsOf the 1997 people with a stage 3 CKD at baseline 266 were cases and 265 controls. Median time to a 50% drop in eGFR was 1.9 years (95% Confi dence Interval (CI) 1.1-2.8), and median follow up for the controls was 3.3 years (95% CI 2.8-4.0).In a multivariate logistic regression model the following baseline factors were signifi cantly associated with being a case after adjustment for base-line eGFR: diabetes duration (OR per 5 years = 1.30, 95% CI 1.14-1.48), and systolic blood pressure (OR per 10 mmHg= 1.15, 95% CI 1.05-1.26). Lipids and HbA1c were not associated with a drop in eGFR 50%.Stratifi cation with risk factors for renal progression could improve clinical trial power; the role of the predictors reported here now needs validation in additional datasets.

Supported by: SUMMIT Consortium Funded by the IMI-JU

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555-PClinicopathological Risk Factors for the Prognosis of Diabetic Nephro pathy in Japanese Patients With Type 2 DiabetesNOSE CHIKAKO, MIHO SHIMIZU, KENGO FURUICHI, TADASHI TOYAMA, AKINORI HARA, KIYOKI KITAGAWA, YASHUNORI IWATA, SHUICHI KANEKO, TAKASHI WADA, Kanazawa, Japan

We evaluated the predictive impact of albuminuria, glomerular fi ltration rate (GFR), and renal pathological factors for the prognosis of Japanese pa-tients with type 2 diabetes.Two hundred and sixty Japanese patients with histologically proven diabetic nephropathy (164 males and 96 females) were examined. Patients were classifi ed by urinary albumin excretion and esti-mated GFR (eGFR) at the time of renal biopsy. Renal events were assessed by requirement for dialysis or 50% reduction of eGFR. Predictors of renal events, cardiovascular events and mortality were examined by the Cox pro-portional hazards regression.A mean follow-up period was 7.9 years. At the time of renal biopsy, the number of cases with normoalbuminuria (<30mg/day) or urinary protein (-) or (±), microalbuminuria (30-299mg/day) or urinary protein (+), and macroalbuminuria ( 300mg/day) or urinary protein (2+) were 47, 50 and 163, respectively. The proportion of subjects with low eGFR (<60ml/min/1.73m2) was 36% in patients with normoalbuminuria, 42% in pa-tients with microalbuminuria, and 71% in patients with macroalbuminuria. During the observational periods, 116 patients experienced renal events, 62 patients experienced cardiovascular events, and 45 patients experienced death. Regarding of renal events, cardiovascular events, and mortality, mac-roalbuminuria was the main predictor. Patients with macroalbuminuria and low eGFR had a 16.3-fold higher risk of renal events, a 5.7-fold higher risk of cardiovascular events and a 13.8-fold higher risk of death than that of normoalbuminuric patients with preserved eGFR ( 60ml/min/1.73m2). Patho-logical risk factors for renal events were atherosclerosis, nodular lesions, exudative lesions, mesangiolysis, diffuse lesions, and interstitial fi brosis.These results suggest that macroalbuminuria was the main predictor of re-nal events, cardiovascular events, and mortality of diabetic nephropathy in Japanese patients with type 2 diabetes.

556-PPhycocyanin and Phycocyanobilin from Spirulina Platensis Amelio-rate Diabetic Nephropathy via Attenuating Oxidative StressJING ZHENG, TOYOSHI INOGUCHI, YASUTAKA MAEDA, MARK MACARTY, SHUJI SASAKI, MASAKAZU FUJII, NORIKO IKEDA, KUNIHISA KOBAYASHI, NORIYUKI SONODA, RYOICHI TAKAYANAGI, Fukuoka, Japan, Encinitas, CA

In recent years, bilirubin has been paid attention to as an endogenous strong antioxidant. We and other investigators have reported that bilirubin and its precursor biliverdin may have benefi cial effects on diabetic vascular complications, including nephropathy, via its antioxidant effects. Here, we investigated whether phycocyanin derived from Spirulina platensis, a blue-green algae, and its chromophore phycocyanobilin, which has a chemical structure similar to that of biliverdin, protect against oxidative stress and renal dysfunction in db/db mice, a rodent model for type 2 diabetes. Oral administration of phycocyanin (300 mg/kg) for 10 weeks protected against albuminuria and renal mesangial expansion in db/db mice, and normalized tumor growth factor- and fi bronectin expression. In parallel, phycocyanin normalized oxidative stress makers such as urinary levels of 8-hydroxy-2’-deoxyguanosine and 8-iso-prostaglandin F2 , and renal dihydroethidium (DHE) staining and normalized the expression of NAD(P)H oxidase compo-nents (Nox4, p22phox and p47phox). Oral administration of phycocyanobilin (15 mg/kg) for 2 weeks also showed similar antioxidant effects. In cultured renal mesangial cells, phycocyanobilin as well as bilirubin and biliverdin in-hibited NADPH oxidase-dependent superoxide production in a dose depen-dency evaluated by the Lucigenin assay. Phycocyanobilin also inhibited an-giotensin II-induced intracellular reactive oxygen species in mesangila cells evaluated by DHE staining. In conclusion, oral administration of phycocyanin and phycocyanobilin may offer a novel and feasible therapeutic approach for preventing diabetic nephropathy.

557-PDifferential Effect of Vitamin C Defi ciency on the Pathogenesis of Diabetic NephropathyHIROSHI OKADA, TAKAFUMI SENMARU, YOSHITAKA KONDO, AKIHITO ISHIGA-MI, HIROSHI OBAYASHI, MAI ASANO, MASAHIRO YAMAZAKI, MICHIAKI FUKUI, GOJI HASEGAWA, NAOTO NAKAMURA, Kyoto, Japan, Tokyo, Japan

Vitamin C (VC)can be attributed to its biological functions as a cofactor for a number ofenzymes and as an antioxidant. It can alsoparticipate in oxida-tive protein folding in the endoplasmic reticulum (ER). Inthis study, we inves-tigated the effect of VC defi ciency on the pathogenesis of diabetic nephrop-

athy using senescence marker protein-30 (SMP30)/glucolactonaseknockout mouse, the VC-defi cient animal model. Diabetes was inducedusing strepto-zocin in male SMP30 Y/- mice (KO) and wild-type, SMP30 Y/+, mice (WT) at 8 weeks of age. Diabetic (DM) or control (C) mice were divided into 2 groups; a VC-supplemented (VC (+)) group and a VC-defi cient (VC (-)) group. After 12 weeks of diabetes, the renal histology and expression (qRT-PCR) of markers of ERstress, oxidative stress, infl ammation and fi brosis were estimated.It was noteworthy that cortical fi brosis area (Sirius red stain, x1005 fi elds/N) was markedly increased only in KO-DM-VC(-) (200003.2±32707.2μmm2,P<0.0001). There were no differences among the other groups including KO-C-VC(-)(45276.4±9933.2~74770±12901.7μmm2). On the other hand, in %mesangium/glomerular area (PAS stain, 10 glomeruli/N),there were no sig-nifi cant differences among the groups. MCP-1 expression wassignifi cantly increased in KO-DM-VC(-) and KO-DM-VC(+) compared to the othergroups (P<0.0001). The expression of p67phox, PPAR , and TGF- were increased in DM groups of both strains, and PPAR expression in KO-DM-VC(-)was signifi cantly lower than KO-DM-VC(+) (P<0.03). These results suggest that thereis a distinct difference in the pathogenesis between glomerular lesion andinterstitial fi brosis (tubulointerstitial lesion) of diabetic nephropathy. Themechanisms induced by VC defi ciency are likely to be complex includin-goxidative stress, inactivation of some enzymes, and mitochondrialinjury. This study points to potential new therapeutic approaches for advanced-stage of diabetic nephropathy.

558-PIL-1B and TNF-A Genes may be Associated With Microalbuminuria Onset in T1DM Pediatric Patients from BrazilMARCELA A. URURAHY, KARLA S. SOUZA, YONARA M. OLIVEIRA, MELINA B. LOUREIRO, HEGLAYNE P. SILVA, FRANCISCO P. FREIRE-NETO, JOÃO F. BEZERRA, RAUL H. BORTOLIN, SONIA Q. DOI, RICARDO F. ARRAIS, ROSARIO D. HIRATA, MARIA G. ALMEIDA, MARIO H. HIRATA, ADRIANA A. REZENDE, Germantown, MD, Natal, Brazil, São Paulo, Brazil

Accumulating evidence indicates that immunologic and infl ammatory mechanisms play a signifi cant role in development and progression of dia-betic nephropathy. In this way, polymorphisms in pro-infl ammatory cytok-ines genes may be associated with renal injury in diabetic patients. Thus, the objective of present study was to investigate the association between IL-1B (rs1143634 and rs16944) and TNF-A (rs1800629) polymorphisms with microalbuminuria in type 1 diabetic children and adolescents assisted at a pediatric hospital (HOSPED/UFRN) in Natal-RN/Brazil. This study included 130 type 1 diabetic patients (T1DM group) and 132 normoglycemic subjects (NG group) aged between 6 and 20 years. Metabolic control was evalu-ated by glucose and glycated hemoglobin. Albumin to creatinine ratio (ACR) was calculated. Polymorphisms was determined by allelic discrimination technique in real time PCR using TaqMan® pre-designed SNP assays from Applied Biosystems. IL-1B and TNF-A mRNA RNA expression were also evaluated by real time PCR. Glucose and glycated hemoglobin were signifi -cantly increased in diabetic individuals compared to NG, indicating a poor metabolic control of these patients. Enhanced ACR was observed in T1DM group when compared to NG, suggesting an initial stage renal injury. IL-1B (rs16944) and TNF-A (rs1800629) polymorphisms were signifi cantly associ-ated with ACR in those patients (p=0.0022 and p=0.0092, respectively). IL-1B rs1143643 polymorphism was signifi cantly associated with glycated hemo-globin values (p=0.0208), showing that this polymorphism is contributing to a poor glycemic control, which would induce an infl ammatory process. This could be evidenced by the signifi cantly higher IL-1B mRNA expression in T1DM patients when compared to NG (p=0.001) and could lead to renal injury. These results suggest a possible association of IL-1B (rs1143634 and rs16944) and TNF-A (rs1800629) polymorphisms with microalbuminuria on-set in T1DM patients.

Supported by: CNPq, CAPES

559-PUrinary Type IV Collagen is an Important Predictor for the Incidence of Microalbuminuria in Young Patients With Type 1 DiabetesMIWA MORITA, YASUKO UCHIGATA, Shimane, Japan, Tokyo, Japan

Urinary type IV collagen is the main component of glomerular basement membrane and mesangial matrix. Recently, we have demonstrated that el-evated levels of urinary type IV collagen are associated with kidney function decline in patients with type 1 diabetes (T1D). There were some reports by cross-sectional design that an increase in urinary type IV collagen occurs before an incidence of microalbuminuria. However, there were no longitudi-nal reports. Therefore, we aimed to clarify whether urinary type IV collagen is a predicator for the incidence of microalbuminuria in patients with T1D.

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A longitudinal observational cohort study was conducted, which included normoalbuminuric patients diagnosed with T1D before the age of 30 years, who were < 40 years old at the start of observation. Patients with < 1 year follow-up period or < 18 years old were excluded. Overall, 225 patients were enrolled (mean [±SD) age: 25±5 years, men: 32.9%). Microalbuminuria was defi ned as 30 urinary albumin-creatinine ratio (ACR) < 300 mg/g. The primary endpoint was the incidence of microalbuminuria. During the mean follow-up period of 8.3±3.5 years, 14 patients reached the endpoint. In the univariate Cox regression analysis, elevated logarithmically-transformed urinary type IV collagen-creatinine ratio (T4C) was identifi ed as a prognostic factor for the endpoint (p < 0.001). In the multivariate analysis using conven-tional risk factors as independent variables, including HbA1c, blood pressure and logarithmically-transformed ACR, elevated T4C was also identifi ed as a prognostic factor for the endpoint (p = 0.022). When patients were classifi ed into 2 groups by median of T4C, patients with high T4C levels had a higher risk for the endpoint than those with low T4C in both the univariate and multivariate Cox regression analysis (p = 0.008 and 0.017). In conclusion, urinary type IV collagen may be an important predictor for the incidence of microalbuminuria in young patients with T1D.

560-PMannan-Binding Lectin and Activation of Complement in Model of Type 1 DiabetesJAKOB A. ØSTERGAARD, METTE BJERRE, FREDERIK DAGNAES-HANSEN, TRO-ELS K. HANSEN, STEFFEN THIEL, ALLAN FLYVBJERG, Aarhus, Denmark

Blood levels of mannan-binding lectin (MBL) are increased in type 1 diabetes patients. High MBL levels are associated with the development of diabetic nephropathy in humans. In animals, a direct effect of MBL on diabetic kidney changes is observed. We hypothesized that MBL level and detrimental complement activation increase as a consequence of diabetes.To evaluate the effects of diabetes in mice, we measured MBL before and 6 weeks after multiple low-dose streptozotocin-induced diabetes. Liver MBL expression by RT-PCR was used to explain changes in circulating MBL levels. MBL elimination was estimated by half-life of intravenously injected recom-binant human MBL. Complement activation was measured by immunohis-tochemistry in glomeruli and by C5a concentration in plasma.Mice have two MBLs, MBL-A and MBL-C. Diabetes led to a 3.6-fold increase in MBL-C con-centration, P<0.001 (95% confi dence interval (CI95%: 2.9;4.5) compared with 0% in controls, P=0.93 (CI95% -7;9%). MBL-C changes differed signifi cantly in the two groups, i.e., effect-modifi cation (P<0.001). A minor difference in change in MBL-A was initially observed between diabetic and control ani-mals (P = 0.02). However, in separate analyses, MBL-A did not change in diabetic animals (P = 0.12) or in controls (P = 0.12).In accordance with the ob-served changes in circulating MBL levels, liver expression of MBL-C mRNA was increased in diabetes by 28% (CI95%: 8;53%), P = 0.006. MBL-A ex-pression did not differ between diabetic and control animals.The half-life of recombinant human MBL was signifi cantly prolonged in mice with diabetes compared with controls, 14.3 hours (CI95%: 11.3;19.4 hours) vs. 11.8 hours (CI95%: 11.0;12.9 hours), respectively (P = 0.017). Complement activation in plasma and glomeruli did not differ between groups.We conclude that our fi ndings support previous clinical observations and furthermore show that MBL increases as a direct result of diabetes. This change may be explained by alternations both in MBL production and turnover.

561-PMechanism of Cell Matrix Accumulation in Diabetes: Tuberin Phos-phorylation/Defi ciency Enhances Cell Matrix Protein Accumula-tionSAMY L. HABIB, MUKESH YADAV, ANTHONY VALENTE, San Antonio, TX

Tubular cells are a primary target of hyperglycemia and chronic exposure to elevated blood glucose levels contribute to the tubulointerstitial changes seen in overt diabetic nephropathy. The mechanisms by which hyperglyce-mia contributes to matrix expansion and fi brosis are not known. Defi ciency in tuberin resulted in 27% increased in kidney mass compared to wild type rats. Increase the basal levels of phospho-tuberin as well as defi ciency in tu-berin is associated with increased fi bronectin expression in kidney cortex of TSC2+/- rats compared to wild type rats as well as in TSC2+/- renal primary proximal tubular cells compared to TSC2+/+ cells isolated from rats. Phos-phorylation of tuberin is associated with increased fi bronectin expression in kidney cortex of diabetic rats compared to control rats. Insulin treatment prevented these changes in diabetic animals. Pre-treatment RPTE cells with Akt inhibitor or rapamycin signifi cantly reduced HG-induced fi bronectin ac-cumulation. Introduction of TSC2 cDNA into tuberin-defi cient cells abolished

fi bronectin protein expression while downregulation of tuberin by siRNA resulted in signifi cant decrease of fi bronectin in cells treated with HG. In addition, rapamycin treatment signifi cantly decreased fi bronectin accumula-tion in the kidney cortex of tuberin-defi cient mice (TSC2+/-). Transcriptional activity of fi bronectin was measured in PRPT cells exposed and pretreated with actinomycin D before exposed to high glucose. Treatment cells with actinomycin D signifi cantly decreased HG-induced increased in mRNA of fi -bronectin expression. In addition pretreated the cells with rapamycin prior exposed to HG signifi cantly decreased the promoter activity of fi bronectin. These indicate that tuberin is a major molecule that regulates kidney hyper-trophy and cell matrix proteins by modulating protein synthesis.

Supported by: AHA and Merit Review Award (S.L.H.)

562-PInfl ammatory Stress Increase Human Mesangial Foam Cell Forma-tion by Increasing SCAP Glycosylation in GolgiYANG YUAN, ZI L. SUN, ZHONG X. RUAN, NanJing, China, London, United Kingdom

Diabetic nephropathy caused by infl ammatory stress is associated with lipid accumulation in glomeruli. This study was designed to investigate whether interleukin-1 (IL-1 ) increase lipid accumulation in human mesan-gial cells (HMCs) via LDL receptor pathway by increasing SREBP cleavage-activating protein (SCAP) transcription and its glycosylation in Golgi.HMCs were treated with IL-1 . Intracellular cholesterol content was assessed by Oil Red O staining and cholesterol enzymatic assay. Expression of mRNA and protein of molecules controlling cholesterol homeostasis in the treated cells was examined by real-time quantitative PCR and western blotting, respec-tively. Golgi enzymes activity was determined using enzyme-based method. SCAP translocation was detected by confocal microscopy.IL-1 increased cholesterol accumulation in HMCs. Exposure to IL-1 increased expression and abnormal translocation of SCAP from ER to Golgi even in the presence of a high concentration of LDL. The increased SCAP translocation carried more transcription factor SREBP-2 to the Golgi for activation by cleavage which enhanced gene transcription of HMGCoA reductase and LDL recep-tor. Furthermore, IL-1 enhanced SCAP glycosylation by upregulating Golgi mannosidase activity. This prolonged the half-life and enhanced recycling of SCAP between the ER and the Golgi. The effects of IL-1 were blocked by inhibitors of Golgi mannosidases.Infl ammatory stress increased lipid synthesis and uptake, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP by Golgi enzymes in HMCs. These data imply that Golgi enzymes inhibitors might have a potential renal protective role in prevention of mesangial foam cell formation.

Supported by: sKidney Research UK (RP372008), Natural Science Foundation (BK2011601)

563-PRenoprotective Advantage of an L/N-Type Calcium Channel Blocker Compared With L-Type Calcium Channel Blockers in Type 2 Diabetic Patients With Early-Stage NephropathySHINYA FUKUMOTO, EIJI ISHIMURA, KOJI TAKEDA, YOHEI MIMA, KOKA MO-TOYAMA, TOMOAKI MORIOKA, KATSUHITO MORI, TETSUO SHOJI, MASANORI EMOTO, YOSHIKI NISHIZAWA, MASAAKI INABA, CLEARED STUDY INVESTIGA-TORS, Osaka, Japan

We evaluated the antiproteinuric advantage of cilnidipine, an N/L-type calcium channel blocker (CCB), compared with L-type CCBs in early-stage diabetic nephropathy. The study was a multicenter, non-randomized cross-over trial pre-registered as a CLEARED study (UMIN ID: 000000835). Par-ticipants were 90 type 2 diabetic patients exhibiting either normo- or mi-croalbuminuria, and undergoing CCB treatment for 6 months prior to study entry. The CCB at the time of entry was continued for the fi rst 6 months (Treatment period 1). Treatment was subsequently switched from cilnidipine to an L-type CCB, or vice versa, for the second 6-month observation period (Treatment period 2). During period 1, the L-type CCB group (n=69) showed a signifi cant increase of urinary albumin excretion (UAE) over time (53.2±7.2 mg/gCr (mean±SEM) 96.3±18.4 mg/gCr, p<0.01), while the cilnidipine group (n=21) showed no signifi cant elevation. During period 2, switching of the treatment from the L-type CCB to cilnidipine resulted in signifi cant reduc-tion of the UAE (96.3±18.4 mg/gCr 59.2±10.3 mg/gCr, p<0.01), whereas switching from cilnidipine to the L-type CCB resulted in no signifi cant change in the UAE. Multivariate logistic regression analysis showed that cilnidipine was a signifi cant factor reducing the risk of UAE-increase (OR: 0.324 (95%CI, 0.107-0.983) for period 1, OR: 0.246 (95%CI, 0.074-0.823) for period 2) inde-pendent of age, sex, blood pressure, RAS inhibitor, and HbA1c. This study demonstrated that, in patients with early-stage diabetic nephropathy, the antiproteinuric effect of cilnidipine, but not the L-type CCBs, was sustained

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even in patients treated for a long time. In addition, the renoprotection can be anticipated after switching from an L-type CCB to cilnidipine, but not vice versa.


Guided Audio Tour: New Ideas on Clinical Diagnosis and Mechanisms of Neuropathy (Posters 564-P to 571-P), see page 13.

& 564-PPeripheral Neuropathy in the SEARCH for Diabetes in Youth Cohort: A Pilot StudyEVA L. FELDMAN, CATHERINE L. MARTIN, RONNY A. BELL, ABIGAIL LAUER, JAS-MIN DIVERS, DANA DABELEA, DAVID J. PETTITT, SHARON SAYDAH, BARBARA LINDER, CATHERINE PIHOKER, DEBRA A. STANDIFORD, BEATRIZ L. RODRIGUEZ, RODICA POP-BUSUI, FOR THE SEARCH FOR DIABETES IN YOUTH GROUP, Ann Ar-bor, MI, Winston-Salem, NC, Aurora, CO, Santa Barbara, CA, Atlanta, GA, Bethesda, MD, Seattle, WA, Cincinnati, OH, Honolulu, HI

Estimates of the prevalence of diabetic peripheral neuropathy (DPN) in adult populations range from 30 to 70%. Data on DPN in children, adoles-cents and young adults, however, are limited. In a cross-sectional pilot study, DPN was assessed using the Michigan Neuropathy Screening Instrument (MNSI), a validated DPN screening tool, in a subset of SEARCH for Diabetes in Youth Study participants The MNSI includes a 15-item, self-administered questionnaire and a structured examination for foot abnormalities, distal vibration perception and ankle refl exes. An MNSI questionnaire (MNSIQ) score of > 7 is positive for DPN; an MNSI exam (MNSIE) score of > 2 is positive for DPN.The MNSIQ and MNSIE were completed in 413 and 418 SEARCH participants respectively; 53% were female; 83% had type 1 diabe-tes (T1D). For youth with T1D, mean age was 15.6 years (standard deviation [SD] 4.4 years] with duration of 6.2 years (SD=0.9); for youth with Type 2 diabetes (T2D), mean age was 21.6 years of age (SD=3.2), with duration of 7.6 years (SD=1.8). Mean A1C was similar for T1D and T2D in a subset of subjects (n=305) with A1C measured within 3 months: [8.8% (SD=1.9) vs. 8.7 (SD=3.1), p=0.34].Two SEARCH subjects (<1%) met the MNSIQ categorical threshold for DPN. Mean MNSIQ score was 1.1 (SD=1.5) and was higher for T2D vs. T1D [1.8 (SD=1.6) vs. 1.0 (SD=1.4), p<0.001]. A cumulative logit model showed positive association between A1C and MNSIQ scores (p=0.04). Mean MNSIE score was 0.8 ± 1.2 and was higher for T2D (1.5[SD=1.5]) vs. T1D [0.7(SD=1.1), p<0.001]. DPN prevalence by MNSIE (e.g., score > 2) was 11.3% (T2D 26.0% vs. T1D 8.2%; p<0.001) and was associated with older age (p < 0.001).DPN prevalence measured by MNSIE among T2D SEARCH participants approached rates reported in adult diabetes populations. Our fi ndings suggest that children, adolescents and young adults with diabetes are not only at risk for DPN, but that many already show measurable signs of DPN based on the MNSIE.


& 565-PEarly Detection of Nerve Fiber Loss by In Vivo Corneal Confocal Mi-croscopy in Recently Diagnosed Type 2 Diabetic PatientsDAN ZIEGLER, ANDREY ZHIVOV, STEPHAN ALLGEIER, KARSTEN WINTER, NIKO-LAOS PAPANAS, IRIS ZIEGLER, SABINE PESCHEL, BERND KOEHLER, OLIVER STACHS, RUDOLF F. GUTHOFF, MICHAEL RODEN, GDC STUDY GROUP, Düsseldorf, Germany, Rostock, Germany, Karlsruhe, Germany, Leipzig, Germany

Corneal confocal laser scanning microscopy (CCM) has emerged as a non-invasive technique to quantify the subbasal nerve plexus (SNP), but it is un-clear whether it allows for detection of small nerve fi ber pathology at early stages of type 2 diabetes. We assessed parameters of CCM, corneal sensa-tion, and peripheral nerve function in 50 patients with recently diagnosed type 2 diabetes (age: 58.5±8.5 [mean±SD] years, diabetes duration: 2.5±1.8 years, BMI: 32.1±5.1 kg/m², HbA1c: 6.6±0.9%) and 28 healthy subjects of similar age. Intraepidermal nerve fi ber density (IENFD) in 3-mm punch biop-sies from the distal leg was quantifi ed in a subset of 29 subjects with and 24 without diabetes. Image data acquired from automatic CCM focus scans were processed with digital image processing algorithms to eliminate mo-tion artifacts and reconstruct images of the SNP devoid of anterior corneal mosaic deformations. Nerve fi bers were segmented and analyzed by a newly developed software. Corneal nerve fi ber density (CNFD) was reduced in pa-tients with diabetes (0.022±0.007 vs. control: 0.028±0.006 μm/μm²; P=0.001) as was nerve branch density (328±163 vs. 417±170/mm²; P=0.026), number of connecting points (37.1±13.5 vs. 50.2±19.2/mm²; P=0.003), while a trend to-

wards reduction was noted for nerve fi ber thickness (2.51±0.39 vs. 2.67±0.37 μm; P=0.065). In the entire population, CNFD and number of connecting points correlated with IENFD (both r=0.39; p=0.004). CNFD was reduced below the 5th percentile in 28% of the diabetic patients. Similar numbers of patients showed concomitantly abnormal CNFD and normal IENFD and vice versa. No difference between patients and controls was noted for corneal sensation. In conclusion, CCM detects early nerve fi ber loss in recently diagnosed type 2 diabetes, but not necessarily in the same patients as skin biopsy, suggest-ing a patchy pattern of small fi ber neuropathy which is detectable in the cornea even before the lower limbs are affected.

& 566-PThe Cingulum Bundle is Altered by Type 2 DiabetesWOUTER S. HOOGENBOOM, VERONICA L. FLORES, NICOLAS R. BOLO, DONALD C. SIMONSON, ALAN M. JACOBSON, MARTHA E. SHENTON, GAIL MUSEN, Bos-ton, MA, Mineola, NY

The cingulum bundle (CB) is a major white matter tract that connects the frontal and parietal lobes with parahippocampal and adjacent tempo-ral regions, and is involved in memory, executive function, and emotion. It also plays a role in connecting the default mode network (DMN) and the frontal and temporal regions that support memory -- a cognitive process often impaired in T2DM. In addition, altered connectivity in the DMN has been identifi ed in T2DM and this may be an early biomarker for Alzheimer’s disease (AD). We used diffusion tensor imaging (DTI) to measure diffusion in the CB of 13 T2DM (age = 57±6 yrs, BMI = 30±5 kg/m2, HbA1c = 7.5±1.9 %, FPG = 135±51 mg/dl, IQ = 114±12) and 17 control subjects (age = 54±7 yrs, BMI = 28±4 kg/m2, HbA1c = 5.6±0.4 %, FPG = 83±6 mg/dl, IQ = 104±14). After regions-of-interest (ROI) were placed on a color-by-orientation map for each individual, we calculated DTI-derived diffusion values from the resulting ROI seed-based tractography to assess overall white matter health [fractional anisotropy (FA, trace)], as well as pathology related to axonal integrity (axial diffusion) and axonal myelination (radial diffusion). People with T2DM had lower FA (-5.5% in left CB: T2DM 345±16 vs controls 365±38; and -8.4% in right CB: T2DM 315±28 vs controls 344±27), lower axial diffusion (-1.3% in left CB: T2DM 1148±35 vs controls 1163±47; and -3.1% in right CB: T2DM 1109±39 vs controls 1144±53), and higher radial diffusion (+3.1% in left CB: T2DM 667±32 vs controls 647±50; and +2.3% in right CB: T2DM 679±43 vs controls 664±47), statistically signifi cant for FA and axial diffusion in the right CB (p<0.05, IQ-corrected). Also, axonal integrity was correlated with performance on delayed-recall and letter-fl uency in controls, but not in T2DM. Our fi ndings suggest that defi ciencies in axonal health underlie al-tered diffusivity in the CB in cognitively intact people with T2DM, which may provide a neuroimaging biomarker for AD.

Supported by: National Institute of Aging (5R01AG34165-2)

& 567-PMeasurement of Gastric Emptying in Diabetic Patients With Ra-diopaque Markers: Correlation With Scintigraphy and Upper GI SymptomsEVA A. OLAUSSON, HAKAN GRUNDIN, MATS ISAKSSON, CHRISTINA BROCK, PER-OVE STOTZER, HASSE ABRAHAMSSON, STIG ATTVALL, MAGNUS SIMRÉN, Gothenburg, Sweden, Aalborg, Denmark

Upper gastrointestinal (GI) symptoms and delayed gastric emptying are common in insulin treated diabetes mellitus (DM). Scintigraphy, the current gold standard to measure gastric emptying, is expensive and not widely available. Emptying of radiopaque markers (ROM) from the stomach using fl uoroscopy is an easy and inexpensive method, available in all hospitals.In this study we validated ROM emptying in insulin treated DM and assess the correlation with gastric scintigraphy and upper GI symptoms.On the same day we measured gastric emptying of a standard meal using scintigraphy (omelet labeled with 15 MBq 99mTc, and 150ml water) and emptying of twen-ty spheric ROM (density 1.27 g/cm3 and 4 mm diameter) using fl uoroscopy in diabetic subjects. To investigate upper GI symptoms the subjects completed the validated questionnaire, Patient Assessment of Upper Gastrointestinal Symptom Severity Index, PAGI-SYM.We included 115 patients with insulin treated DM age 52±12 (mean± SD) (range 25-69) years; 59 females. The mean duration of diabetes was 26±14 (range 2-63) years. 86 (75%) patients had delayed gastric emptying rate with the scintigraphy, whereas 29 pa-tients had delayed emptying of ROMs. Of these 29 subjects, 28 also had delayed scintigraphic emptying, whereas 58 of the patients with a normal ROM test had delayed gastric emptying with scintigraphy, yelding a sen-sitivity of the ROM test 33 %, whereas the specifi city 97%. The strongest correlations between gastric emptying and upper GI symptoms were found for scintigraphic emptying and nausea/vomiting (r=0.30; p<0.001) and post-

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prandial fullness/early satiety (r=0.34; p<0.0001).In summary, the sensitivity of the ROM emptying test had a high specifi city but rather low sensitivity.We found a strong correlation between scintigraphic emptying and nausea/vomiting and postprandial fullness/early satiety.

Supported by: Health Care in the West Region of Sweden

& 568-PRAGE Defi ciency Improves Post-Injury Sciatic Nerve Regeneration in Type 1 Diabetic MiceJUDYTA K. JURANEK, MATTHEW S. GEDDIS, FEI SONG ZOU, YU SHAN, JOSE GARCIA, XIAOPING (JENNIFER) SHEN, BIN CHENG, ANDREAS POLLREISZ, PRA-TIK KOTHARY, ROSA ROSARIO, LOUIS WEIMER, THOMAS H. BRANNAGAN, ANN MARIE SCHMIDT, New York, NY

Diabetes is of one the most prevalent metabolic, non-communicable dis-orders of today, becoming the main public health challenge worldwide. One of the major complications, affecting 30 - 50 % of all diabetic patients, is a peripheral diabetic neuropathy (PDN). Although molecular mechanisms un-derlying the pathogenesis of the disease are yet not fully understood, it has been shown that one of the possible contributors to the disease might be RAGE (Receptor for Advanced End-Glycation products). Studies showed that in normoglycemia peripheral RAGE contributes to effective axonal regenera-tion prompted by acute crush injury while in diabetes, intact peripheral nerve studies showed that RAGE signaling is a deteriorating factor. In the present study we hypothesized that in diabetes, RAGE contributes to progressive neuropathy and that upon superimposed acute injury, RAGE-dependent neuronal dysfunction would worsen, thereby attenuating neurite outgrowth and further suppressing effective axonal regeneration and re-innervation of target tissues. To validate the hypothesis we have set non-diabetic and diabetic (streptozotocin injected) groups of transgenic RAGE defi cient (RKO) and wild type (WT) C57 BL6 mice, performed sciatic nerve injury two months after diabetes induction, 21 days post surgery performed conduction veloc-ity tests and collected nerve tissue for morphological, immunohistochemical and mRNA analysis. Results depicted on fi gure 1. The fi ndings of our study provide evidence that RAGE suppresses effective axonal regeneration in diabetic peripheral nerve after acute injury.

& 569-PGLP-1 Analogs Represent a Novel Treatment Strategy for Diabetic EncephalopathyMASAHIRO OKOUCHI, NAOTSUKA OKAYAMA, GOJI SHIGEKI, TADASHI IWAMA, TETSUYA KATSUNO, KAORU ASADA, KOHEI HATTORI, AKITOMO GOTO, YASU-TAKA KAMIYA, TSUNEO OHNO, TAKASHI JOH, Inazawa, Japan, Nagoya, Japan

Diabetic encephalopathy, characterized by cognitive impairment in pa-tients with diabetes, involves increased neuronal apoptosis and impaired adult neurogenesis in the hippocampal regions, followed by hippocampal atrophy. Recent evidence indicates that GLP-1 analogs cross the blood-brain barrier and increase neural stem/progenitor cell proliferation in the hippocampal regions. Therefore, we fi rst compared hippocampal atrophy on MRI between 400 type 2 diabetic patients and 400 control subjects, and determined the important factors affecting hippocampal atrophy. In vitro, rat pheochromocytoma cells used commonly as neuronal cell models were ex-posed to 1 mM methylglyoxal (MG) with or without 3.3 μg/ml GLP-1, and the intercellular signaling of GLP-1 protection against neuronal apoptosis were determined. Additionally, by using immunohistochemistry method, we inves-tigated whether the number of progenitor cells or young neurons in the hip-pocampal regions was infl uenced by treatment with GLP-1. Diabetic patients

had more hippocampal atrophy than control subjects. Poor glycemic control, and marked glycemic fl uctuation were associated with hippocampal atrophy, while the use of GLP-1 analogs was negatively associated with hippocampal atrophy. In vitro, GLP-1 induced phosphorylation of Akt, mTOR, p70S6K, the upregulation of GCL, the inhibition of caspase-9 and -3 activation, and pro-tected against MG-induced neuronal apoptosis. We also found an increase in progenitor cells or young neurons in the hippocampal regions after GLP-1 analogs treatment. These results suggest that poorly glycemic control or marked glycemic fl uctuation were closely associated with hippocampal atro-phy in diabetic patients. Additionally, GLP-1 analogs treatment may prevent hippocampal atrophy through protecting neuronal apoptosis and increasing neural stem/progenitor cell proliferation in the hippocampal regions, and thus represent a promising treatment modality for diabetic encephalopathy.

& 570-PEffects of Hyperglycemia on the Expression of Neprilysin (NEP) and Substance P Receptor (NK1R), and the Activity of MMP2 and MMP9 in Adult Dermal FibroblastsALLISANDRA MOWLES, GENER AUGUSTIN, FRANK W. LOGERFO, LEENA PRAD-HAN-NABZDYK, Boston, MA

Impairment in wound healing is a major complication of diabetes. Fibro-blasts play an important role in the wound healing process, especially in the remodeling phase. Previously we have shown that hyperglycemia impairs Adult Dermal Fibroblast (ADF) migration and proliferation while Substance P (SP) mitigates these effects. In vitro ADF cultured in DMEM were treated with D-glucose (5mM, 10mM or 30mM) and co-treated with SP (100nM) for 24 hours or 5 days. NEP and NK1R protein expression was assessed by west-ern blot. MMP2 and MMP9 activity was assessed via zymography. Data are expressed as mean fold change±SEM compared to 5mM glucose (N=3-6). Compared to 5mM glucose, 30mM glucose signifi cantly increased NEP pro-tein expression at 5 days (1.0±0 vs. 2.08±1.27). There were no differences in NK1R protein expression between different treatment groups; however, a trend towards up-regulation is observed for ADF treated with 10mM glucose when compared with other treatments. Compared to 5mM glucose, 30mM glucose signifi cantly decreased MMP2 (1.0±0 vs. 0.64±0.036) and MMP9 (1.0±0 vs. 0.602±0.079) activity at 5 days and this was not restored by SP co-treatment. In conclusion, these data suggest that hyperglycemia increases the expression of NEP, the enzyme that breaks down SP whereas it does not alter the expression of SP receptor NK1R in AFB. Hyperglycemia also alters the activity of MMP2 and MMP9, which cannot be restored by addition of SP suggesting that the previously observed increase in AFB migration and proliferation upon co-treatment with SP is most likely not through MMP2 or MMP9.

Supported by: NIH (5R01NS066205-02)

& 571-PInhibition of Chymase Protects Against Brain Oxidative Stress and Cognitive Dysfunction in Diabetic db/db MiceEIICHI HIRATA, NORIYUKI SONODA, YOHEI MINAMI, TAKAHIRO KATO, YOSHI-HIRO SEKI, AKIRA MONJI, RYOICHI TAKAYANAGI, TOYOSHI INOGUCHI, Fukuoka, Japan

Accumulating evidence suggests that the tissue renin-angiotensin system may be involved in the progression of diabetic complications. Chymase is a potent local angiotensin II-forming enzyme. We have reported that a causal role of chymase in diabetic nephropathy and the therapeutic effectiveness of chymase inhibition (Am J Physiol 2010), and that NAD(P)H oxidase activa-tion , consequently elevation of oxidative stress and infl ammatory cascade might be causative pathogenic mechanisms in diabetes-associated cognitive impairment in middle-aged diabetic rodent model (ADA 2011). Therefore, we speculated that chymase-dependent ANG II production may play an impor-tant role in the enhancement of oxidative stress in diabetic brain, thus con-tributing to the development of diabetes-associated cognitive impairment. In the present study, we report the therapeutic effectiveness of chymase-specifi c inhibitior(TEI-F00806) for cognitive function and its association with oxidative stress using diabetic db/db mouse model.Overproduction of ROS assessed by dihydroethidium staining and elevation of lipid peroxidation products in 20-week aged db/db mice brain were partially reduced by TEI-F00806. Furthermore, Upregulation of NAD(P)H oxidase components includ-ing Nox 2 and pro-infl ammatory cytokines in db/db mice was supressed by TEI-F00806, whereas downregulation of anti-infl ammatory cytokines was corrected by TEI-F00806. However,there was no deposition of amyloid beta in either db/db and db/+ or the chymase inhibitor administrated db/db mice brain. In correlation with these fi ndings, impairment of working memory in db/db mice assessed by radial arm water maze test was partially restored

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by TEI-F00806.This study showed for the fi rst time that chymase inhibition may protect against oxidative stress, and cognitive dysfunction in diabetes. These fi ndings suggest that chymase offers a new therapeutic target for diabetes-associated cognitive impairment.

572-PIntermittent High Glucose, More than Constant High Glucose, En-hances Apoptosis Related to Oxidative Stress in Schwann CellsLIAN Q. SUN, YING Y. CHEN, XUE WANG, BING XUE, XIAO J. LI, JU M. LU, Beijing, China

To explore the effect of intermittent high glucose (IHG) on Schwann cells (SCs) apoptosis induced by oxidative stress, Rat SCs were primarily cultured for 2 days in media containing different glucose concentrations: 5.6mM, 50mM, or 8h alternating 5.6 mM or 50 mM glucose and osmotic controls. Apoptosis was studied by Annexin-V and TUNEL analysis. Intracellular ROS generation and mitochondrial transmembrane potential were detected by fl ow cytometry analysis. The concentration of 8-OHdG was detected as oxidative stress marker. Real-time PCR was performed to analyze the ex-pression levels of Bax and BcL-2. Western blot were performed to analyze the expression levels of some important transcription factors. Our data indi-cated that IHG induced high levels of ROS production and mitochondrial dys-function, which triggered high frequency of SCs apoptosis. IHG up-regulated Bax expression and release of cytochrome c and AIF nuclear translocation, but down-regulated the expression of BcL-2. In addition, treatment with IHG induced activation of caspase-3 and -9 and mitigated the cleavage of PARP. More importantly, the cytotoxic effect of IHG was signifi cantly more potent than that of constant high glucose (HG) and the cytotoxicy is not related to the changes of osmolarity. Our results suggested that IHG induced SCs apoptosis in both caspase-dependent and caspase-independent pathways by activating the mitochondrial pathway related to oxidative stress. These fi ndings suggest that variability in glycemic control could be more deleteri-ous to SCs than a constant high concentration of glucose.

Supported by: Postdoctoral Science Foundation of China (No. 20090461435)

573-PCardiovascular Autonomic Neuropathy in the SEARCH for Diabetes in Youth Cohort: A Pilot StudyRODICA POP-BUSUI, CATHERINE L. MARTIN, RONNY A. BELL, ABIGAIL LAUER, JASMIN DIVERS, DANA DABELEA, DAVID J. PETTITT, SHARON SAYDAH, BAR-BARA LINDER, CATHERINE PIHOKER, DEBRA A. STANDIFORD, BEATRIZ L. RO-DRIGUEZ, EVA L. FELDMAN, FOR THE SEARCH FOR DIABETES IN YOUTH STUDY GROUP, Ann Arbor, MI, Winston-Salem, NC, Aurora, CO, Santa Barbara, CA, Atlanta, GA, Bethesda, MD, Seattle, WA, Cincinnati, OH, Honolulu, HI

Data regarding cardiovascular autonomic neuropathy (CAN) in young people with diabetes are limited. Several measures of CAN [resting heart rate (HR), R-R variability at rest and during deep breathing] were assessed in a standardized manner (ANSAR system, ANSAR Inc. PA) in a pilot study that included 222 subjects from the SEARCH for Diabetes in Youth study (a large U.S. population-based study in people diagnosed with diabetes be-fore age 20). Analyses used the Wilcoxon two-sample test for continuous outcomes and Chi-square test for categorical outcomes.The mean age of the pilot study cohort was 17.4 ± 4.9 years; 78% with type 1 diabetes (T1D) (age 16.1 ± 4.4 years, duration 6.2 ± 0.9 years, 53% female), and 22% with type 2 diabetes (T2D) (age 22.1 ± 3.1 years, duration 8.0 ± 1.9 years, 59% female). Resting HR and ratio of sympathetic/parasympathetic activity were higher among T2D vs. T1D participants (77.1 vs. 72.0, p=0.004 and 1.6 vs. 1.1, p=0.003). E/I ratio, a measure of R-R variability during deep breathing, was lower in T2D participants (1.3 vs. 1.4, p=0.001). Higher resting HR was

negatively associated with age (p=0.003) and with higher recent A1C value, which was obtained within 3 months of CAN testing on a subset of 146 sub-jects (p = 0.023). Lower E/I ratios were negatively associated with older age (p = 0.029) and diabetes type (p = 0.028).In this cross-sectional pilot study of young people with diabetes measures of CAN were associated with older age and with diabetes type. The lack of normal values for the CAN measures in an age-matched healthy population limits our ability to defi ne the preva-lence of CAN among SEARCH participants. These data suggest vulnerability in this cohort of young people with diabetes for CAN-associated compli-cations, placing them at higher cardiovascular risk. Studies are ongoing to better defi ne the prevalence, risks and outcomes associated with CAN in SEARCH participants.


574-PLower Heart Rate Variability is Associated With Increased Arterial Stiffness in Youth With Type 1 Diabetes: The SEARCH CVD StudyMAMTA JAISWAL, Aurora, CO

Lower heart rate variability is associated with increased arterial stiffness in youth with type 1 diabetes: the SEARCH CVD study.Lower heart vari-ability (HRV), a marker of subclinical cardiac autonomic neuropathy (CAN), and increased arterial stiffness (AS), a marker of subclinical cardiovas-cular disease, have been shown to develop and progress silently in youth with type 1 diabetes (T1D), which might predispose them prematurely to cardiovascular events. The SEARCH CVD study assessed the associations between measures of HRV and measures of AS in youth with and without T1D. Participants were 402 youth with T1D (mean age 19 years, duration 9.8 years, 87% non-Hispanic white (NHW) and 206 youth without T1D (mean age 19 years, 75% NHW). SphygmoCor device was used to measure HRV using SDNN (Standard deviation of NN interval), HF (high frequency) and LF (low frequency) power variables, AS measured by pulse wave velocity (PWV), and augmentation index adjusted to a heart rate of 75/min (AIx75). Brachial dis-tensibility (BrachD), was measured on a Dynapulse instrument. Multivariate linear regression analyses explored the associations between markers of reduced HRV (lower SDNN, lower HF and higher LF) and increased AS (higher PWV and AIx75 and lower BrachD), adjusting for age, sex and race/ethnic-ity, separately among youth with and without T1D. Among youth with T1D, lower SDNN was associated with higher PVW in the carotid-femoral seg-ment (p=0.0009), higher AIx75 (p =0.01) and lower BrachD (p= 0.03). Lower HF and higher LF were also associated with lower BrachD among youth with T1D (p=0.005 for each). No signifi cant associations were observed among youth without T1D. The results were similar after additional adjustment for lipid levels (LDL,HDL, triglycerides), systolic and diastolic blood pressure and BMI. Our results suggest a parallel evolution of subclinical CAN and AS in youth with T1D and point toward a common mediator, likely represented by hyperglycemia.

575-PImproved Diagnostic Accuracy of the LDIfl are in Diagnosing Clini-cal Neuropathy Using Age-Related Centile ChartsPRASHANTH VAS, SANJEEV SHARMA, GERRY RAYMAN, Ipswich, United King-dom

The Laser Doppler Imaging fl are (LDIfl are) is a non-invasive test of small fi bre function assessed on the dorsum of the foot involving skin heating and measurement of the axon-mediated fl are area using a laser Doppler imager. We have previously used ROC analysis to determine sensitivity and specifi ci-ty in detecting neuropathy [based on the Neuropathy Disability Score(NDS)]. However, having now established normative reference ranges we believe these will further improve the diagnostic accuracy. This study therefore compares the sensitivities and specifi cities derived by these two methods.The normative centiles were determined by assessing the LDIfl are in 94 (m=45;f=49) healthy controls (HC) between the ages of (20-80 years). LDI-fl ares were also determined in 66 diabetes individuals with (n=31, NDS>3) and without clinical neuropathy (n=35, NDS<3). Standard ROC analysis was used to generate the sensitivity and specifi city. Additionally, sensitivity and specifi city based on the normative centiles, were determined by defi ning as abnormal any LDIfl are areas below the 5th centile for ageAmong HC, there was an age dependant decrease in small fi bre function (r=-0.42, p<0.0001). There was no signifi cant correlation with gender, height, and weight. The ROC analysis suggested an optimal threshold cut off of <3.7 c m2. This gave a sensitivity of 76%, specifi city of 84%, positive predictive value (PPV) of 77%, and negative predictive value (NPV) of 87%. Using 5th centile cut-offs, the sensitivity was similar at 77% but there was improved specifi city (90%), PPV (80%) and NPV (91.1%).Although both analytic methods demonstrate

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excellent sensitivity and specifi city for the LDIfl are method to detect clini-cal neuropathy, the application of centile derived normative values further enhances the diagnostic accuracy. We would recommend the use of centile charts in future studies of small fi bre function.

576-PInterplay of Sorbitol Pathway of Glucose Metabolism, 12/15-Lipox-ygenase, and Mitogen-Activated Protein Kinases in the Pathogen-esis of Diabetic Peripheral NeuropathyROMAN STAVNIICHUK, HANNA SHEVALYE, HIROKO HIROOKA, JERRY L. NA-DLER, IRINA G. OBROSOVA, Baton Rouge, LA, Mie, Japan, Norfolk, VA

The interactions among multiple pathogenetic mechanisms of diabetic pe-ripheral neuropathy largely remain unexplored. Increased activity of aldose reductase (AR), the fi rst enzyme of the sorbitol pathway, leads to accumula-tion of cytosolic Ca++, essentially required for 12/15-lipoxygenase (12/15-LO) activation. The latter, in turn, causes oxidative-nitrosative stress, an impor-tant trigger of MAPK phosphorylation. We therefore evaluated the interplay of AR, 12/15-LO, and MAPKs in diabetic peripheral neuropathy. In study 1, male control and streptozotocin (STZ)-diabetic mice were maintained with or without the AR inhibitor fi darestat, 16 mg kg-1d-1, for 12 wks. In study 2, male control and STZ-diabetic wild-type (C57Bl6/J) and 12/15-LO-defi cient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofl uorometric assays) as well as 12(S) hydroxyeicosatet-raenoic concentration (ELISA), a measure of 12/15-LO activity, in the sciatic nerve and spinal cord. 12/15-LO expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fi darestat-treated diabetic mice. 12/15-LO gene defi ciency prevented diabetes-associated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly acti-vated in diabetic wild-type and 12/15-LO-/- mice. These fi ndings identify the nature and tissue specifi city of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its man-agement.

Supported by: NIH (DK074517, DK077141, DK081147)

577-PInsulin Treatments Ameliorate Tau Hyperphosphorylation in a Rat Model of Type 2 DiabetesYANG YAN, YUAN GANG, Wuhan, China

Brain insulin could regulate brain glucose metabolism, and accelerate the memory and cognition.The lack of brain insulin is one of the mechanisms of Alzheimer’s disease (AD), the most common form of dementia.It has been proved that type 2 diabetes (T2D), the characteristics of which is insulin resistance/hyperinsulinmia, has the potiential to increase the risk of AD. However, the underlying mechanism is obscured. We evaluted the effects of abnormal brain insulin on pathophysiological changes in the brain with streptozocin (STZ) induced T2D rat model with a high protein, high glucose and high fat diet. Our data showed that brain insulin in T2D rats were signifi -cantly decreased compared with control group(CTL)(1.2±0.2 vs 2.9±0.4,uIU/ml, p<0.01). Hyperphosphorylated tau protein, which represented of AD-like changes was presented in the brains of T2D rats. And glycogen synthase kinase-3 (GSK-3 ) was activated in T2D brain also. Furthermore we found that administration of insulin in abdomen decreased the level of blood glu-cose, but could not change the hyperphosphorylation of tau or insulin defi -cency in brain. In additional, intranasal insulin treatment could reduce AD-like changes in brain, and light the activities of GSK-3 . Taken together, our present data indicated that insulin is defi ency in type 2 diabetic brain, which is a key factor involved Alzheimer’s disease, and intranasal insulin treatment could reverse the AD-like changes in T2D.

Supported by: National Natural Science Foundation of China (81100582)

578-PAutonomic Nervous System and Cystatin C Assessment in Type 1 Diabetes MellitusTRIANTAFILLOS DIDANGELOS, EFSTRATIOS MORALIDIS, FOTIOS ILIADIS, ANES-TIS ZANTIDIS, CHARALAMPOS MARGARITIDIS, ANNA GOTZAMANI-PSARRAK-OU, APOSTOLOS HATZITOLIOS, Thessaloniki, Greece

Aim: This study investigates if there is an association between Cystatin-C measurement, as index of renal function, and autonomic function assess-ment in patients with type 1 diabetes mellitus (T1DM).Patients-methods:

Forty nine patients (21 female), aged 36±10 years (range 19-62), with a dura-tion of T1DM 19±6 years (range 7-31) and receiving only insulin were enrolled prospectively. Participants were evaluated for autonomic dysfunction with Autonomic Function Tests (AFT) [mean circular resultant (MCR),Valsalva maneuver (Vals), postural index (PI), orthostatic hypotension (OH)] assess-ment and cardiac 123I metaiodobenzylguanidine (MIBG) imaging [with the ratio of the heart to upper mediastinum count density (H/M) at 4 hours post-injection calculated].Within one month patients underwent glomerular fi ltration rate measurement with 51Cr-EDTA (expressed in ml/min/1.73m2), Cystatin-C (CC, mg/l) and Serum Creatinine (SC, mg/dl) measurements as indices of renal function.Results: The values of examined variables were as follows [mean±1SD (range)]: GFR 98±17 (61-137), MCR 40±29 (5-108), Vals 1.55±0.30 (1.11-2.29), PI 1.33±0.17 (1.03-1.81), OH 5±9 (0-30), CC 0.78±0.14 (0.49-1.06), SC 0.74±0.16 (0.51-1.12) and H/M 1.65±0.20 (1.30-2.34).GFR cor-related signifi cantly with age (r=-0.377, p=0.011), duration of diabetes (r=-0.514, p=0.000), Vals (r=0.377, p=0.010), OH (r=-0.448, p=0.002), number of abnormal AFT (r=-0.366, p=0.012), CC (r=-0.362, p= 0.017) and SC (r=0.465, p=0.001). MCR, PI and the H/M showed no signifi cant correlation with GFR. CC showed signifi cant correlation with GFR and SC but not with AFT and MIBG.Conclusions: In present study in T1DM patients CC is associated with GFR and serum creatinine but not with AFT (which predominantly address the parasympathetic system) and MIBG measurements (which refl ect sym-pathetic dysfunction).

579-PSeverity of Retinopathy, Nephropathy and Peripheral Neuropathy Correlates With Autonomic Dysfunction in Type 1 Diabetes: The DAN-StudyJESPER FLEISCHER, SIMON L. CICHOSZ, KLAUS S. JENSEN, POUL ERIK JAKOB-SEN, KNUD YDERSTRAEDE, ELISABETH GULICHSEN, PERNILLE HOEYEM, ESBEN LAUGESEN, TROELS K. HANSEN, HANS NYGAARD, EBBE ELDRUP, HANS HENRIK LERVANG, LISE TARNOW, NIELS EJSKJAER, Aarhus, Denmark, Copenhagen, Den-mark, Aalborg, Denmark, Odense, Denmark, Herlev, Denmark, Gentofte, Denmark

OBJECTIVE: Clinical correlates and predictors of diabetic cardiovascular autonomic neuropathy (CAN) include the presence of retinopathy, nephropa-thy and peripheral neuropathy. The objective of this study was to elucidate the association of tests for subclinical CAN with the presence of these com-plications.RESEARCH DESIGN AND METHODS: We compared a selected co-hort consisting of 290 type 1 diabetes patients from the DAN-study (Danish multi-center study focusing on CAN) and 89 sex- and age-matched healthy volunteers. The presence of CAN was quantifi ed by measuring heart rate variability (HRV) in time and frequency domains during cardiovascular re-fl ex tests (valsalva ratio [VT], response to standing [RT] and deep breathing [E:I]) and during 5 minutes of supine resting. In order to describe possible associations between severity of complications and measures of autonomic dysfunction, multivariate analysis was performed.RESULTS: In all tested pa-rameters, HRV analysis revealed signifi cant differences (P<0.05) between the control and the type 1 diabetes group. After adjusting for diabetes duration, sex, age, pulse pressure and heart rate, autonomic dysfunction remained signifi cantly correlated with severity of retinopathy, nephropathy and peripheral neuropathy in individuals with type 1 diabetes patients. For retinopathy, subgroup analysis showed that only spectral analysis of active tests (response to standing and deep breathing) were signifi cant different from all stages of P<0.05). CONCLUSIONS: Our results support the use of a combination of different measures and tests for early detection of auto-nomic dysfunction. This study corroborates that the severity of retinopathy, nephropathy and peripheral neuropathy correlates with autonomic dysfunc-tion.

580-PCorrelation between Small Fibre Neuropathy Assessed by the LDIfl are Technique and the SUDOSCAN® in Type-1 DiabetesSANJEEV SHARMA, PRASHANTH R. VAS, GERRY RAYMAN, Ipswich, United Kingdom

Assessing small fi bre neuropathy in a busy clinical setting is a challenge. The SUDOSCAN® (Impeto Medical, France), a new device for quickly assess-ing sudomotor function may have such applicability. This study assesses the SUDOSCAN® in patients with type-1 diabetes (T1DM), comparing its results with those of the Laser Doppler Imaging (LDI) fl are technique -a non-invasive method shown to be sensitive and specifi c for assessing C-fi bre function.39 subjects with T1DM [mean age 42.3 (SD=11.3)], 23 with (MV+) and 16 with-out (MV-) microangiopathy, and 42 healthy controls [HC, mean age 47.1 (SD=15.9)] were studied. The LDIfl are involves heating dorsal foot skin and measuring the axon-mediated fl are area with a laser Doppler imager. The

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EZSCAN® measures sudomotor function by assessing electrochemical skin conductance (ESC) utilizing reverse iontophoresis and chronoamperometry in the hands, feet and forehead.LDIfl ares were reduced in T1DM compared to HC (6.5 ± 3.5 v 9.6 ± 1.9 cm2; p<0.001). ESC in the feet was also reduced in T1DM (70.3 ±1 9.6 v 77.4 ± 7.7 μS; p=0.029). Furthermore, MV+ compared with MV- subjects had lower LDIfl ares (4.3 ± 2.5 v 9.8 ± 1.98; p<0.0001) as well as ESC in the feet (64.4 ± 23.5 v 77.1 ± 8.9; p=0.046). In contrast, neither LDIfl are nor ESC responses differed between in MV- and HC groups in keep-ing with our previous observation that C-fi bre function is not impaired in the absence of microvascular disease. Across all subjects the methods correlat-ed (r=0.2; p=0.02), as well as within the T1DM group (r=0.364; p=0.03). There was no correlation within HC (r=-0.21; p=0.2).In conclusion, SUDOSCAN®

results correlate with those of the LDIfl are technique; an established marker of small fi bre function. Furthermore, as with the LDIfl are technique, abnor-mal small fi bre function was found in MV+ but not in MV- patients. Because it is simple and quick to perform, the SUDOSCAN® may serve as a useful tool in assessing small fi bre neuropathy in the busy clinic setting.

581-PSuppression of Rho/Rho-Kinase Pathway in Diabetic Rats Provides a Therapeutic Effect on Diabetic NeuropathyYASUSHI KANAZAWA, JUNKO TAKAHASHI-FUJIGASAKI, NAOKO TAKA-BAYASHI, KUMIKO ISHIBASHI, SHO ISHIZAWA, KEIICHIRO MATOBA, DAIJI KAWANAMI, TAMOTSU YOKOTA, KAZUNORI UTSUNOMIYA, Tokyo, Japan

Rho-kinase is a serine-threonine kinase, a downstream effector of a small GTPase protein Rho. Rho/Rho-kinase pathway participates in various bio-logical events, such as cell adhesion. We previously found that excessive enhancement of Rho and Rho-kinase activity related to pathogenesis of nerve defi cits of the diabetic rats. In the animal models, when a selective Rho-kinase inhibitor, fasudil, is administrated from onset of diabetes, fasudil inhibits development of both motor nerve conduction velocity (MNCV) and sensory defi cit. In this study, we aim to examine whether fasudil could be effective, when it is started after development of the diabetic nerve defi -cits.Sprague-Dawley rats were rendered diabetic with streptozotocin, and divided in the 3 groups, normal control, diabetic, and diabetic rats treated with fasudil. Fasudil administration started after 4 weeks of induction of diabetes, and the rats were maintained totally for 8 weeks. To evaluate the sensory defi cit, the latency of the hindpaw withdrawal evoked by ther-mal stimulation was determined weekly. After 8 weeks, MNCV of sciatic nerve was examined, and RhoA and Rho-kinase activities were estimated biochemically using resected sciatic nerves.RhoA and Rho-kinase activities were enhanced signifi cantly in the diabetic rats and were attenuated by ad-ministration of fasudil. Both MNCV and sensory defi cits of the diabetic rats were improved after the 4 weeks administration of fasudil. In the previous study, we found that enhanced Rho-kinase activity signifi cantly altered Rho-kinase related adhesion structures in Schwann cells, and the alteration was restored by fasudil. This might be one of the underlining mechanisms for the effectiveness of fasudil. This study showed that fasudil was effective, even when it was started after development of the nerve defi cits. This also indi-cates that disturbance of the Rho-kinase related adhesion could be restored by fasudil, if it is once induced by diabetic condition.

582-PGlycemic State has Real-time Impact on Beading Size and Fre-quency while Requiring Several Years to Infl uence Nerve Fibers by Corneal Confocal Microscopy (CCM)FUKASHI ISHIBASHI, Hiroshima, Japan

Hyperglycemia induces swelling and then, degeneration of mitochondria (Mt) by over-produced reactive oxygen species, initiating neuron death. Beading in CCM represents Mt. It remains to be settled what happens fi rst upon hyperglycemia or glycemic control among corneal nerve (CN) struc-tures_fi ber density (CNFD) and length (CNFL), branch density (CNBD) and length (CNBL), tortuosity grade (TG), and beading frequency (BF) and size (BS)_as identifi ed by CCM. BS was assessable by enlarging and smoothing of beads using S-Spline Max algorithm. We clarifi ed impact of prior hyperg-lycemia on BS and BF in 38 type 1 diabetic patients (T1DM, HbA1c;7.3±0.1%, follow-up period;11.5±1.2yrs) compared with 38 age-matched controls, and impact of strict glycemic control (HbA1c;10.3±0.4 6.9±0.1% during 11.8±0.4 M) on BS and BF in 32 type 2 diabetic patients (T2DM). Compared with controls, T1DM had lower BF (22.4±0.73 vs 30.4±0.14/0.1 mm, p<0.001), larger BS (10.7±0.12 vs 9.7±0.15 μm2, p<0.001), lower skewness of BS distri-bution (0.30±0.032 vs 0.61±0.052, p<0.001), reduced CNFD (p<0.001), CNFL (p<0.04), and increased TG (p<0.001). HbA1c at CCM, 1-3 M, or mean HbA1c 1 yr before CCM was an independent predictor for BF (p=0.0442-0.005) and BS

(p=0.049-0.004), whereas CNFD (p=0.02-0.003) and CNFL (p=0.042-0.002) inversely correlated with mean annual HbA1c 7-10 yrs before CCM. Glycemic control in T2DM increased BF (18.6±0.51 20.7±0.34, p<0.001) and CNBD (p<0.01) and decreased BS (9.6±0.13 8.4±0.09, p<0.001) and TG (p<0.001) without infl uencing CNFD and CNFL. BS decreased correlated (p=0.012-0.031) with HbA1c 1-3 M before last CCM, and BF increased inversely cor-related (p=0.012-0.035) with HbA1c 8-10 M before last CCM. In conclusion, change in glycemic state was quickly refl ected in BS and BF and preceded changes in CNFD and CNFL, suggesting that changes in bead (Mt) play a pivotal role in subsequent changes in nerve fi bers.

583-PSUDOSCAN: A Simple, Rapid, and Objective Method of Diagnosing Diabetic Autonomic NeuropathySOLOMON TESFAYE, TOM CASH, ADITHYA SANKAR, JOSIE REEVES, JENNI-FER DAVIES, GANESH RAO, IAIN WILKINSON, DINESH SELVARAJAH, Sheffi eld, United Kingdom

Diabetic cardiac autonomic neuropathy (CAN) is a serious complication of diabetes and carries up to a fi ve-fold increased risk of mortality. Current methods of detecting CAN are based on a battery of cardiovascular refl ex tests (CARTS) are impractical for widespread use in busy clinical practice. SUDOSCAN is a new and rapid method of assessing sudomotor function by measuring electrochemical skin conductance (ESC) through reverse ionto-phoresis. The aim of the present study was to evaluate if SUDOSCAN can reliably diagnose CAN.Methods: 41 subjects with type 1 diabetes and 13 healthy volunteers (HV) underwent detailed assessments including clini-cal, neurophysiological (NISLL+7) and 5 CARTs (O’Brien protocol). Based on CARTs subjects were divided into CAN [3 abnormal CART results], Subclini-cal CAN [1-2 abnormal results] and No-CAN [0 abnormal results]. All subjects underwent the SUDOSCAN test. Participants placed their hands and feet on electrodes, and an incremental low direct current (<4V) was applied to the anode for 2min. ESC (μSiemens) as a measure of sudomotor function was obtained from the voltage and current and an autonomic risk score was cal-culated.Results: The results show that SUDOSCAN can detect established CAN having corrected for age and BMI.Conclusion: SUDOSCAN a simple, rapid (<5min) and objective method of screening that doesn’t require special patient preparation or medical personnel training may serve as screening test for CAN. It could help adhere to ADA’s recommendation of annual AFT for all diabetic patients which is currently not fulfi lled as current tests are cumbersome and time consuming.

HV NoCAN

SubclinicalCAN

CAN p-value p-valueadjusted

Age 41±18 43±13 55±16 50±14 0.068 —BMI (Kg/M2) 23.9±3.7 28.4±5.9 29.6±5.7 30.3±6.0 0.018 —Systolic BP 132±18 140±11 140±14 139±17 0.503 0.409ESC hands (μS) 62±15 59±12 62±18 61±15 0.893 0.886ESC feet (μS) 78±6 76±6 78±9 60±26 0.003 0.004ESC feet:hands ratio 1.3±0.3 1.4±0.3 1.3±0.3 1.0±0.4 0.045 0.044Autonomic risk score (%) 22±14 30±15 37±18 39±13 0.022 <0.001

Supported by: Impeto Medical

584-PThe Functions of Glucose-Responsive ATP-Sensitive K (KATP) Chan-nels in Peripheral Nervous SystemsTATSUHITO HIMENO, HIDEKI KAMIYA, YUSUKE SEINO, KEIKO NARUSE, TETSUJI OKAWA, JIRO KATO, MASAKI KONDO, ATSUSHI FUJIYA, EITA UENISHI, SHIN TSUNEKAWA, YOJI HAMADA, YUTAKA OISO, SUSUMU SEINO, JIRO NAKA-MURA, Nagoya, Japan, Nagakute, Japan, Kobe, Japan

Background:KATP channels in pancreatic -cells are metabolic sensors that determine glucose-responsive membrane excitability in the regulation of insulin secretion. The KATP channel is an octameric protein consisting of two subunits: the pore-forming inward rectifi er K+ channel member Kir6.1 or Kir6.2, and the sulfonylurea (SU) receptor SUR1 or SUR2. In central nervous systems, glucose responsive neurons share the same molecular composi-tion Kir6.2 and SUR1 with -cells. Here we investigated functions of KATPchannels and potential physiological effects of SU agents in peripheral ner-vous systems (PNS).Research design and methods:The expressions of Kir6.1, Kir6.2 and SURs in PNS were determined with RT-PCR, immunohistochem-istry (IHC) and western blotting (WB). Kir6.2-/- mice (KO) and wild type mice (WT) were used in this study. In 4- and 24-week old mice, current percep-tion thresholds (CPTs), thermal plantar test (TPT), and motor and sensory

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nerve conduction velocities (MNCVs and SNCVs, respectively) were evalu-ated. Dorsal root ganglion (DRG) neurons derived from WT were cultured with or without glibenclamide (GB) or tolbutamide (TB) for 24hs, and were immunostained with an anti-neurofi lament M antibody to evaluate neurite outgrowth.Results:RT-PCR, IHC and WB revealed the expression of Kir6.2 in PNS. The expression of SUR2 in PNS was confi rmed with RT-PCR. CPTs in KO were signifi cantly increased compared with those in WT, indicating hypoal-gesia. In TPT, paw withdrawal latencies in KO were delayed compared with those in WT (WT: 6.1 ± 1.3 s, KO: 11.1 ± 5.3). SNCVs in KO were signifi cantly delayed (WT: 34.8 ± 9.2 m/s, KO: 25.0 ± 2.6). The neurite outgrowth of DRG neurons was remarkably reduced in the presence of GB (303.6 ± 68.2 μm) or TB (403.1 ± 46.9) compared with that in the absence of these compounds (698.2 ± 91.1).Conclusions:These results suggest that KATP channels could have important physiological roles in the functions of PNS.

585-PDiabetic Autonomic Neuropathy and Cutaneous Circulation in Sub-jects With Type 2 Diabetes MellitusIOANNA ELEFTHERIADOU, PINELOPI GRIGOROPOULOU, STAVROS LIATIS, ALEX-ANDER KOKKINOS, VASILIKI ARGIANA, DESPOINA PERREA, NICHOLAS KATSIL-AMBROS, NICHOLAS TENTOLOURIS, Athens, Greece

Transcutaneous oxygen tension (TcPO2) maps the oxygen supply available for the skin tissue and is an indicator of the microvascular function. Patients with type 2 diabetes (T2DM) have lower TcPO2 values in comparison with subjects without diabetes. Previous data showed that diabetic individuals with peripheral arterial disease (PAD) and peripheral neuropathy (PN) have lower TcPO2 values in comparison with patients without these complica-tions. In the present study we examined the effect of cardiac autonomic neuropathy (CAN) on cutaneous circulation assessed by determination of TcPO2 in patients with T2DM. A total of 100 patients with T2DM were recruited (mean age 66.5±8.7 years, duration of diabetes 14.0±10.8 years). TcPO2 was measured on the dorsum of the feet between the fi rst and second metatarsal heads. Diagnosis of CAN was based on the battery of the four autonomic function tests. Diagnosis of PAD was based on the absence of triphasic waveform in the posterior tibial artery, while of PN on neuropa-thy disability score and vibration perception threshold. A total of 20 sub-jects had CAN. Subjects with CAN had more often PAD and PN and lower TcPO2 levels in comparison with those without CAN (43.6±15.0 vs. 49.1±11.1 mmHg, p=0.033). Univariate regression analysis showed that diabetes dura-tion, HbA1c, presence of PAD, presence of PN and presence and severity of CAN were signifi cantly and negatively associated with TcPO2. Multivariate analysis demonstrated that after adjustment for gender, age, duration of diabetes and HbA1c, severity of CAN and presence of PAD was signifi cantly and negatively associated with TcPO2. The same analysis showed that after controlling for gender, age, diabetes duration and HbA1c, severity of CAN and presence of PN were signifi cantly associated with lower TcPO2 levels. In conclusion, presence and severity of CAN is associated with reduction in cutaneous perfusion irrespective of the presence of PAD or PN.

586-PThe Effect of Aliskiren on Cardiovascular Autonomic FunctionRAELENE E. MASER, M. JAMES LENHARD, PAUL KOLM, Newark, DE

The renin-angiotensin-aldosterone system (RAAS) and autonomic nervous system (ANS) regulate the cardiovascular system. Dysfunction of the ANS results in increased risk of mortality. Autonomic neuropathy is a multifacto-rial pathogenic process (e.g., metabolic insult; neurovascular insuffi ciency). Pharmacological agents directed at components of the pathogenic process have shown varied results. Aliskiren, a direct renin inhibitor, may offer an ad-vantage over other mediators of the RAAS as renin catalyzes the rate-limiting step of the RAAS. We investigated the effect of aliskiren in a double-blind, placebo-controlled study. Nerve function was assessed by refl ex tests (e.g., RR-variation during deep breathing (assesses parasympathetic function)) and power spectral analysis (PSA). RR-variation was measured by vector analysis (i.e., mean circular resultant (MCR) and expiration/inspiration (E/I) ratio). Sub-jects (n=60, 55% men, 48% type 2, aged 51±12 (mean±SD) yrs, duration16±12 yrs) were randomly assigned to 300mg/day aliskiren (n=30) or placebo (n=30) for 6-wks. Measures of ANS were analyzed by linear mixed effect models comparing change in outcome from baseline to follow-up for aliskiren vs. pla-cebo. There was a signifi cant interaction (aliskiren x visit) for MCR (p<0.003) and E/I ratio (p<0.003) indicating improvement in MCR and E/I ratio for those on aliskiren. Table 1 shows the mean±SD for MCR and E/I ratio at baseline and follow-up for aliskiren vs. placebo. Measures of PSA, however, did not differ for baseline vs. follow-up in either group. Those on aliskiren did show improve-ment in some measures of paraysmpathetic nerve function.

Table 1. Tests of RR-variation During Deep Breathing for Aliskiren vs. PlaceboBaseline Follow-up p-value

MCR for those on Aliskiren 41.8±19.7 50.8±26.1 <0.001MCR for those on Placebo 38.2±23.6 37.5±24.1 NSE/I ratio for those on Aliskiren 1.22±0.12 1.28±0.15 =0.001E/I ratio for those on Placebo 1.21±0.14 1.20±0.14 NS

Supported by: Delaware INBRE, NIH Grant 2 P20 RR016472-11 from the NCRR

587-PCardiovascular Autonomic Neuropathy in Type 1 and Type 2 Diabe-tes and Associations With Albuminuria, Retinopathy, Peripheral Neuropathy, Pulse Pressure and Obesity: The DAN-StudyJESPER FLEISCHER, KNUD YDERSTRAEDE, ELISABETH GULICHSEN, POUL ERIK JAKOBSEN, HANS HENRIK LERVANG, EBBE ELDRUP, HANS NYGAARD, LISE TAR-NOW, NIELS EJSKJAER, Aarhus, Denmark, Odense, Denmark, Gentofte, Denmark, Aalborg, Denmark, Herlev, Denmark

OBJECTIVE: To identify the presence of subclinical cardiovascular au-tonomic neuropathy (CAN) in a cohort of individuals with diabetes in four outpatient clinics in Denmark and to evaluate the association between CAN and other risk factors.RESEARCH DESIGN AND METHODS: The DAN-Study is a Danish multi-center study focusing on diabetic autonomic neuropathy. Over a period of twelve months, 382 type 1 and 271 type 2 individuals with diabetes were tested for CAN. Patients were randomly recruited and test-ed during normal visits to outpatient clinics at four Danish hospitals. The presence of CAN was quantifi ed by measuring cardiovascular refl ex tests (valsalva, response to standing and deep breathing) and analysis of heart rate variability in time and frequency domain. In order to describe possible associations, multivariate analysis with CAN as the dependent variable was performed.RESULTS: Besides heart rate above 80 (p 0.001) and age above 60 years (p<0.02) in both type 1 and type 2 patients multiple ordinal logistic regression analysis revealed that in Type 1 diabetes patients CAN was as-sociated with microalbuminuria (p=0.015), macroalbuminuria (p=0.005), and proliferative retinopathy (p=0.021). Among Type 2 diabetes patients CAN was associated with high pulse pressure (p<0.001) and BMI above 35 kg/m2 (p=0.025).CONCLUSIONS: In this cross-sectional observational study CAN is associated with proliferative retinopathy, micro- and macroalbuminuria in type 1 diabetes patients, whereas in Type 2 diabetes patients CAN is as-sociated with pulse pressure and obesity.

588-PSkin Intrinsic Fluorescence is Associated With Nerve Fiber Morpho-logy and Conduction Velocity in Persons With Diabetic NeuropathyBAQIYYAH CONWAY, MICHAEL E. MAY, ILIZA MYERS, JOHN D. MAYNARD, KAY ARTIBEE, WILLIAM J. BLOT, AMANDA PELTIER, Nashville, TN, Albuquerque, NM

Advanced glycation end products (AGEs) are thought to play a role in dia-betic neuropathy (DN) and are observed in both myelinated and unmyelinated diabetic nerve fi bers. Certain dermal collagen AGEs fl uoresce and thus skin intrinsic fl uorescence (SIF) can be quantifi ed and act as a novel marker of colla-gen AGEs. We hypothesized that alterations in intrapapillary dermal myelinat-ed nerve endings (IME) and sensory mechanoreceptos (Meissner corpuscles, MCs) may correlate with AGEs as indicated by SIF. We compared SIF with morphology data from skin biopsies in individuals with DN, (6 type 1 and 8 type 2 diabetes) and healthy controls (HC, n=9). We also compared SIF with nerve conduction studies (NCS) in DN cases, HC, and 5 non-diabetic individuals with idiopathic neuropathy (neuropathy controls, NC). Immunohistochemistry was performed on 2 and 3 mm skin punches from the lateral index fi nger and distal leg, respectively. Spearman correlations were used to compare SIF with age adjusted skin morphology data and age and height adjusted NCS data. Mean age and diabetes duration was 54.0 and 20.3 yrs, respectively, in those with DN, while mean age were 53.8 and 58.8 in HC and NC, respectively. Average MC density was 10.9 ± 8.0 MCs/ mm2 and 18.4 ± 8.0 MCs/ mm2 in HC (p=0.07). Intraepidermal nerve fi ber density (IENFD) in the distal leg was 4.7 ± 4.8 fi bers/mm in DN and 12.0 ± 4.7 in HC (p=0.001). SIF was higher in DN compared to HC (p=0.03) but not compared to NC (p=0.31). SIF was inversely associated with MC density (r= -0.93, p=0.0009) and IME density (r= -0.93, p=0.0007) in DN only. SIF was not associated with IENFD in DN cases or healthy controls. SIF showed a strong inverse relationship with peroneal motor amplitude (r=-0.74, p=0.009) in DN only. In conclusion, noninvasively measured SIF suggests an association between myelinated fi ber and mechanoreceptor loss and elevated AGEs, but not unmyelinated fi ber loss.

Supported by: NINDS K23 NS056009, Vanderbilt CTSA Grant 1 UL1 RR024975 from NCRR/NIH

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589-PRetinoic Acid Increases Nerve Growth Factor and Prevents Neu-ropathy in OLETF RatsJONG SUK PARK, MIN KYUNG KIM, JIYOON HA, YOUNGMI LEE, EUN JIN KWON, JIWOON KIM, SHIN AE KANG, CHUL WOO AHN, KYUNG RAE KIM, Seoul, Re-public of Korea

Retinoic acid is known to increase the endogenous expression of nerve growth factor (NGF) which regulates neuronal differentiation and regenera-tion. The aim of this study was to investigate the effects of all trans retinoic acid (ATRA) on diabetic neuropathy in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes.20 OLETF rats were re-ceived with an oral dose of 10 mg/kg/day ATRA, and 20 OLETF rats and 10 Long-Evans Tokushima Otsuka (LETO) rats were received with the cellulose as a vehicle once a day for 16 weeks. At the end of the treatment, glucose levels and contents of NGF in serum and sciatic nerve were measured. Neu-rometer test was conducted to examine improvements of the current stimu-lus thresholds. The neurite growth from dorsal root ganglion (DRG) cells ex-posed to NGF were assessed and mRNA of NGF and NGF receptors p75NGFR

and trkA in response to ATRA concentration in DRG cells were measured by RT-PCR and real-time PCR.Fasting glucose levels were signifi cantly higher in the ATRA-non-treated OLETF rats (178.5 ± 38.3 mg/dL) than in the ATRA-treated OLETF rats (151.0 ± 25.1 mg/dL) (P<0.01) or in the LETO rats (105.4 ± 14.4 mg/dL) (P<0.01). Contents of NGF in LETO rats were 1423.50 + 503.21 pg/mL in serum and 243.2 ± 41.9 pg/g in nerve; in ATRA-non-treated OLETF rats the values were 683.71 ± 169.89 pg/mL in serum and 150.0 ± 28.8 pg/g in nerve; and in ATRA-treated OLETF rats, 1855.17 ± 667.34 pg/mL in serum and 377.9 ± 61.5 pg/g in nerve (P<0.05). A signifi cant decrease of threshold at 2000, 250 Hz was observed in ATRA-treated OLETF rats than in ATRA-non-treated OLETF rats. Neurite outgrowth and length in DRG cells increased in NGF and ATRA dose dependent. But ATRA did not increase the expression of the NGFR, p75NGFR and trkA mRNA levels.Our results have shown that ATRA treatment increases serum and nerve contents of NGF in OLETF rats and infl uence nerve cell regeneration by promoting the synthesis of the NGF and suggest that ATRA has a potential therapeutic role for diabetic neuropathy.

590-PEvaluation of Diabetic Neuropathy Using Pupillary Light Refl ex Test in Patients With Diabetes MellitusTAKESHI KUROSE, SHUUICHI FUJIWARA, KOIN WATANABE, TAKANORI HYO, DAISUKE YABE, YUTAKA SEINO, Osaka, Japan

Since cardiovascular autonomic neuropathy has been postulated as a risk of sudden death and affects the mortality of diabetic patients, reliable and easy bedside test for evaluation of diabetic neuropathy is needed. In this study we examined the effi cacy of pupillary light refl ex test in the evaluation of diabetic neuropathy. A total of 282 (189 male, 93 female) patients were recruited in this study. The pupillary light refl ex test was performed using a portable infrared video-pupillography system (Iriscorder Dual C10641®;Hamamatsu Photonics). The measured parameters are as follows: D1, ini-tial diameter before light stimulus (mm); D2, minimum diameter after light (mm); CR, constriction ratio [CR=(D1 - D2)/D1]; VC, the maximum velocity of constriction (mm/s). Mean age was 62, mean duration of diabetes was 11.2 years, and mean HbA1c level was 8.6 %. CR was signifi cantly associated with coeffi cient of variation of R-R interval of 100 heart beat at rest (CV R-R) (p<0.01), however, CR showed no association with the frequency of orthostatic hypotension. On the other hand, VC was signifi cantly associ-ated with both of CV R-R and orthostatic hypotension. Accordingly VC may represent the autonomic neuropathy more precisely than CR. We have also evaluated vibration perception threshold using C-64 tuning folk as a marker of somatic nerve dysfunction. VC showed signifi cant correlation with vibra-tion perception threshold (p<0.01). From the regression analysis of vibration sensation and VC, VC which corresponds to the lower limit of normal sensa-tion of vibration was 3.52. The frequency of abnormal Achilles tendon refl ex in groups of less than 3.6 VC was more than the group of over or equal to 3.6 VC (p<0.01). Since the somatic nerve dysfunction appears earlier than autonomic neuropathy, cut-off point of 3.6 VC may represent the early stage of autonomic neuropathy. These results suggest that VC may be a good and easy test for the detection of early stage of diabetic neuropathy.

591-PThe Functions of Gastric Inhibitory Polypeptide Signals in Periph-eral Nervous Systems in MiceTETSUJI OKAWA, HIDEKI KAMIYA, TATSUHITO HIMENO, NORIO HARADA, JIRO KATO, KEIKO NARUSE, ATSUSHI FUJIYA, YUSUKE SEINO, AYAKO FUKAMI, SHIN TSUNEKAWA, YOSHITAKA HAYASHI, YOJI HAMADA, YUICHIRO YAMADA, NO-BUYA INAGAKI, YUTAKA SEINO, YUTAKA OISO, JIRO NAKAMURA, Nagoya, Ja-pan, Nagakute, Japan, Kyoto, Japan, Akita, Japan, Osaka, Japan

The gastric inhibitory polypeptide (GIP) is one of the incretin hormones secreted from the intestine on ingestion of glucose or nutrients to stimu-late insulin secretion. GIP exerts its action by binding to a specifi c receptor (GIPR) that belongs to the G-protein coupled receptor families. In addition to insulinotropic effects, GIP has been reported to play several roles in various tissues and organs. Here we investigated the effect of GIP signals on periph-eral nerve functions.The presence of GIPR in dorsal root ganglions (DRGs) was evaluated by immunohistochemistry (IH), westen blotting (WB) and RT-PCR. DRG neurons were isolated from wild type (WT) and GIPR knockout mice (KO), and were cultured in the presence or absence of a human recombi-nant GIP. Then the neurite outgrowth of DRG neurons was determined. In the animal model experiments, 5-month old KO and age-matched WT were used. Peripheral nerve functions were evaluated by current perception thresholds (CPTs), thermal plantar test (TPT), and motor and sensory nerve conduction velocity (MNCV and SNCV, respectively). Sciatic nerve blood fl ow (SNBF) and plantar skin blood fl ow (PSBF) were also evaluated.The expression of GIPR in DRG neurons was confi rmed by IH, WB and RT-PCR. GIP signifi cantly promoted the neurite outgrowth of DRG neurons derived from WT in a dose dependent manner. On the other hand, the neurite outgrowth of DRG neu-rons derived from KO was signifi cantly reduced compared with that from WT. KO showed hypoalgesia (TPT; WT: 9.1±0.9 s, KO: 15.8±2.1, p < 0.0001), and delayed MNCV and SNCV (MNCV; WT: 47.7±1.5 m/s, KO: 36.4±9.2, p < 0.0001, SNCV; WT: 46.2±1.4, KO: 29.0±5.2, p < 0.0001). There were no differ-ences in SNBF or PSBF between WT and KO.Our fi ndings indicate that GIP signals would play important roles in maintaining peripheral nerve functions, which might be mediated through their direct actions on DRG neurons and axons.

592-PCardiovascular Autonomic Neuropathy is Associated With Arte-rial Stiffness and Carotid Intima-Media Thickness in Patients With Type 2 DiabetesSU JIN OH, WON CHUL HA, HYUN SHIK SON, TAE SEO SOHN, Uijeongbu, Re-public of Korea

Cardiovascular autonomic neuropathy (CAN) represents a signifi cant cause of morbidity and mortality in patients with diabetes and is associ-ated with a high risk of cardiac arrhythmia and sudden death. The aim of this study is to evaluate the association between CAN and arterial stiffness and carotid intima-media thickness (CIMT) in patients with type 2 diabe-tes (T2DM).CAN, pulse wave velocity (PWV), ankle-brachial index (ABI), and CIMT were evaluated in a cross-sectional sample of 268 patients (126 men and 146 women) with T2DM. CAN was assessed by Ewing’s method which employs fi ve non-invasive tests of autonomic function.Among 268 patients, CAN was found in 48 patients (24%). Compared to the patients without CAN, those with CAN were signifi cantly older and had signifi cantly longer duration of diabetes, lower GFR, higher hs-CRP, mean CIMT, PWV, and ABI. The CAN scores correlated positively with mean CIMT and PWV (P<0.05). Using multi-variate regression analysis, CAN score independently predicted mean CIMT in patients with T2DM after adjustment for age.In conclusion, CAN was as-sociated increased arterial stiffness and CIMT in patients with T2DM. CAN may infl uence the development of cardiovascular disease through arterial stiffness and CIMT in T2DM.

593-PRole of Cholesterol in Neuronal Cell Function in DiabetesKENJI FUKUI, C. RONALD KAHN, Boston, MA

Diabetes is associated with neurologic and cerebral complications, includ-ing increased decline in cognitive function with the age, higher prevalence of depression and increased risk of Alzheimer disease. Recently we have shown that this is due, at least in part, to a reduction of SREBP-2 activity leading to reduced brain cholesterol synthesis and reduced synapse forma-tion. To determine the effect of reduced cholesterol content on neuronal cells, we have mimicked the effect of diabetes to reduce cholesterol in GT1-7 neuronal cells by 3 approaches: treatment with the cholesterol-depleting agent methyl-beta-cyclodextran (MBCD), treatment with the HMG-CoA reductase inhibitor and cholesterol lowering drug simvastatin; and stable

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knockdown of the regulator of cholesterol synthesis, SREBP2. Each of these treatment reduced cholesterol content by 20-40%. The resultant choles-terol depleted neuronal cells exhibited impaired proliferation (69.7±5.0% of control, p<0.01) and a parallel decrease in DNA synthesis (63.4±5.7% of control, p<0.01). Cells treated with MBCD or symvastatin also showed de-creased insulin and IGF-1 stimulated phosphorylation of AKT and ERK, with no change in expression of these proteins or the insulin receptor. NGF and BDNF induced phosphorylation of AKT and ERK was also suppressed in cho-lesterol depleted cells. When cholesterol was chronically depleted by stable knockdown of SREBP2, insulin, IGF-1 and NGF stimulated phosphorylation of phosphorylation of AKT was decreased to the same extent as acute cho-lesterol depletion with MBCD or simvastatin, but ERK phosphorylation was increased. Cholesterol depleted cells also showed a tendency to reduced secretion of GnRH following stimulation of cAMP. These result suggest that reduction of cholesterol in neurons by multiple methods, including reduced SREBP-2 as in diabetes and treatment with statins, impairs neuronal cell proliferation and function and alters insulin signaling. These effects could contribute to the CNS dysfunction observed in diabetes.

594-PSudomotor Function for Screening of Peripheral NeuropathyRAMAKRISHNAN SANTOSH, RAJITHA K. AFSAR HUSAIN, ABHILASH PILLAI, JEAN PAUL DESLYPERE, Hyderabad, India, Red Hill Forum, Singapore

Sensorimotor dysfunction is mainly considered for the diagnosis of periph-eral neuropathy (PN). Autonomic small C-Fibers innervating sweat glands are thin, long and unmyelinated and thus can be damaged very early in the diabetes process. Sweat dysfunction leading to abnormal skin conditions including dryness and fi ssures could increase diabetic foot risk. A consen-sus statement of the ADA has suggested that sudomotor function should be included in diagnostic tests for the detection of neuropathies in diabetes but lack of simple, and quick methods have restricted the widespread of such examination. The aim of this study was to evaluate SUDOSCAN™ a new quick, non-invasive, quantitative method to measure sudomotor dysfunction as a screening tool for diabetic peripheral neuropathy.68 patients with type 2 diabetes (age 48 ± 12 years, BMI 27 ± 5 kg/m2) were measured for vibration perception threshold (VPT) using a biothesiometer with 15 V as cut-off value for diagnosis of PN. Retinopathy status and serum creatinin level were also assessed as indicator of microvascular lesions. Sudomotor dysfunction was assessed by measuring electrochemical sweat conductance (ESC). Pa-tients place their hands and feet (area with highest sweat gland density) on large stainless-steel plates where a low voltage (<4V) is applied. ESC results from chloride movements and electrochemical reaction in response to electric stimulation.Based on biothesiometer examination 19 patients had peripheral neuropathy. Using biothesiometer as reference, measurement of sweat dysfunction had 95% sensitivity, 95% negative and 37% positive predictive value for diagnosis of PN. Among the false positives 17 (out of 31) had diabetic retinopathy and/or high serum creatinin level indicating mi-crovascular lesions.Sweating status using SUDOSCAN™ a simple, quick and quantitative method may be used as a screening tool of diabetic peripheral neuropathy. Relevance of the method could be explained by earlier damages in small C-fi ber when compared to large fi bers.

595-PReproducibility of a Rapid Point-of-Care Sural Nerve Conduction TestXUAN KONG, BONNIEJEAN BOETTCHER, KENNETH SNOW, SHAI GOZANI, Waltham, MA

Nerve conduction studies (NCS) are the most accurate and reproduc-ible test for diabetic peripheral neuropathy (DPN). They are used in clini-cal settings and as an outcome measure in neurotherapeutic clinical trials. However, NCS have not been widely adopted in the clinical management of diabetes due to expense, limited availability, and complexity. A 1-minute, low cost, sural nerve conduction test is now available as a point-of-care (POC) biomarker of DPN. In order to be clinically useful, this test must have high reproducibility so small changes in nerve function are reliably identifi ed. The objective of this study was to quantify the reproducibility of POC sural nerve conduction.Seventeen subjects (15 male, age 27-64) without periph-eral neuropathy were evaluated in two sessions 7-14 days apart. In each session, sural conduction velocity (CV) and amplitude (Amp) were measured twice in each leg. The device was removed and repositioned with every test. The device corrects CV for skin temperature below 30 oC. The primary outcome measure was variance component analysis and the correspond-ing coeffi cient of variation (CoV).A total of 136 tests were conducted. CV was 55.83.5 m/s and Amp was 14.34.2 V. Between-session CoV was 3.6%

and 8.8% for CV and Amp, respectively. Within-session CoV was 2.4% (CV) and 8.2% (Amp). The session-test CoV, which quantifi es the total variation between sessions, was 4.3% (CV) and 12.0% (Amp). Log-transforming the Amp reduced its CoV to 5.4%. Averaging the left and right limbs reduced the session-test CoV to 3.4% (CV), 8.5% (Amp), and 3.8% (log(Amp)).A 1-minute POC sural nerve conduction test demonstrated high reproducibility in a non-neuropathic cohort. Reproducibility can be improved by log transforming the amplitude and combining bilateral data. These results are comparable to benchmark studies in which measurements were performed manually in controlled multi-center clinical trials.

596-PCorrelation of Prolonged QTc Interval With Multi-Cardiovascular Risk Factors in Patients With Type 2 Diabetes MellitusZHANGRONG XU, HUI REN, AIHONG WANG, YUZHEN WANG, Beijing, China

Objective: To investigate the relationship between the prolonged QTc interval and the multi-cardiovascular risk factors in patients with T2DM.Methods: Medical data of 3940 T2DM patients were analyzed. After pa-tients who may have cause of abnormal QTc were excluded, a total of 3426 T2DM patients who had underwent diabetic complication assessment and ECG recording were enrolled. They were divided into normal QTc interval group(group A, QTc <0.44 s, n = 2157) and prolonged QTc interval group(group B, QTc 0.44s, n = 1269). The multi-cardiovascular risk factors were com-pared between the two groups and further analyzed.Results: Compared with group A, the patients in group B were older[(55.6±11.0) vs (54.6±10.9) yrs], had longer diabetes duration, more females (51.5% vs 41.5%), short-er body height[ (1.64±0.08) vs (1.65±0.08) m], bigger BMI [(26.2±3.6) vs(25.8±3.2) kg/m2], bigger WC[(89.7±9.6) vs (88.2±9.5) cm], bigger hip circum-ference[(95.4±7.3) vs (94.4±6.6)cm], higher WHR [(0.94±0.07) vs (0.93±0.08)],higher BP[(134.8±20.3) /(76.9±10.2) vs (130.1±19.1) /(74.5±9.6) mmHg], higher heart rate[(82.9±10.2) vs (70.7±10.1)beats/min], higher HbA1c [(8.0±2.0)% vs(7.7±1.9)%], higher post-meal glucose[(13.7±5.1) vs (12.8±4.8) mmol/L], higher fasting insulin[(9.4±8.6) vs (8.2±7.5)IU/ml], higher TC[(5.1±1.1) vs (5.0±1.1) mmol/L], higher TG [(2.4±2.6) vs (2.2±2.5) mmol/L], and higher Alb/Cr [15(8,36) vs 12 (7, 27) mg/g, median(P25, P75)]. All these indices were signifi cantly different between the two groups(P<0.05). Logistic regression analysis showed that factors including age, sex, BMI, waist circumference, WHR,heart rate and Cr, were signifi cantly associated with prolonged QTc interval.Conclusions: More than one third of T2DM patients present with prolonged QTc interval and more severe muslti-cardiovascular risk factors.

597-PUpregulation of S100B Protein in Astrocytes Cultured Under High Glucose ConditionKEVIN K. YUE, MAN TAK CHU, Hong Kong, China

Diabetes mellitus (DM) is characterized by hyperglycemia and diabetic complications. Recently it has been reported that there is an increase risk of developing neurodegernative diseases like Alzheimer disease (AD) in diabetic patients. Activation of astrocytes has been shown to be involved in neuronal dysfunction, and S100B is a calcium binding protein that is pri-marily expressed in astrocytes within central nervous system. In addition, modulation of astrocytic activities and increase of S100B content were found in different brain regions upon onset of neurodegenerative diseases. Therefore, the correlation between DM and neuronal dysfunction was inves-tigated, and more specifi cally the levels of S100B protein and glial fi brillary acidic protein (GFAP), a marker of astroctye activation, were determined in astrocytes cultured under high glucose condition.Primary astrocyte cultures and D1TNC1 type 1 astrocyte cells were treated with high glucose levels (12 mM, 25 mM and 50 mM) for different time periods, with normal glucose level (5.5 mM) as control. GFAP S100B and GFAP mRNA and protein levels were then determined by real time PCR and immunoblotting.Astrocytic cul-tures were shown to have a signifi cant increase of 20% in GFAP protein expression after challenged with hyperglycemia stress for 3 and 4 days but not in earlier time points. Similar results were obtained from primary astro-cytes. Up-regulation of S100B mRNA levels were found in both astrocyte cultures, with an increase of 23% and 27% in D1TNC1 type 1 astrocytes and primary astrocytes respectively. From western analysis, the increase of S100B protein in these cells were 30% and 33% respectively. Furthermore, up-regulation of S100B was found to coincide with the changes in GFAP expression.These results therefore imply that high glucose stress leads to up-regulation of S100B protein and activation of astrocyte, and may play a role in neuronal dysfunction resulting in diabetic cerebral vasculopathy and other neurodegenerative diseases.

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599-PIntraepidermal Nerve Fiber Density Decreases in Advance of Large Fiber Neuropathy of Diabetic PatientAIKO ARIMURA, TAKAHISA DEGUCHI, HIROSHI TAKASHIMA, YOSHIHIKO NISH-IO, Kagoshima, Japan

Diabetic polyneuropathy (DP) consists of small and large fi ber neuropathy. The large fi ber neuropathy can be assessed with nerve conduction study (NCS) and the fi ndings of NCS have been used for the diagnosis or staging of the DP. The small fi ber neuropathy, however, has not been fully investigated in relation to the staging of the DP.Using intra-epidermal nerve fi ber density (IENFD) and CVR-R as quantitative markers for the small fi ber neuropathy, we compared them with the fi ndings of NCS.A total 44 patients with dia-betes were subjected to analyses. All patients underwent full neurological examination, skin biopsy to evaluate the IENFD, NCS, and measurement of CVR-R.The 44 diabetic patients with DP were classifi ed into stage I - V ac-cording to the criteria by the Diabetic Neuropathy Study Group in Japan. No signifi cant differences in age, BMI, A1C, or duration of diabetes were found among each stage of the patients. Findings of NCS such as motor and sensory nerve conduction velocity, their action potential, and F-wave latency were impaired as the stage progressed. Similarly, IENFD and CVR-R decreased signifi cantly with progressing the stage. However, the patients at the early stages such as stageIand II showed signifi cant decreases in the IENFD (40% and 60%, respectively). Similarly, 10% of patients at stage I and 20% of those at stage II showed signifi cant decreases in the CVR-R.Diabetic small neuropathy assessed with IENFD and CVR-R developed in accordance with the progression of the stage of DP. However, signifi cant proportion of the patients with normal NCS showed decreases in IENFD and CVR-R sug-gesting that small fi ber neuropathy may develop in advance of the large fi ber neuropathy.

600-PPrevalence of Autonomic Dysfunction and its Risk Factors in Pa-tients With Impaired Glucose Tolerance (IGT)RIDDHI PATIRA, SHARAD SHARMA, DEEPAK VYAS, MAHESH GUPTA, NAVNEET AGARWAL, Gwalior, India, Agra, India

Autonomic dysfunction is well known in diabetic population. There is evidence that autonomic neuropathy may start even earlier in IGT(defi ned by American Diabetes Association as two-hour plasma glucose value dur-ing a 75 g oral glucose tolerance test between 140 and 199 mg/dL), lead-ing to increased incidence of cardiovascular complications. Variables like Hypertension, BMI>25, poor glycemic control and smoking may also act as addendum to autonomic neuropathy in patient with IGT. Our aim was to as-sess prevalence of autonomic dysfunction and variables like Hypertension and BMI in patients with IGT. We randomly selected 72 patients with IGT from diabetic screening program in Gwalior, India. Autonomic neuropathy test was based on criteria by Ewing et al (blood pressure response to stand-ing, Valsalva test, 30th:15th beat ratio and deep breathe test). 32 (44.4%) were found to have defi nite parasympathetic damage i.e. minimum two parasympathetic function test results abnormal. Deep breathe test which is predictor of parasympathetic abnormality, was present in highest num-ber of patients 60 (83.33%) suggesting that this test may be able to detect autonomic dysfunction earlier than other tests. However, this can be only confi rmed with further studies where diagnosis of autonomic dysfunction can be made before performing this test. We also found that prevalence of autonomic neuropathy in IGT patents with BMI>25 was 27/51(52.94%). Similarly, prevalence of autonomic neuropathy in hypertensive IGT pateints was 21/36 (58.33%). We conclude that autonomic dysfunction occurs more widely in IGT than was hitherto suspected and the autonomic neuropathy is more prevalent in IGT patients with BMI>25 and Hypertension.

601-PHyperglycemia Alters the Apoptotic Pathway via Heat Shock Pro-tein, and Insulin-Like Growth Factor Binding Protein Alterations in the Mammalian AmygdalaJESSICA R. HAYDEN, EDWARD P. SAENZ, REJEANA M. STEPHENS, CARLOS A. GARCIA, Kingsville, TX

Investigators have reported hyperglycemia alters the functioning of the central nervous system (CNS). Abnormalities including cognitive impair-ments are well documented in diabetics. Cognitive defi cits may be tied to decreases in brain volume. The amygdala, an important brain region that regulates emotional responses and memory, contains receptors for leutiniz-ing hormone, vasopressin and somatostatin may be impacted by hypergly-cemia. Early events, prior to clinically signifi cant CNS changes, may include alterations in apoptotic protein levels. The purpose of the current work is to test the hypothesis that hyperglycemia induces apoptotic pathways in the cells of the mammalian amygdala by altering the concentrations of heat shock proteins (Hsp) and both insulin-like growth factor proteins (IGF) and insulin-like growth factor binding proteins (IGFBP) of streptozotocin (STZ)-induced diabetic Wistar rats. Age and sex-matched rats were separated into two groups (n=3), control and diabetic (STZ: 60 mg/kg dissolved in 0.5ml 0.05M citrate buffer, pH 4.2). Three months after the onset of diabetes, both groups were sacrifi ced and brains dissected. The amygdala was col-lected for biochemical analysis. The content of Hsp-27, Hsp-60, Hsp-70, IGFI, IGF II, and IGFBP1-IGFBP6 was measured by an apoptosis antibody array in amygdala tissue homogenates. The levels of Hsp-27 (43%), Hsp-60 (48%) and Hsp-70 (36%) decreased in the diabetic amygdala along with decreases in IGFI (38%), IGFII (42%) and all of the IGFBPs tested. The results suggest an alteration of apoptotic pathways in the amygdala are stimulated by hy-perglycemia. Cellular survival mechanisms mediated by HSPs, IGFs, and IGFBPs were down regulated pointing to an increase in cell death due to apoptosis. This study provides novel therapeutic molecular pathways that could be targets for future drug intervention for diabetics that experience cognitive defi cits.

Supported by: HRSA H9CRH22885 and a University Research Award

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602-PSympathovagal Imbalance and Abnormal Cerebrovascular Reserve in Impaired Glucose Tolerance (IGT) and Type-2 DiabetesDINESH SELVARAJAH, TIM HUGHES, ELAINE CACHIA, SOLOMON TESFAYE, IAIN WILKINSON, Sheffi eld, United Kingdom

Type 2 DM imposes a 2-3 fold risk of stroke and poorer outcomes. The abil-ity to alter blood fl ow in response to haemodynamic stress helps maintain adequate brain perfusion. Impaired vascular reactivity is associated with increased stroke risk and poor recovery. This study examines the relation-ship between cardio-vagal autonomic function and cerebrovascular hae-modynamic abnormalities in diabetes and prediabetes.Method: Each group [T2DM with no history of stroke, IGT and healthy volunteers (HV)] underwent standard tests of cardiovascular autonomic function (O’Brien’s protocol) and barorefl ex sensitivity. Quantitative measures of internal carotid artery fl ow (ICA-F ml/s) and reactivity (ICA-R) were determined using MR Phase Contrast Angiography. MR data was acquired before and 30min after IV ad-ministration of 1g acetazolamide (ACZ), a potent intracerebral vasodilator. ICA-R = percentage change in ICA-F.Results: There were no signifi cant group differences in autonomic tests. T2DM subjects had signifi cantly lower ICA-F [pre-ACZ 4.3(1.0) ANOVA p=0.01; post-ACZ 6.1(1.5), ANOVA p=0.002] com-pared with IGT [ICA-F pre-ACZ 4.9(0.9); post-ACZ 6.7(1.0)] and HV [ICA-F pre-ACZ 4.3(1.0); post-ACZ 6.9(1.1)].Mean ICA-R was signifi cantly lower in both T2DM [43.1(18.3)] and IGT [40.1(19.8)] compared with HV [58.6(15.2), ANOVA p=0.01].There was signifi cant positive correlation between ICA-R and LF:HF ratio ( =0.45, p=0.04) and relative proportion of LF ( =0.46, p=0.03) and negative correlation with proportion HF ( =-0.46, p=0.03).Conclusions: This study provides new insights into the pathophysiology of cerebrovascular disease.We have demonstrated signifi cantly lower ICA-R in IGT and asymp-tomatic T2DM subjects. Moreover, abnormal sympatho-vagal balance was strongly associated with ICA-R reduction. Future work should examine if ab-normal sympatho-vagal balance results in worse outcomes and if targeted risk factor modifi cations can ameliorate this.

603-PThe Role of Acupuncture in the Management of Painful Diabetic Neuropathy (PDN): A Randomized Clinical TrialADAM GARROW, MEI XING, JOANNE VERE, BARBARA VERRALL, LIFEN WANG, EDWARD JUDE, Salford, United Kingdom, Ashton-under-Lyne, United Kingdom, Manchester, United Kingdom

In this single-blind placebo controlled Randomised Controlled Trial we examined the role of acupuncture as an additional treatment in theman-agement of Painful Diabetic Neuropathy (PDN). A total of 59 primary and secondary care patients with Type 1 or Type 2 diabetes and a clinical di-agnosis of PDN were recruited. 28/59 (47%) were randomly allocated to received a 10-week course either of real (53%) or sham (47%) acupuncture. 5 standardised acupuncture points on the lowerlimb of each leg used in the study: Liv-3, Kid-3, Sp-6, Sp-10, St-36. The Principal Outcome Measure was the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS) scale. Secondary outcomes were lower-limb pain intensity (VAS); Sleep Problem Scale (SPS); Measure Yourself Medical Outcome Profi le (MYMOP); SF36 and resting Blood Pressure (BP). Over the 10-week treatment period, patients receiving active acupuncture showed statistically signifi cant improvements in VAS -14 (-26,-3.5): MYMOP -0.89 (-1.4,-0.3) SPS -2.5 (-4.3,-0.82) and Rest-ing Diastolic Blood Pressure -5.2 (-10.4,-0.14). In contrast, there was little change in those receiving sham acupuncture. A signifi cant moderate treat-ment effect in favour of active acupuncture was detected in MYMOP scores -0.66 (-0.96 to -0.35). Non-signifi cant improvements were also seen for the LANSS Pain Scale -0.37 (-2.2 to 1.4) Resting Diastolic BP -0.50 (-3.0 to 1.99) and the Sleep Problem Scale -0.51 (-2.2 to 1.4). Acupuncture treatment was well tolerated with no appreciable side effects. Further randomised trials are needed to confi rm the clinical and cost-effectiveness of acupuncture in the treatment of Painful Diabetic Neuropathy.

Supported by: UK NIHR Research for Patient Benefi t Scheme

604-PImpact of the Glyco-Metabolic Control on Erectile Dysfunction in Patients With Type 2 Diabetes MellitusGIUSEPPE DEROSA, CARMINE TINELLI, ANGELA D’ANGELO, ELENA FOGARI, ALDO BONAVENTURA, PAMELA MAFFIOLI, Pavia, Italy

The aim of this study was to evaluate the impact of the glyco-metabolic control on erectile dysfunction (ED) in patients with type 2 diabetes mel-litus. We evaluated 88 males affected by type 2 diabetes mellitus, with a mean age of 62.78±9.26 years. We administered patients the IIEF (Interna-tional Index of Erectile Function) questionnaire to assess erectile function,

organ function, sexual desire, and satisfaction level during and after the sexual intercourse. We also administered SAS (self-rating anxiety scale) and SDS (self-rating depression scale) questionnaires to evaluate anxiety and depression. We also evaluated some parameters such as BMI, abdominal circumference, glycated hemoglobin (HbA1c), fasting plasma glucose (FPG), fasting plasma insulin (FPI), HOMA index, lipid profi le, testosterone, free tes-tosterone, dihydrotestosterone, and sex hormone binding globulin (SHBG).The IIEF questionnaire showed that in the examined sample there were 50 patients (56.8%) affected by ED, and 38 patients (43.2%) without ED. Com-paring the two groups, 57.9% of patients without ED, and 70.0% of patients with ED were smokers, and the difference between the two groups was signifi cant (p<0.05). Furthermore, 23.7% of patients withoutED, and 38.0% of patients with ED had a history of chronic ischemic heartdisease (p<0.05 between the two groups). Patients with ED were older, and, surprisingly, had lower levels of HbA1c. Furthermore, patients with EDhad higher levels of FPI, and lower levels of testosterone and dihydrotestosterone.In conclusion our study showed that the prevalence of ED in Italian males with type 2 diabetes mellitus with mean age of 62 years, is about 56% and it is linked to higher levels of FPI, but lower levels of HbA1c, and lower levels of free testosterone and dihydrotestosterone.

605-PExecutive Function and Diabetic Peripheral NeuropathyJASON L. RUCKER, STEPHEN D. JERNIGAN, JOAN M. MCDOWD, PATRICIA M. KLUDING, Kansas City, KS, Kansas City, MO

There is growing evidence that cognitive impairments in executive function (EF) contribute to the functional defi cits associated with diabetes mellitus (DM). We examined EF in adults with DM and diabetic peripheral neuropathy (DPN) as compared to age-matched control subjects. Twenty subjects with DM and DPN (DM; 8 female; 58.4 ± 6.2 years) and 20 control subjects (CN; 14 female; 54.9 ± 6.1 years) completed the study. Subjects were administered Beck’s Depression Inventory (BDI) and the Mini Mental Status Examination (MMSE). EF was assessed via measures of attention (digit span), working memory (reverse digit span), shifting (Trial Making Test), verbal organiza-tion (letter and category fl uency), perceptual organization (Rey-Osterrieth Complex Figure; ROCF), and dual task performance (Timed Up and Go with mental subtraction; cTUG). Severity of DPN was quantifi ed via tibial and per-oneal nerve conduction testing. The CN group had greater overall cognition (MMSE; p=0.001), lower BMI (p=0.02), and less depression (BDI; p<0.001). The DM group performed worse on letter fl uency (p=0.003), category fl u-ency (p=0.001), and the ROCF (p<0.001). The DM group also required more time to complete the TUG (p<0.001) and cTUG (p<0.001); however cognitive and motor dual task costs were not signifi cantly different. Reduced peroneal nerve amplitude was signifi cantly associated with lower MMSE scores (r=0.51) and greater cognitive dual task costs (r=0.51), while depression was signifi cantly associated with lower MMSE score (r=-0.46) and a longer TUG (r=0.54) and cTUG time (r=0.52). Obesity (BMI) and glycemic control (HbA1c) were not signifi cantly correlated with any cognitive or functional measure. Our data indicate that adults with DM and DPN exhibit verbal and perceptual organization defi cits. As problems with such executive functions have been linked to impairments in many aspects of functional ability, future research should examine how these and other cognitive factors, such as depression, impact function in this population.

606-PGastrointestinal Symptoms in Patients With Insulin Treated Diabe-tes Mellitus and the Correlation With Mental and Physical HealthEVA A. OLAUSSON, JAN BRUN, GISELA RINGSTROM, CHRISTINA BROCK, KLAS BUNNER, PETER FORS, MAGNUS SIMRÉN, STIG ATTVALL, Gothenburg, Sweden, Aalborg, Denmark, Kungalv, Sweden, Alingsas, Sweden

In this study we characterized the spectrum of GI symptoms in patients with insulin treated DM and how these correlate with physical and mental health.We invited 801 patientswith insulin treated DM attending two diabe-tes outpatient clinics to participate. They were asked to complete a ques-tionnaire to rate the severity of twenty GI symptoms during the previous two weeks (Patient Assessment of Upper Gastrointestinal Symptom Sever-ity Index (PAGI-SYM). A proportion of the subjects also completed question-naires to assess quality of life (QOL) (SF-36), and anxiety and depression (HAD).475 patients returned completed questionnaires (response rate 59%) (age 49±15 (mean±SD) years; 266 males). In the non-responder group a larger male predominance was observed (65 vs. 56%; p=0.009), but there was no age difference between the groups. 75 (16%) reported no GI symptoms at all and the majority of patients reported very mild or mild symptoms. Using “at least moderate symptom severity” as a cut-off, the most prevalent GI symp-

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toms were stomach fullness (29.4%), visible abdominal distension (20.5%), postprandial fullness (17.3%), bloating (17.3%) and loss of appetite (10.8%), whereas nausea and vomiting of at least moderate severity was reported by 8.6% and 4% of the patients, respectively.The GI symptom severity was associated with impaired physical and mental QOL, as well as with anxi-ety and depression. Stronger correlations with the severity of GI symptoms were seen for anxiety than for depression, and for physical vs. mental QOL.In summary, GI symptoms are common in DM, and fullness, bloating and distention are particularly common. are the most common GIS in these DM patients, but the vast majority of DM patients with GIS report mild symptom severity. GI symptoms were associated with impaired physical and mental health and paying attention to and treating these symptoms seems to be clinically important.


Guided Audio Tour: Diabetic Retinopathy—Bench to Bedside (Posters 607-P to 614-P), see page 13.

& 607-PDesign, Characterization, and Structure-Activity Relationships of Substituted 2-Amino-4-Phenyl-Thiophenes as Novel Atypical Pro-tein Kinase C Inhibitors for Diabetic Macular EdemaPAUL M. TITCHENELL, JEAN-FRANÇOIS PONS, DAVID A. ANTONETTI, Hershey, PA, Abingdon, United Kingdom, Ann Arbor, MI

Diabetic retinopathy remains a leading cause of blindness worldwide. Growth factors such as vascular endothelial growth factor (VEGF) and in-fl ammatory cytokines such as tumor necrosis factor (TNF) are increased in the diabetic eye and contribute to the loss of the blood-retinal barrier and subsequent macular edema associated with vision loss. Although anti-VEGF therapy improves visual outcome for some patients, there remain a subset of individuals that fail to respond. Additionally, current anti-VEGF therapies require repeat intra-ocular injections. Our laboratory has recently demon-strated that atypical protein kinase C (aPKC) isoforms are required for both TNF and VEGF induced endothelial permeability. Further, a series of small molecules inhibitors of aPKC isoforms that are effective at preventing VEGF and TNF induced retinal permeability were identifi ed from a Chembridge library screen. Here, we describe a detailed drug discovery and medicinal chemistry optimization approach to further characterize and defi ne these novel small molecule inhibitors of aPKC isoforms on a phenyl-thiophene plat-form. Structure-activity relationships (SAR) were defi ned by the synthesis and screening of over 200 phenyl-thiophene analogues for their ability to inhibit aPKC in vitro. From this analysis a detailed SAR model was built and a pharmacophore elucidated that confers inhibitor activity. Lead compounds with favorable physicochemical parameters were further tested for their ability to prevent TNF and VEGF induced permeability in primary culture. Lead compounds were shown to be specifi c, potent, and exhibit high effi cacy for their ability to prevent TNF and VEGF induced permeability. Importantly, 2-amino-4-phenyl-thiophene derivatives may be developed to preserve the blood-retinal barrier and ameliorate the macular edema associated with retinal diseases such as diabetic retinopathy.

Supported by: JDRF (D.A.A.), NIH EY012021 (D.A.A.), FFS

& 608-PThe Role of VLDLR in Down-Regulation of Wnt/ -Catenin Signaling and Diabetic RetinopathyKYUNGWON LEE, JIAN-XING MA, Oklahoma City, OK

Diabetic retinopathy (DR) is a severe ocular complication of diabetes, involving progressive retinal infl ammation, vascular leakage and neovascu-larization (NV). Recent studies revealed that Wnt/ -catenin signaling is im-plicated in the pathogenesis of DR. Retinal levels of -catenin and Wnt co-receptor low-density lipoprotein-related receptor 5/6 (LRP5/6) are increased in humans with DR and in DR animal models. Very low-density lipoprotein receptor knockout (vldlr-/-) mice show up-regulated Wnt/ -catenin signalingwith increased levels of -catenin and LRP6 in the retina. Moreover, absence of vldlr exhibits retinal NV and retinal infl ammation, pathological changes similar to DR, suggesting that VLDLR may play inhibitory roles for DR via suppression of Wnt/ -catenin signaling.In the present study, we dem-onstrated that VLDLR and LRP6 formed heterodimerization (VLDLR:LRP6) through ectodomains using co-immunoprecipitation assay. The soluble VLDLR ectodomain suffi ciently attenuated Wnt3A-induced phosphoryla-tion of LRP6 and TCF/ -catenin transcriptional activity in retinal pigment

epithelial cells. In addition, VLDLR ectodomain accelerated turn-over rate of LRP6, a possible mechanism by which the VLDLR:LRP6 heterodimeriza-tion inhibits Wnt/ -catenin signaling.A monoclonal antibody (mAb) specifi c for the VLDLR ectodomain that blocks VLDLR:LRP6 heterodimerization, en-hanced Wnt/ -catenin signaling via activation of LRP6. Intravitreal injection of the anti-VLDLR mAb stabilized -catenin and increased the expression of Wnt target genes including VEGF and TNF- in the retina. Taken together, these observations demonstrated that the VLDLR:LRP6 heterodimerization represents a mechanism for VLDLR to inhibit Wnt/ -catenin signaling. These fi ndings suggest that VLDLR ectodomain has therapeutic potential for the treatment of DR.

Supported by: NIH (EY018659, EY012231, EY019309, P20RR024215)

& 609-PDefi cient XBP1 Signaling in Endothelial Cells Exacerbates Leuko-stasis and Tight Junction Damage in Diabetic RetinopathySARAH X. ZHANG, JINGMING LI, CHEN CHEN, GUANGJUN JING, JOSHUA J. WANG, Oklahoma City, OK

Endoplasmic reticulum (ER) stress has been widely implicated in chronic infl ammatory diseases, such as diabetes and its complications. X-box bind-ing protein 1 (XBP1) is a central coordinator of cellular responses during ER stress. The objective of this study is to investigat the role of XBP1 in en-dothelial infl ammation and vascular dysfunction associated with diabetic retinopathy (DR). Endothelium-specifi c XBP1 defi cient (XBP1EC-/-) mice were generated by using the Cre-loxp system. Diabetes was induced by intrap-eritoneal injection of streptozotocin (STZ). Expression of infl ammatory cy-tokines and tight junction proteins, leukostasis and vascular permeability were evaluated in diabetic XBP1EC-/- and wild type (XBP1EC+/+) mice. As expected, retinal expression of intracellular adherent molecule-1 (ICAM-1) and vascular adherent molecule-1 (VCAM-1) was signifi cantly increased, in parallel with enhanced leukocytes adhesion to retinal vasculature in diabetic XBP1EC+/+ mice. When compared to XBP1EC+/+ mice, diabetic XB-P1EC-/- mice showed much higher levels of adhesion molecules and greater increase in leukostasis in the retina. In addition, retinal expression of ZO-1, a major tight junction protein, was much lower in XBP1EC-/- mice, indicat-ing impaired blood-retinal barrier. Furthermore, XBP1EC-/- mice developed more severe retinal vascular leakage compared with XBP1EC+/+ mice. Con-sistent with the in vivo results, endothelial cells isolated from XBP1EC-/- mice expressed much higher level of ICAM-1, but lower level of ZO-1 after incubation with pro-infl ammatory cytokine TNF- . This suggests that loss of XBP1 sensitizes endothelial cells to infl ammation and tight junction damage. Taken together, our results indicate that XBP1 plays a critical role in regulat-ing immune homeostasis and barrier function in endothelial cells. Impaired XBP1 signaling may contribute to vascular infl ammation and retinal edema in diabetic retinopathy.

Supported by: NIH EY019949, AHAF, OCAST

& 610-PKaplan-Meier Analysis of Time to First Signifi cant Vision Improve-ment in Two Phase III Trials of Ranibizumab for Diabetic Macular Edema (DME)CARL D. REGILLO, SUNIR J. GARG, JASON HSU, LINDA YAU, YAN ZHENG, J. JILL HOPKINS, JASON S. EHRLICH, LISA TUOMI, Philadelphia, PA, San Francisco, CA

DME is a serious vision-threatening complication of diabetic retinopathy (DR). Vascular endothelial growth factor A (VEGF-A) promotes vascular per-meability and is upregulated in DR. Ranibizumab is a monoclonal antibody fragment (Fab) that binds to and inhibits VEGF-A. The phase 3 trials, RIDE and RISE, enrolled 759 patients with DME randomized 1:1:1 to monthly intravitreal ranibizumab (0.5mg or 0.3mg) or sham. Macular laser was available for all arms starting at month 3. Using the Kaplan-Meier (KM) method, we estimated time to fi rst signifi cant improvement in best corrected visual acuity (BCVA) defi ned as a gain of 15 letters from baseline on the Early Treatment Diabetic Retin-opathy Study (ETDRS) chart or Snellen equivalent of 20/40 or better. Vision worse than 20/40 has been shown to adversely affect patient self-care and independent function. Comparing the ranibizumab and sham KM curves, a signifi cantly shorter time to fi rst improvement in BCVA of 20/40 or better was seen in ranibizumab-treated patients (P < 0.001) within the 24-month controlled treatment period (Table). More than half of the ranibizumab-treated patients fi rst achieved 20/40 vision or better within 2 months of treatment compared to >6 months in sham patients. Over half of the ranibizumab-treated patients who ever gained 15 letters during the study did so after at least fi ve months of treatment, indicating that signifi cant BCVA improvement could be achieved with ongoing therapy even if the initial response was suboptimal.

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Median number of days (95% CI) to fi rst visit with outcome & cumulative

% of patients who ever achieved the outcomea by Month 24

RanibizumabSham (n=257) 0.3 mg (n=250) 0.5 mg (n=252)

Gain of 15 ETDRS letters >720 (NE) 337 (272-426)b 331 (244-427)b

cumulative % with outcome 37% 69% 65%Snellen equivalent of 20/40 or betterc 217 (119-329) 71 (59-120)b 58 (35-91)b

cumulative % with outcome 67% 81% 83%NE = not estimable because less than half of the patients reached the endpoint in the fi rst 24-months.aOutcome not necessarily maintained at month 24.bP < 0.001, obtained from a log-rank test between the treatment arm vs. sham arm.c Proportion of patients with Snellen equivalent of 20/40 or better at baseline: 19.5%, 16.4% and 19.4% in the sham, 0.3 mg and 0.5 mg groups respectively.

& 611-PNovel Lipid Mediator Maresin Isomer Harnesses Mesenchymal Stem Cells to Ameliorate Diabetes Mellitus and Diabetic Retinopa-thy in db/db MiceSONG HONG, HAIBIN TIAN, STEPHANIE R. GROSS, YAN LU, JOSE A. GALINDO, QUANSHENG WANG, BRUCE A. BUNNELL, CHRISTIAN T. SHELINE, New Orleans, LA

Stem cell-based therapies provide promise to cure diabetic mellitus and complications. Lipid mediators derived from docosahexaenoic acid are po-tently anti-infl ammatory and tissue-protective. Herein, we tested the hypoth-esis that Maresin isomer (7S-MaR1; 7S,14R-dihydroxy-4Z,8E,10Z,12Z,16Z,19Z-docosahexaenoic acid), can promote stem cell functions in amelioration of diabetic mellitus and retinopathy. GFP-labeled bone marrow mesenchymal stem cells (MSCs), derived from C57BL/6J mice, were treated with 7S-MaR1 or control medium and then delivered into 16-week old diabetic db/db mice via tail vein every 5 days for 35 days. Compared to control or untreated MSC groups, the treatment with 7S-MaR1-treated-MSCs to db/db mice reduced blood glucose level (522.7 ± 46.3 vs 611.2 ± 14.0 (control) or 632.7 ± 23.1 (un-treated) mg/dl at day 35 of treatment, n = 6, p < 0.05 ); improved glucose tol-erance, increased blood insulin level (1.3 ± 0.1 vs 0.7 ± 0.1, 0.6 ± 0.1 ng/ml, n = 6, p < 0.05); increased insulin+ -cell ratio (77.7 ± 1.8% vs 72.8 ± 1.4%, 68.6 ± 43.2%, n = 60, p < 0.05) and decreased glucogan+ -cell ratio (22.4 ± 1.3% vs 27.0 ± 1.3%, 28.8 ± 2.4%, n = 60, p < 0.05) in islets; promoted vascular-ity, and reduced macrophage infi ltration in pancreas. 7S-MaR1 augmented MSC functions of promoting MIN6 -cell viability and insulin secretion invitro. Additionally 7S-MaR1 promoted MSC paracrine functions of increasing the generation of hepatocyte growth factor and vascular endothelial growth factor. Moreover, 7S-MaR1 enhanced MSC functions to ameliorate diabetic damage on retinal endothelial cells, pericytes, and ganglion cells at early stage of diabetic retinopathy of db/db mice by increasing their density in ret-ina and decreased apoptotic cells in retina compared to control or untreated MSC groups. This work provided novel lead to develop better modality based on 7S-MaR1-prompted-MSCs for human diabetic mellitus and retinopathy.

Supported by: NIH Grant 1-R01-DK087800 (S.H.)

& 612-PA Simple Algorithm to Estimate Time to Development of Sight-Threatening RetinopathyIRENE M. STRATTON, AMANDA I. ADLER, STEPHEN J. ALDINGTON, DAVID TAY-LOR, MARK HISTED, PETER H. SCANLON, Gloucester, United Kingdom, Cambridge, United Kingdom

The American Diabetes Association and UK National Health Service rec-ommend annual screening for diabetic retinopathy (DR) to refer sight threat-ening retinopathy (STDR) to ophthalmology clinics. Using longitudinal data from Gloucester Retinal Screening Service we developed a model based on retinal photographs to inform screening frequency. From 2005, 14,554 patients with non-referable DR (no DR or microaneurysms (MA) only) at 2 consecutive annual screenings were grouped on presence of MA in neither, 1 or both eyes at each screening and followed thereafter for a median 3 years (Table 1). Of 7,246 with no MAs at both screenings, 120 progressed to STDR, 0.7% by 1 year. Of 1,778 with MAs in neither eye at fi rst and 1 eye at second screening, 80 progressed to STDR, 1.9% by 1 year, hazard ratio (HR) 2.9 (95% CI 2.2 to 3.8) compared to those with no MA. Of 1,159 with MA in both eyes at both screenings 299 progressed to STDR, 11% by 1 year, HR 18.2 (14.7 to 22.5) compared with no MA (Fig 1). Those in higher risk groups were likely to progress to serious DR, and those with little or no DR unlikely to progress to STDR in the one year screening interval. Results from 2 an-

nual screenings enable progression risk estimation which informs screening frequency decisions even without other clinical data.

Characteristics of DR categories and progression to sight threatening refer-able retinopathyFirst Screen R0

Both eyesR1

one eyeR1

Both eyesR0 or R1 in either eye

R0 Both eyes

R1 one eye

R1 Both eyes

Second Screen

R0 Both eyes

R0 Both eyes

R0 Both eyes

R1 one eye

R1 Both eyes

R1 Both eyes

R1 Both eyes

N 7246 1266 343 3031 853 656 1159Age (years) 64.5 (13.0) 66.3(12.2) 66.8(12.6) 65.5(12.7) 68.0 (12.2) 64.1(14.1) 63.0 (14.6)proportion male (%)

55.6% 59.4% 53.9% 55.9% 53.6% 55.0% 58.9%

Progressed to referable DR

120 30 12 152 82 108 299

Hazard ratio 1.0 1.5 (1.0 to 2.3)

2.6 (1.4 to 4.7)

3.3 (2.6 to 4.2)

6.0 (4.5 to 7.9)

10.0 (7.7 to 13.0)

18.2 (14.7 to 22.5)

Expected pro-portion with DR 2 years after second screening

1% 2% 3% 4% 7% 12% 21%

& 613-PPuerarin Inhibits the Retinal Pericyte Apoptosis Induced by Ad-vanced Glycation End Products In Vitro and In Vivo by Inhibiting NADPH Oxidase-Related Oxidative StressJUNGHYUN KIM, CHAN-SIK KIM, EUNJIN SOHN, YUN MI LEE, KYUHYUNG JO, JIN SOOK KIM, Daejeon, Republic of Korea

Retinal pericyte loss is one of the histopathological hallmarks of early diabetic retinopathy. Puerarin (4’-7-dihydroxy-8-beta-d-glucosylisofl avone), which is an isofl avone-C-glucoside, causes various pharmacological ef-fects that include anti-hyperglycemic and anti-infl ammatory activities. In the present study, we determined the effi cacy and possible mechanism of puerarin on the advanced glycation end product (AGE)-modifi ed bovine se-rum albumin (BSA)-induced apoptosis of cultured bovine retinal pericytes and rat retinal pericytes in intravitreally AGE-modifi ed rat serum albumin (RSA)-injected eyes. Puerarin signifi cantly inhibited pericyte apoptosis, the generation of reactive oxygen species (ROS) and NADPH oxidase activity by inhibiting the phosphorylation of p47phox and Rac1 which were induced by the AGE-BSA treatment. The puerarin treatment markedly suppressed the activation of nuclear factor-kappaB (NF- B). In addition, the in vivo apoptosis of the retinal pericyte of rats that was stimulated by the intravitreal injec-tion of AGE-RSA was evidently attenuated by the puerarin treatment. These results demonstrate that puerarin may exert inhibitory effects on AGE-induced pericyte apoptosis by interfering with the NADPH oxidase-related ROS pathways and blocking NF- B activation, thereby ameliorating retinal microvascular dysfunction.

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& 614-PTwo New Loci Associated With Onset and Progression of Diabetic Retinopathy Identifi ed by a Genome-Wide Association StudyHETAL S. SHAH, JAN SKUPIEN, JASON NOBLE, AHMED Z. SOLIMAN, PAOLO A. SILVA, GABRIEL D. POZNIK, ADAM M. SMILES, LLOYD P. AIELLO, JENNIFER K. SUN, ANDRZEJ S. KROLEWSKI, ALESSANDRO DORIA, Boston, MA

Diabetic retinopathy (DR) is a leading cause of vision loss in developed countries. Although susceptibility to proliferative DR (PDR) is infl uenced by heritable factors, the genes involved are still unclear. The aim of this study was to identify genetic variants associated with DR onset and progression in subjects with type 1 diabetes (T1D). Using a time-to-event approach, we conducted a genome-wide association study of time from diabetes onset to four DR severity levels (mild, moderate, and severe non-proliferative DR, and PDR), as documented in the medical records of 872 White T1D subjects: 352 with normoalbuminuria, 311 with microalbuminuria, and 209 with protei-nuria. A total of 2.4 M single nucleotide polymorphisms (SNPs), genotyped (Illumina 300K or Affymetrix 500K chip) or imputed (MACH 1.0), formed our GWAS array. Data were analyzed by means of a Cox proportional hazards model stratifi ed by kidney status, applying a robust variance estimator to account for the correlated times to consecutive DR stages and for the clus-tering of events between the two eyes. Using a joint 2 d.f. test of genetic marginal association and gene-environment interaction, with glycemic con-trol (HbA1c below or above the median, 8.3%) as the interactor, two SNPs reached genome-wide signifi cance: rs7668242 on chromosome 4 (p=1.2 x 10-

8) and rs4755904 on chromosome 11 (p=4.2 x 10-8). Both genetic effects were exclusively observed in the high HbA1c stratum, with hazard ratios (HRs) of 2.0 and 2.6, respectively, as compared to HRs of 0.97 and 0.85 in the low HbA1c stratum. Rs7668242 is located in the neural retina-expressed gene GP6MA, and is also near WDR17, a candidate gene for retinal disease. Three other loci approached genome-wide signifi cance on chromosomes 3, 10 and 15. In conclusion, we have identifi ed two new loci potentially contributing to DR onset and progression. Studies are underway to further replicate these fi ndings and dissect the mechanisms underlying these genetic effects.

615-PRod Degeneration Accelerates Cone Photoreceptor Death in Dia-betesYUN-ZHENG LE, KE SHI, Oklahoma City, OK

Diabetic retinopathy (DR) is traditionally considered as a microvascular complication in diabetic retina. However, emerging evidences suggest that the loss of retinal neuron function and the death of retinal neurons are involved in DR. To determine the relationship between rods and cones in diabetes, we utilized a rod-specifi c Bcl-x knockout (KO) mice that had been shown to have impaired stress-induced survival previously and determined whether rod death affected cone survival in diabetic conditional Bcl-x KO mice. In our study, diabetes was induced with streptozotocin. Retinal func-tion was measured with electroretinography (ERG) and retinal morphol-ogy was assessed with hematoxylin & eosin (H&E) stained sections. Cone density was evaluated with immunohistochemistry. Thirty two weeks after inducing diabetes, rod-specifi c Bcl-x KO mice demonstrated accelerated loss of outer nuclear layer (ONL) thickness and rod photoreceptor function, compared with that of wild-type animals. Interestingly, cone photoreceptor function, as measured by photopic ERG, was signifi cantly reduced, compared with wild-type controls. Immunohistochemical analysis of S-opsin and M-opsin, markers of cone photoreceptors, showed that cone density was also signifi cantly reduced in diabetic rod-specifi c Bcl-x KO mice, compared with wild-type animals. Our data showed that BCL-xL, a PI3K-AKT survival path-way downstream target, is involved in rod photoreceptor protection under diabetic condition. Our results also suggest that cone photoreceptors are vulnerable to diabetes-induced rod photoreceptor death. We are currently investigating the underlying mechanism of this observation.

Supported by: R01EY20900, P20RR024215, OCAST Contract HR09-058

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617-PVitamin D Is Not Associated With Diabetic Retinopathy in Patients With Type 1 Diabetes and Type 2 DiabetesJOHANNA M. BRIX, ASTRID DOSSENBACH-GLANINGER, SIMON BRUNNER, EVA KRZIZEK, GERIT H. SCHERNTHANER, GUNTRAM SCHERNTHANER, Vienna, Austria

Diabetic Retinopathy (DR) is the leading cause for blindness in the West-ern world. Hypothesis suggests that Vitamin D defi ciency may be associated with diabetic microvascular disease, but a study in patients carefully clas-sifi ed for stages of DR is lacking. Thus, we investigated Vitamin D levels in patients with type 1 diabetes (T1D) and type 2 diabetes mellitus (T2D) well defi ned for different stages of DR and compared it to healthy controls (CO).Vitamin D levels were determined in 90 T1D and 135 T2D patients, both with and without DR and in 25 CO. Mean age was 41±12 in T1D, 62±10 in T2D and 61±3 years in CO. Patients were classifi ed for DR: 0 - no DR, 1 - mild nonpro-liferative DR, 2- moderate-severe nonproliferative DR, 3 - mild proliferative DR and 4 - high proliferative DR. Patients were staged according to a simpli-fi ed Early Treatment of Diabetic Retinopathy Study classifi cation. Vitamin D was measured as 25-Hydroxy (OH) Vitamin D (LC-MS-MS reference method).Patients with T2D had signifi cantly lower Vitamin D levels than CO (12.9±7.5 ng/ml vs 20.5±7.8 ng/ml; p<0.001). Patients with T1D had signifi cantly higher Vitamin D levels (27.7±11.2 ng/ml vs 12.9±7.5 ng/ml; p<0.001) than patients with T2D, but they were signifi cantly younger (p<0.001). Vitamin D levels did not signifi cantly differ in patients with or without DR and were not related to the stages of DR either in T1D or in T2D (Table 1).In summary, patients with T2D and different stages of DR have signifi cantly lower 25-OH Vitamin D levels compared to healthy controls. However, no association was found between Vitamin D defi ciency and DR in patients with either T1D or T2D.

Table 1Diabetic Retinopathy Stage 0 Stage 1 Stage 2 Stage 3 Stage 4 p-valueType 1 diabetes 28.1±9.9 29.6±10.4 19.5±18.1 27.4±10.8 22.9±17.3 0.54Type 2 diabetes 13.8±8.3 15.1±8.1 9.5±6.1 13.1±4.7 10.9±4.6 0.34

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Increased Hemoglobin AGE Levels in Type 1 Diabetes Patients and Correlation With Vascular Infl ammationANDRZEJ S. JANUSZEWSKI, DANIEL CALANDRO, DAVID N. O’NEAL, CONNIE S. KARSCHIMKUS, ALICIA J. JENKINS, Melbourne, Australia, Footscray, Australia

Hyperglycemia, infl ammation and Advanced Glycation End-Products (AGEs) may promote diabetes complications. Glycemia is refl ected by HbA1c, but clinically useful AGE measures are lacking. Given their lifespan and ac-cessibility Red Blood Cell (RBC) related AGEs are of interest. We quantifi ed hemoglobin (Hb) AGEs by a previously published assay and by our novel as-say of globin autofl uorescence (AF) in a cross-sectional study of 147 (18-73 year old Type 1 diabetic (T1DM) patients (including 54 with and 93 without complications) and 60 age and gender matched healthy non-diabetic con-trols. Renal failure subjects were excluded. Mean±SD diabetes duration was 21±13 years. HbA1c in T1DM and controls was 7.9±1.4% and 5.0±0.3% respectively (p<0.00001).Relative to controls Hb-AGEs were increased in T1D 0.19±0.09 vs. 0.14±0.03 AU, p=0.0003; as was Globin AF 2.37±0.16 vs. 2.19±0.09, p<0.00001. There were no signifi cant differences in either AGE related to T1D complication status nor use of insulin pumps vs. multiple daily insulin injections. Hb-AGEs and globin AF correlated in T1DM (r=0.41; p<0.00001) but not in controls (r=0.05; p=NS). Only Globin-AF correlated with vascular health (mean BP r=0.39; p=0.002 and Small Artery Elasticity r=-0.30; p=0.02 in controls, but not in T1D patients. In T1D Globin AF correlated with measures of vascular infl ammation (ELISA, RnD, Minneapolis, MN): se-Selectin r=0.26; p=0.002; sICAM r=0.23; p=0.007 and sVCAM-1 r=0.19; p=0.03. Neither AGE level correlated with age, HbA1c or renal dysfunction in T1D.Relative to controls, T1D is associated with signifi cantly higher levels in two Hb related AGEs, which correlate with measures of vascular infl am-mation, but not with vascular complications. These simple, low-cost repro-ducible assays may facilitate monitoring of anti-AGE therapies and vascular infl ammation. Longitudinal studies are merited.

619-PCases of Vision-Threatening Diabetic Retinopathy Avoided Using Ranibizumab for Visually-Impairing Diabetic Macular Edema in the United StatesROHIT VARMA, NEIL M. BRESSLER, QUAN DOAN, PAUL LEE, IVAN SUÑER, MARK DANESE, CHANTAL M. DOLAN, ADAM TURPCU, ABRAHAM LEE, JASON S. EH-RLICH, SHOSHANA COLMAN, Los Angeles, CA, Baltimore, MD, Westlake Village, CA, Durham, NC, Tampa, FL, San Francisco, CA

The number of non-Hispanic White and Hispanic persons aged 45 years and older in the US with diabetic retinopathy and center-involved diabetic macular edema (DR with DME) who would avoid vision-threatening DR over a 2-year period following treatment with ranibizumab 0.5 mg was estimated using a Monte Carlo simulation model. The outcome was defi ned as cases that did not have severe non-proliferative DR (NPDR) or proliferative DR (PDR) at baseline and worsened to severe NPDR or PDR (i.e., vision threaten-ing DR) at 2 years. Incident cases of DR with DME were estimated based on: the US population size in 2011, the prevalence of diagnosed diabetes, and the 1-year incidence rate for DR with DME with vision impairment. All insured patients (private and Medicare/Medicaid) were included. Baseline DR severity in the population was assumed to be similar to baseline DR dis-tribution of patients enrolled in the RISE and RIDE clinical trials. DR changes with and without ranibizumab were based on the RISE and RIDE data and were conditioned upon patients’ baseline DR status.The model predicted that 15,293 incident cases of visually-impairing DR with DME were eligible for ranibizumab treatment. Without ranibizumab, 1,263 (95% confi dence interval, 312 to 3,069) new cases of vision-threatening DR were expected over 2 years. Ranibizumab treatment reduced the estimated new cases of vision-threatening DR by 83% (81% to 84%) to 220 (55 to 537) cases, with 1,043 (257 to 2,542) cases avoided. The results suggest that monthly ranibi-zumab could substantially reduce worsening to vision-threatening DR within 2 years after treatment of non-Hispanic white and Hispanic patients. This benefi t may be an underestimation since the model only included incident cases and not prevalent cases of visually-impairing DR with DME.

620-PInteractive Effect of Aldose Reductase Z-4 Microsatellite Polymor-phism and Glycaemic Control on Cataract Development in Type 2 DiabetesYING WANG, ANDREA O. LUK, CALVIN C. PANG, MAGGIE C. NG, VINCENT K. LAM, RICHARD CHOY, SHAO C. LEE, WING YEE SO, DENNIS S. LAM, RONALD C. MA, JULIANA C. CHAN, Hong Kong, China

Aldose reductase (ALR2) of the polyol pathway is a key enzyme implicated in formation of cataracts in diabetes. We examined the interactive effect of the z-4 microsatellite polymorphism of ALR2 gene and glycemic control on risk of cataract in a cross-sectional cohort of Chinese type 2 diabetic patients. The (CA)n microsatellite polymorphism of ALR2 was determined using polymerase chain reaction followed by capillary gel electrophoresis. Cataract was defi ned by presence of lens opacity on direct ophthalmoscopy or history of cataract surgery. A non-linear curve approach was used to iden-tify the threshold of glycated hemoglobin (HbA1c) at which the odds ratio (OR) for cataract started to increase. The association of z-4 allele with cata-ract, above and below this threshold, was assessed using multiple logistic regression analysis.Of the 5,823 patients examined, 28.1% had cataracts. Those with cataracts were older, had longer duration of diabetes and worse metabolic control. After adjusting for conventional risk factors and using non-z-4 carriers with HbA1c<8.0 % as referent group (n=3173), the OR (95% confi dence intervals) for cataract was highest in z-4 carriers with HbA1c 8.0 % [1.43(1.05-1.96), n=244], compared to non-z -4 carriers with HbA1c 8.0 [1.27(1.10-1.47), n=1836] and z-4 carriers with HbA1c<8.0% [1.01(0.77-1.29), n=420, Ptrend<0.001). This interaction remained signifi cant after additional adjustments for drug use (Ptrend=0.002) and renal function (Ptrend=0.01). In type 2 diabetic patients with suboptimal glycemic control, z-4 microsatellite polymorphism of ALR2 gene was independently associated with increased susceptibility to cataracts.

Supported by: Innovation and Technology Fund (ITS/487/09FP), and the CUHK Focused Investment

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DIABETIC DYSLIPIDEMIA

622-PImpaired Flow-Mediated Vasodilatation and Diabetic Retinopathy in Patients With Type 2 Diabetes MellitusSEUNG-HYUN KO, JUNG-MIN LEE, HYUK-SANG KWON, SUNG-DAE MOON, YONG-MOON PARK, YU-BAE AHN, Suwon, Gyeonggi-Do, Republic of Korea, Seoul, Republic of Korea

Endothelial dysfunction plays an important role in the development of atherosclerosis and vascular complications in type 2 diabetic patients. We investigated the relationship between the endothelial-dependent vasodila-tion and diabetic retinopathy (DR) in patients with type 2 diabetes.167 pa-tients with type 2 diabetes (90 men and 77 women) were enrolled. Patients were excluded if they had a history of cardiovascular disease, acute infec-tions, aged more than 75 years, uncontrolled diabetes (HbA1c > 10.0%), or uncontrolled hypertension (> 160/ > 100 mmHg). We assessed endothelial dysfunction by fl ow-mediated vasodilation (FMD) method of the brachial ar-tery using high-resolution ultrasound, and changes in vasodilation (%FMD) were expressed as percent change over baseline values. The presence of DR was also assessed.The mean age was 54.1 ± 8.6 years. The mean diabetic duration and HbA1c level were 7.3 ± 6.0 years and 7.5 ± 1.0%, respectively. The median value of FMD was 3.85% (0-7.89). Twenty-nine patients (17.4%) had DR, and the %FMD was signifi cantly lower in patients with DR than without DR (0.65% (-0.33-4.20) vs. 4.87% (0.60-8.58), P < 0.001). Groups were divided according to FMD tertiles (T1 1.35, T2=1.35-5.95, T3 > 5.95%). The prevalence of DR decreased across increasing tertiles of %FMD. After adjusting for patients’ age, sex, diabetes duration, use of insulin, antihyper-tensive, anti-platelet, or lipid lowering medications, systolic blood pressure, fasting and 2-hour plasma glucose, HbA1c, and urinary albumin excretion, participants with a reduced %FMD were more likely to have DR. (Odds ratio 11.37 [95% confi dence interval (CI) 2.14 - 60.31]), comparing the lowest and highest tertiles of %FMD).Endothelial dysfunction was an independent pre-dictor of DR, and this was most visible when the endothelial dysfunction was the most severe. Our study provides insights into the possible mechanism of endothelial dysfunction’s infl uence on the development of DR.

623-PCurrent Prevalence and Correlates of Retinopathy in Patients With Type 2 DiabetesGIUSEPPE PUGLIESE, ANNA SOLINI, ENZO BONORA, EMANUELA ORSI, CECILIA FONDELLI, GIANPAOLO ZERBINI, ROBERTO TREVISAN, MONICA VEDOVATO, GABRIELLA GRUDEN, FRANCO CAVALOT, MAURO CIGNARELLI, LUIGI LAVIOLA, SUSANNA MORANO, ANTONIO NICOLUCCI, GIUSEPPE PENNO, THE RENAL IN-SUFFICIENCY AND CARDIOVASCULAR EVENTS (RIACE) STUDY GROUP, Rome, Italy, Pisa, Italy, Verona, Italy, Milan, Italy, Siena, Italy, Bergamo, Italy, Padua, Italy, Turin, Italy, Foggia, Italy, Bari, Italy, Santa Maria Imbaro, Italy

The natural history of diabetic complications, including diabetic retinopa-thy (DR), is changing due to improved diabetes care. This study was aimed at assessing current prevalence of DR and its association with risk factors and complications, particularly chronic kidney disease (CKD), in subjects with type 2 diabetes mellitus (T2DM). Patients with T2DM from the Renal Insuffi ciency And Cardiovascular Events (RIACE) Italian Multicenter Study (n=15,773), visiting consecutively 19 hospital-based Diabetes Clinics in years 2007-2008, were examined. DR was assessed by fundoscopy. CKD was defi ned based on albuminuria and glomerular fi ltration rate, as estimated from serum creatinine. Major acute cardiovascular disease (CVD) events were adjudicated based on hospital discharge records. Any retinopathy was observed in 22.2% (3,497) of patients; 12.4% had non-advanced DR, whereas 9.8% had advanced DR (non-proliferative 4.2%, proliferative 4.2%, maculopathy 1.3%, blindness 0.1%). Non-advanced and advanced DR were independently associated with HbA1c, diabetes duration and treatment (par-ticularly with insulin) hypertension, any previous CVD, albuminuria and the albuminuric CKD phenotypes and, inversely, with age and age at diabetes diagnosis. Maculopathy alone was also associated with female sex, but not with HbA1c, hypertension and age. Triglycerides were independently associ-ated with presence versus absence of CKD in subjects with advanced retin-opathy. Other correlates differed according to the CKD phenotype, with male sex, hypertension, and previous CVD associated with the albuminuric forms, female sex with the nonalbuminuric phenotype, and age with reduced GFR, independent of albuminuria. Data from this large cohort show that retin-opathy is present in less than one fourth of subjects, with a relatively high prevalence of advanced forms, and correlates with surrogate indexes of chronic hyperglycaemia, hypertension, cardiovascular events, and albuminu-ria in subjects with T2DM.

Supported by: Italian Society of Diabetology (Fo.Ri.SID)


Guided Audio Tour: Diabetic Dyslipidemia (Posters 624-P to 631-P), see page 17.

& 624-PEnteral Glucose Enhances Chylomicron Production in Healthy HumansCHANGTING XIAO, SATYA DASH, LINDA SZETO, GARY F. LEWIS, Toronto, ON, Canada

Overproduction of VLDL by the liver is an important contributor to diabetic dyslipidemia. We and others have recently shown that intestinal lipopro-tein (chylomicron) production is also increased in insulin resistance and is similarly regulated by several nutrient and hormonal factors that control hepatic lipoprotein production. High carbohydrate diets and hyperglycemia are associated with hypertriglyceridemia, believed to be mainly due to VLDL hypersecretion, whereas chylomicron secretion is not known to be modifi ed by the addition of glucose to ingested fat. Here we examined the effects of lipid, with or without added glucose, delivered directly into the duodenum, on hepatic and intestinal lipoprotein metabolism in humans. Seven normolip-idemic, normoglycemic men underwent 2 separate studies each, 4-6 weeks apart, in random order. During each study the subjects received a constant infusion of lipid (20% Intralipid, 60ml/h) plus either normal saline (60ml/h) (NS study) or glucose (25% dextrose, 60ml/h) (G study) through a nasoduo-denal tube for 15 hours. TG-rich lipoprotein (TRL) apoB-100 and B-48 kinetics were assessed with a primed, constant infusion of deuterated leucine under pancreatic clamps. Compared with lipid infusion alone, intraduodenal co-infusion of glucose increased plasma and TRL TG concentrations by 41% and 50%, respectively. TRL apoB-48 concentrations (NS=1.4+/-0.5, G=3.3+/-0.9 mg/L, P=0.04) and production rates (NS=0.04/-0.02, G=0.25+/-0.10 mg/kg/day, P=0.004) were markedly increased with glucose co-infusion. TRL apoB-100 concentrations (NS=37.8+/-7.7, G=47.5+/-6.3 mg/L, P=0.19) or production rates (NS=7.8+/-2.6, G=10.1+/-2.2 mg/kg/day, P=0.21) were not signifi cantly affected by treatments. These results show that enteral glucose enhances chylomicron particle production and may have important implications for di-etary recommendations especially for individuals predisposed to hypertrig-lyceridemia, such as those with insulin resistance and type 2 diabetes.

Supported by: Canadian Institutes of Health Research

& 625-PAPOA5 Genotype Modulates 2-Year Changes in Lipid Profi le in Re-sponse to Weight-Loss Diet Intervention: The Pounds Lost TrialXIAOMIN ZHANG, QIBIN QI, FRANK B. HU, FRANK M. SACKS, LU QI, Boston, MA

Apolipoprotein A5 gene (APOA5) is a major gene involved in lipid metabo-lism and modulated by dietary factors. A new variant rs964184 in APOA5 was recently identifi ed to be associated with lipids in genome-wide association studies (GWAS). We aimed to examine whether APOA5 variant might modify changes of lipid levels in response to 2-year weight-loss diet intervention in a randomized trial. We genotyped APOA5 rs964184 in 734 overweight or obese adults who were randomly assigned to one of four diets differing in the percentages of energy derived from fat, protein and carbohydrate for 2 years. We evaluated the change of fasting serum concentrations of total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density li-poprotein cholesterol (HDL-C), and triglyceride (TG) from baseline to 2 years of follow-up. After 2-year dietary intervention, we found signifi cant interac-tion between the APOA5 rs964184 polymorphism and dietary fat intake (low vs high) modulating changes in TC, LDL-C and HDL-C concentrations (P for interactions=0.010, 0.007 and 0.021, respectively). In low-fat intake group (20% of energy derived from fat), carriers of the risk allele (G allele) exhibited a greater reduction in TC and LDL-C than the non-carriers (Padd=0.040 and 0.042); the genotype effects were not observed in high-fat group (40% of energy derived from fat). Analyses in the white participants showed similar results. Our data suggest that a long-term low dietary fat intake may im-prove lipid profi le in the APOA5 rs964184 risk allele carriers.

Supported by: HL071981, RR-02635

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& 626-PPlasma Angiopoietin-Like Protein 4 is Increased in Patients With Type 2 Diabetes and the Metabolic Syndrome in the Presence of Low Grade Infl ammationNATHANJA TJEERDEMA, JACQUELINE JONKER, SANDER KERSTEN, PATRICK C. RENSEN, JOHANNES W. SMIT, Leiden, The Netherlands, Wageningen, The Neth-erlands

Angiopoietin-like 4 protein (Angptl4) is an adipocytokine that plays a role in lipid metabolism by inhibition of lipoprotein lipase and has been asso-ciated with dyslipidemia. Experimental studies suggest that Angptl4 also plays a role in infl ammation, but it is unknown how Angptl4 levels are infl u-enced by infl ammation.Therefore the objective of this study was to assess plasma Angptl4 in T2DM, in subjects with MetS in the presence or absence of low grade infl ammation, and in healthy controls (Controls).A total of 268 male subjects (T2DM n=67, MetS with low grade infl ammation, n=67, MetS without low grade infl ammation n=67 and Controls n=67) were included in this study. All subjects were matched for age and waist circumference. The IDF-criteria for the MetS were used and low grade infl ammation was defi ned as serum CRP 1.8 mg/L. As Angptl4 levels are not normally distrib-uted, comparisons between groups were performed with Wilcoxon signed rank tests adjusted for the number of comparisons.In all patients combined, mean(SD) age was 56.8(5.8) years and mean waist circumference was 105(9) cm. Angptl4 [median(interquartile range)] was increased in subjects with T2DM [6.4(5.2-7.8) ng/mL, P<0.001] and in subjects with MetS with low grade infl ammation [6.8(5.3-8.1) ng/mL, P<0.001] compared to healthy con-trols [4.0(2.8-5.6) ng/mL]. This elevation was independent of obesity as sub-jects were matched for waist circumference. Patients with the MetS with-out infl ammation did not show elevated levels of Angptl4 [3.8(2.8-5.2) ng/mL, P=0.751].In conclusion, increased plasma Angptl4 levels are observed in subjects with T2DM and MetS with, but not without, low grade infl amma-tion. We hypothesize that systemic infl ammation plays an important role in the association between Angptl4 in MetS and T2DM. Further studies into the causal relationships are required.

& 627-PRelationship Between Lipoprotein Kinetics and Lipoplysacharide Distribution Among Lipoproteins in Normal Subjects and Patients With Type 2 DiabetesBRUNO VERGÈS, CHRISTELLE VACHOU, LAURENCE DUVILLARD, EMMANUEL FLORENTIN, JEAN-MICHEL PETIT, MARIE-CLAUDE BRINDISI, REMY BURCELIN, Dijon, France, Toulouse, France

Lipopolysacharides (LPS) are components of gram-negative bacterial membrane that are associated in plasma with lipoproteins. LPS binding with lipoproteins may promote their catabolism and then reduce their pro-infl am-matory effects. However, data on LPS distribution among lipoproteins are scarce and the relationship between LPS distribution and lipoprotein metab-olism remains unknown. This prompted us to perform an in vivo lipoprotein ki-netic study in 31 individuals (16 with type 2 diabetes [T2DM], 15 controls) and to analyze the relationship between lipoprotein kinetics and LPS distribution among VLDL, LDL and HDL.Plasma LPS levels were not different between T2DM patients and controls but LPS distribution was different between the 2 groups with, in T2DM vs. controls, higher proportion of LPS-VLDL (31±7 vs. 21±11%,p=0.001), LPS-HDL (28±9 vs. 20±10%,p=0.03), free LPS (10±4 vs. 7±4%,p=0.04) and lower part of LPS-LDL (30±13 vs. 51±16%,p=0.001). In T2DM patients, VLDL catabolism was decreased when VLDL production and HDL catabolism were increased. In multivariate analysis, for the whole stud-ied population: LPS-VLDL was associated with LPS-HDL (p<0.0001) but not with apoB kinetics; LPS-LDL was associated with LPS-VLDL (p=0.003) and VLDL catabolism (p=0.008) but not with LPS-HDL; LPS-HDL was associated with free LPS (p<0.0001) and LPS-VLDL (p=0.041) but not with HDL kinetics; free LPS was only associated with LPS-HDL.In conclusion, our data indicate that an important LPS transfer occur in vivo between free LPS and HDL as between HDL and VLDL whereas LPS-LDL is mainly derived from VLDL ca-tabolism and may represent a catabolic pathway for LPS. T2DM patients show a signifi cant decrease of LPS bound to LDL that may represent an im-paired LPS catabolic pathway. Further studies are needed to see whether these modifi cations of LPS distribution among lipoproteins in T2DM patients may increase intra-vascular pro-infl ammatory state.

& 628-PHyperadiponectinemia is Strongly Linked to Elevated HDL-Choles-terol (HDL-C) in Type 1 Diabetes (T1D)ROSSANA M. CALDERON, ANGELA SZETO, THAMER ALESSA, ARMANDO MEN-DEZ, RONALD B. GOLDBERG, Miami, FL

Although HDL-C levels are often increased in T1D, the basis for this fi nd-ing and its clinical signifi cance are not well understood. Adiponectin (APN) levels have been shown to correlate strongly with HDL-C, suggesting a mechanistic relationship between the two. Since APN levels are increased in T1D, we investigated whether elevated APN is related to increased HDL-C in T1D. We studied 114 T1D subjects attending our Diabetes Clinic (diabetes duration 25.8±1.4 yrs [mean±SEM], HbA1c 7.8±0.4%) and 64 controls (CON). T1D were slightly older (42.3±1.4 vs 37±1.4yrs; p<0.05) and had a higher BMI (26.3±0.4 vs 24.5±0.4 Kg/m2; p<0.01) than CON. Both HDL-C (64±2 vs 52±2 mg/dL; p<0.005) and APN (13.9±6.5 vs 10.8±4.0 μg/ml; p<0.001) were higher in T1D than CON, and women (W) had higher HDL-C, apo A-I and APN than men (M) in both groups (Table). 44% of T1D W and 50% of M had HDL-C >90th

percentile of the CON group values (W>76; M>54 mg/dL).

T1 Diabetes ControlsMen (n=53) Women (n=61) Men (n=31) Women (n=33)

BMI (kg/m2) 27.2 ± 0.5b 25.6 ± 0.6 25.1 ± 0.4 24 ± 0.8Triglyceride (mg/dL) 97 ± 7.4 88 ± 6.6 125 ± 14.5 91 ± 8.1a

HDL-C (mg/dL) 56 ± 2b 71 ± 2a,b 45 ± 2 59 ± 2a

apo A-I (mg/dL) 153 ± 3.4b 183 ± 4.3a,b 137 ± 2.8 162 ± 4.4a

APN (μg/mL) 10.2 ± 0.6 16.9 ± 0.9a,b 9.6 ± 0.6 11.5 ± 0.6a p<0.05, M vs W within Group; b p<0.05, T1D vs CON between genders.

APN was correlated with HDL-C and apo A-I in both T1D (r=+0.56, p<0.001 and r=+0.42, p<0.001, respectively) and CON (r=+0.65, p<0.001 and r=+0.41, p<0.001). In step-wise regression analysis APN remained associated with HDL-C in both T1D W and M after adjusting for age, diabetes duration, HbA1c, serum creatinine, CRP, BMI, and triglyceride (r2 M=0.08, p<0.05; W=0.20, p<0.001). Our results demonstrate that APN levels are strongly linked to HDL-C values in T1D, supporting the notion that the hyperadiponectinemia of T1D is in part responsible for the frequent fi nding of elevated HDL-C in this condition. The implications of these fi ndings and its impact on cardiovascular risk remain to be determined.

& 629-PEffects of AMR101 on Lipid and Infl ammatory Parameters in Patients With Diabetes Mellitus-2 and Residual Elevated Triglycerides (200-500 mg/dL) on Statin Therapy at LDL-C Goal: The ANCHOR StudyELIOT A. BRINTON, CHRISTIE M. BALLANTYNE, HAROLD E. BAYS, JOHN J. KASTELEIN, RENE A. BRAECKMAN, PARESH N. SONI, Salt Lake City, UT, Houston, TX, Louisville, KY, Amsterdam, The Netherlands, Bedminster, NJ

AMR101 is a novel omega-3 fatty acid agent containing 96% pure icosa-pent ethyl, the ethyl ester of eicosapentaenoic acid. The phase 3 ANCHOR study evaluated the effi cacy and safety of AMR101 in patients with residual high fasting TG levels (200-500 mg/dL) despite optimized LDL-C (40-100 mg/dL) on statins. Of 702 patients randomized to AMR101 4 g/d, 2 g/d, or place-bo for 12 weeks, 514 (73%) had diabetes mellitus-2. Intent-to-treat analysis of the effects of AMR101 on median placebo-adjusted percent change from baseline in endpoint parameters was performed in 3 subgroups: total (all subjects with diabetes), well-controlled diabetes, and less-controlled dia-betes (</> median HbA1c; see Table for 4 g/d results). Baseline parameters were similar among all groups except hsCRP, which was higher in the total and less-controlled diabetes groups. AMR101 signifi cantly reduced TG, non-HDL-C, apo B, and RLP-C in all diabetes groups, LDL-C in the total diabetes group, and hsCRP in the total and less-controlled diabetes groups (Table). Interestingly, decreases in hsCRP and apo B appeared to be far greater in pa-tients with less-controlled diabetes. Importantly, there were no signifi cant increases in FPG, HbA1c, insulin, or HOMA-IR in any group. In conclusion, in patients with diabetes and mixed dyslipidemia, AMR101 4 g/d signifi cantly improved lipid and lipid-related parameters without worsening glycemic control, with possibly greater effects among those with less-controlled diabetes.

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Median Placebo-adjusted Percent Change in Lipid and Infl ammatory Parame-ters From Baseline to Study End in Patients With Diabetes Treated With AMR101 4 g/day% Change in: Well-controlled

Diabetes*, PLess-controlledDiabetes**, P

Total Diabetes, P

TG (n=78, 87, 165) -21.0<0.0001 -24.8<0.0001 -23.2<0.0001LDL-C (n=78, 87, 165) -6.60.0831 -5.70.1304 -6.30.0227Non-HDL-C (n=78, 87, 165) -11.30.0019 -18.0<0.0001 -14.4<0.0001hsCRP (n=75, 85, 160) -4.00.7372 -34.60.0002 -21.50.0028RLP-C (n=28, 32, 60) -26.70.0468 -21.30.0364 -25.00.0024Apo B (n=75, 85, 160) -6.10.0170 -12.8<0.0001 -9.5<0.0001P-values are from Wilcoxon rank-sum test.*HbA1c < median of 6.8%. **HbA1c

median of 6.8%.Abstract abbreviations: Apo B=apolipoprotein B; FPG=fasting plasma glucose; HbA1c=hemoglobin A1c; non-HDL-C=non-high-density lipopro-tein cholesterol; HOMA-IR=homeostasis model index insulin resistance; hsCRP=high-sensitivity C-reactive protein; LDL-C=low-density lipoprotein cho-lesterol; RLP-C=remnant-like particle cholesterol; TG=triglyceride.

Supported by: Amarin, Inc.

& 630-PEstimation of Plasma Free Fatty Acids through Integrated Analysis of Exhaled Breath in Obese and T2DM AdultsSTACY R. OLIVER, MATTHEW K. CARLSON, ROBERT NEWCOMB, SIMONE MEIN-ARDI, DONALD R. BLAKE, PIETRO R. GALASSETTI, Irvine, CA

Volatile organic compounds (VOCs) represent ideal, non-invasive markers of endogenous biochemical processes; however, clinically usable metabolic tests from VOCs has proven challenging. We previously estimated plasma glucose, insulin, and free fatty acids (FFAs) by integrating exhaled VOCs. If developed, a breath-based lipid test would greatly improve the diagnosis and management of obesity and diabetes (OW, T2DM).Therefore we stud-ied 10 T2DM (5M, 44±3yrs) and 5 OW subjects (5F, 41±5yrs) during 4 hours of controlled glycemic and insulinemic fl uctuations (hyperglycemia of ~220 mg/dL; euglycemic-hyperinsulinemia). Plasma, room air and exhaled gases were sampled at 12 different time points.Concentrations of ~100 VOCs were determined by gas chromatography and matched with plasma FFAs. Multi-linear models to reconstruct plasma FFAs concentrations for each subject were generated by least squares regression on several subsets of exhaled VOCs. The gas clusters yielding the strongest correlations with plasma FFAs were acetone, CH3I, CH2Br2, n-butane in OW (r = 0.927); and n-butane, m\p-xylene, i-pentane, ethylbenzene, MeONO2 in T2DM (r = 0.943). Additional gas combinations were identifi ed allowing broad range applicability across both groups.Our data show the feasibility of accurately estimating plasma FFAs in OW and T2DM subjects during glucose and insulin fl uctuations via in-tegrated analysis of multiple exhaled gas profi les. Our observations indicate the potential of this methodology for non-invasive diagnosis and monitoring of metabolic variables relevant to obesity and diabetes.

Supported by: NIH (1UL1RR031985, K24 DK085223)

& 631-PActivation of GPR119 Reduces the Appearance of Labeled Choles-terol in an Oral Fat Tolerance TestKATHLEEN K. BROWN, MELANIE K. SHADOAN, DALLAS K. CROOM, ANDREW J. CARPENTER, DEREK J. NUNEZ, ANDREW A. YOUNG, PAUL L. FELDMAN, Re-search Triangle Park, NC

Agents which lower cholesterol in circulation by impeding intestinal ab-sorption offer an alternative to statins for reduction in cardiovascular risk. GPR119, identifi ed as a sensor of fatty acid derivatives, is present in the gastrointestinal tract, but effects on lipid homeostasis have not previously been described. To track the appearance and accumulation of chylomicrons in circulation, fasted male rats were gavaged with radiolabeled cholesterol in olive oil. Triton was injected intravenously to block LPL-mediated clearance and thus enable tracking of cholesterol appearance over the next 5 hours. Effects are reported as counts, relative to those at corresponding time points in vehicle-treated rats (=100% in Fig). Ezetimibe used as a positive control, decreased tracer appearance to 19 ± 3.4 % of control by 4 hrs. Each in a struc-turally diverse set of GPR119 agonists tested (30 mg/kg p.o. at t=-2 hr) slowed appearance of labeled cholesterol, suggesting the effect was GPR119-specif-ic. The relative decreases for all agonists compared to vehicle control were 50 ± 8.1, 51 ± 11.3, 55 ± 9.6, 63±15.4 and 58±10.2 (mean±SEM, n=5-8/group) for GSK263, GSK706, Arena, Prosidion and MBX respectively. These previously unreported effects of GPR119 agonists are predicted to have therapeutic ben-efi t in the setting of dyslipidemia, metabolic syndrome and T2DM.

632-PA Role for GPR119 in Lipid and Lipoprotein MetabolismKATHLEEN K. BROWN, MANDY L. BERGQUIST, MELANIE K. SHADOAN, ANDREW J. CARPENTER, ANDREW A. YOUNG, PAUL L. FELDMAN, DEREK J. NUNEZ, ANI-TA VAN DEN HOEK, HANS M. PRINCEN, Research Triangle Park, NC, Leiden, The Netherlands

Activation of GPR119 on islet and -cells and enteroendocrine cells in the gastrointestinal (GI) tract coordinates insulin secretion and the release of gut peptides to promote the disposal of meal-derived nutrients. At present, the signifi cance of its role as a fatty acid sensor in the GI tract is unclear. Fe-male APOE3Leiden.hCETP transgenic mice, fed a western diet, were used to investigate the effects of acute (single dose) or chronic (5 weeks) treatment with a potent and selective GPR119 agonist, on lipid homeostasis. For each study, groups (n=8/group), matched for total cholesterol (TC), triglycerides (TG) and age, were treated with either vehicle (V) or GSK2041706 (GSK706) at 10 or 30 mg/kg/d. To measure acute effects on TG appearance, overnight fasted mice received a single gavage of V or GSK706. Mice received 200 μl of olive oil 2hr later (with [3H]-triolein and [14C]-oleic acid) immediately after iv injection of Triton (T) to block TG hydrolysis. Blood was collected at 0, 1, 2, 3 and 4h to measure plasma radioactivity and TG. To assess VLDL production, overnight fasted mice received a single gavage of V or GSK706, were anesthetized 1.5hr later and injected iv with 0.1 mL PBS containing 100 μCiTran35S to measure de novo ApoB synthesis. Mice received an iv injec-tion of T 30 min later, and samples were collected at 0, 15, 30, 60 and 90 min to measure plasma TG. After 90 min, mice were sacrifi ced and blood was collected for isolation of VLDL. There were no acute treatment related ef-fects on TG absorption, hepatic VLDL-TG secretion or de novo ApoB synthe-sis. However, there were changes in lipid and lipoprotein parameters with chronic dosing manifested by decreased FFA (24% and 30%, p<0.001, @ 10 and 30 mg/kg) and decreased serum TC and TG (36% and 37%, p<0.05, @ 30mg/kg) compared to V. TC in the VLDL/LDL peak on FPLC was decreased by 27% and 39% (p<0.05, @ 10 and 30 mg/kg). There were no effects on CETP mass or activity or on post-heparin plasma lipolytic activity. This suggests a therapeutic role for GPR119 in regulation of plasma TC and TG.

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633-PFasting and Postprandial Overproduction of Apolipoprotein B48-Containing Lipoproteins in Juvenile Zucker Fatty Rats: Impact of a Cinnamon Extract on EnterocytesBOLIN QIN, RICHARD A. ANDERSON, Beltsville, MD, Spring Hill, TN

Emerging evidence suggests that the overproduction of intestinal apoli-poprotein (apo) B48 is associated with obesity and insulin resistance. We investigated the metabolism of apoB48-containing lipoproteins in male juve-nile Zucker lean (Zl) and fatty (Zf) rats. At 5 weeks of age, Zf rats had higher body weight and epididymal white adipose mass compared to Zl rats, and elevated area under the curve of glucose response to an oral glucose chal-lenge. After an overnight fast, rats were subjected to an oral olive oil loading test. The fasting and postprandial triglyceride (TG) levels and TG-rich lipo-proteins (apoB48) in Zf rats were signifi cantly higher compared with Zl rats. The small intestinal mRNA expression of insulin signaling genes, including insulin receptor (Ir), and insulin receptor substrates-1&2 (Irs1 and 2 ) were lower. The gene expression related to lipogenic signaling and cholesterol metabolism including sterol regulatory element binding protein (Srebp)-1c, microsomal triglyceride transfer protein (Mtp) and scavenger receptor type B class I (Scrbi ) were higher; and the mRNA levels of ATP-binding cassette transporters G (Abcg )5, ATP-binding cassette transporters A (Abca1), Nie-mann-pick C1-like (Npc1l )1 and peroxisome proliferator-activated receptor (Ppar)g were down regulated in Zf rats compared to the Zl rats. An aqueous cinnamon extract (CE, 10μg and 100μg/mL) reversed the expression of mul-tiple dysregulated genes in the primary enterocytes of Zf rats and inhibited the oversecretion of apoB48 into the media. In summary, obesity-induced systemic insulin resistance is associated with compromised signaling path-ways related to apoB48 production in the small intestinal tissue of juvenile Zf rats and CE is benefi cial in the control of intestinal apoB48 metabolism.

Supported by: USDA/ARS CRADA No.58-3K95-7-1184 with Integrity Nutraceuti-cals, Inc.

634-PCrosstalk between Salivary Gland Chronic Infl ammation and the En-docrine Function of Adipocytes in Insulin ResistanceYOSUKE SHIKAMA, QUINKAI LI, YUKIKO BANDO, NANAKO AKI, CHISATO KO-SUGI, MAKOTO FUNAKI, Tokushima, Japan

In insulin resistance, free fatty acids (FFAs) in plasma are increased due to adipocyte dysfunction, which inhibits insulin action in the muscle and liver and contributes to a development of type 2 diabetes (T2D). Emerging evidence suggests that, among FFAs, saturated fatty acids (SFAs) are po-tent activators of toll-like receptors (TLRs), which initiate a proinfl ammatory response in these organs, such as activation of mitogen-activated protein kinase (MAPK) or nuclear factor- B (NF- B). Epithelial cells in salivary glands also express TLRs. Interestingly, it has been reported that sjögren syndrome (SS), in which salivary gland epithelial cells exhibit chronic infl ammation due to autoimmunity and secrete interleukin (IL)-6, is frequently accompanied by T2D or dyslipidemia.In this study, we investigated the effect of SFAs on a proinfl ammatory response in salivary gland epithelial cells and whether or not adipocyte function could regulate it. Here we report that treatment with SFAs, but not with unsaturated fatty acids, activated p38 MAPK and NF- B, which led to an increased secretion of IL-6 in a human salivary gland cell line, HSY cells. HSY cells expressed both AdipoR1 and AdipoR2 recep-tors, and PPAR 1. SFA-induced secretion of IL-6 from HSY cells was inhibited by globular adiponectin or simvastatin, but not by full-length adiponectin. These results suggest a putative crosstalk between chronic infl ammation of salivary glands and the endocrine function of adipocytes, which affect other organs by adipocytokine secretion in insulin resistance.

635-PDifferential Effects on Lipid Profi les between Alogliptin and Sita-gliptin in Newly Diagnosed, Drug Naïve Subjects With T2DMEIJI KUTOH, YASUHIRO UKAI, Tokyo, Japan

Different DPP-4 inhibitors show signifi cant structural differences. As an initial step towards fi nding any differences in their pharmacological actions, effects on glycemic and other parameters were compared between sita-gliptin (sita) and alogliptin (alo). Newly diagnosed, drug naive subjects with T2DM were assigned to either 25-50mg/day sita (n=21) or 12.5-25mg/day alo (n=22) monotherapy. At 3 months, the levels of these parameters were compared with those at baseline. This study is a head-to-head comparison. At baseline, the levels of the measured parameters were similar between these two groups. At 3 months, similar glucose lowering effi cacies were observed in these groups (HbA1c: from 10.46 to 8.32%, p<0.001 for sita and from 10.4 to 8.63%, p<0.002 for alo). In contrast to this, distinct regulatory

patters were observed with lipid profi les. Briefl y, in the alo group, signifi -cant reductions of non-HDL-C (-9.8%, p<0.005) and LDL-C (-9.2%, p<0.05) levels were observed. Changes of ( )HbA1c levels were weakly correlated with those of ( )non-HDL-C (R=0.291) or LDL-C (R=0.327). By contrast, non-HDL-C and LDL-C levels had little, if any, changes in the sita group. Other parameters including TG, HDL-C, FFA and body weight had no change in both groups. Besides these lipid parameters, HOMA-B increased in both groups (+56.3% for alo and +89.7% for sita) with signifi cant inter-group differences (p<0.05). Both drugs are well tolerated and no clinically signifi cant adverse events were observed. In conclusion, this study suggests that: 1) Both sita-gliptin and alogliptin are effective and safe as an initial therapy for T2DM. 2) Alogliptin, but not sitagliptin, can ameliorate atherogenic lipid profi les. Further, the atherogenic lipids may be associated with the glycemic effect during alogliptin treatment. 3) Although similar glucose lowering effi cacies were observed, sitagliptin had a higher degree of enhancing beta-cell func-tion than alogliptin.

636-PCardiovascular Risk Management in Diabetes PatientsDOO MAN KIM, SHIN GON KIM, WON YOUNG LEE, CHONG HWA KIM, CHUL SIK KIM, DONG HYEOK CHO, GYU JANG WON, YOUNG JOO KIM, Seoul, Repub-lic of Korea, Gyeonggi-do, Republic of Korea, Gwangju, Republic of Korea, Daegu, Republic of Korea

Current guidelines on cardiovascular (CV) disease prevention recommend targeted management after assessment of CV risks using many of available method even if the patient is asymptomatic. This study was performed to explore how CV high risk is differently detected between 2 distinct meth-ods: non-invasive test (NIT) and risk calculation and how the awareness of CV high risk impacts physician and patient behavior for risk management in diabetes patient.A prospective, observational study was carried out in 22 hospitals in Korea. 622 type 2 diabetes patients aged 40 years were as-sessed by Carotid Ultrasound (CUS) and United Kingdom Prospective Diabe-tes Study (UKPDS) risk engine. CV high risk from CUS was defi ned as carotid intima-media thickness 1mm or plaque presence. Before and 6months af-ter the test, patients completed a questionnaire on health-related behaviors and physicians collected data on treatment patterns via chart review.Among 622 (mean age: 60.02±9.50 years), 271 (43.5%) and 66 (10.6%) patients were stratifi ed as CV high risk from CUS and UKPDS, respectively. Approximately 40% of patients at moderate and low risk from UKPDS were determined as high risk from CUS. The awareness of high risk from CUS altered physi-cian’s treatment patterns (P=0.021) for managing major CV risk factors: blood pressure (BP) and lipid. Along with CUS, risk levels by UKPDS also impacted physician’s behavior: more changes of treatment pattern in high than in low risk level for BP (12.0 vs 15.6 %) and lipid (13.8 vs 21.6 %). Patients noted increased health-related behaviors: smoking cessation and dietary changes (P<0.005, respectively) in 6 month follow up than before CUS and bigger in high risk.This study identifi ed NIT could detect more CV high risk patients than risk calculation. However, awareness of CV high risk itself has a posi-tive impact on physician and patient behavior, regardless of assessment methods. Therefore, assessing CV risks using varied methods with the pa-tient could be better for risk management in diabetes patients.

Supported by: Pfi zer Pharmaceuticals Korea Ltd.

637-PA Multi-Centric, Prospective, Randomized, Double Blind Study to Evaluate the Safety and Effi cacy of 2mg and 4mg of ZYH1 Compared to Placebo in Hypertriglyceridemia With Type II Diabetes Not Con-trolled With Atorvastatin TherapyRAJENDRAKUMAR H. JANI, DHIRAJ GAMBHIRE, GUNJAN JARIWALA, Mumbai, India

ZYH1 is a new, predominantly PPAR alpha agonist and developed by Zy-dus Cadila. It demonstrated favorable anti-dyslipidemic and safety profi le in phase 1 and 2 studies. In this randomized, double blind, placebo control, ICH GCP compliant phase 3 study, either 2mg ZYH1, 4mg ZYH1 or placebo was given orally once daily (OD) for 12 weeks to subjects with diabetic dyslipi-demia (DD). All the subjects were on Atorvastatin (Ato) 10 mg OD for mini-mum of 4 weeks before enrolment and had Triglyceride (TG) 200-500 mg/dl. ZYH1 2mg + Ato 10mg, ZYH1 4mg + Ato 10mg and placebo + Ato 10mg reduced TG by 44.9%, 47.2% and 25% respectively. Other lipid markers and fasting plasma glucose were also corrected favorably in ZYH1 arms (Table). The frequency of mild to moderate adverse events was comparable in ZYH1 2 mg (11%), ZYH1 (16%) and Placebo (9.8%). Gastritis was frequent in ZYH1 (4%). ZYH1 plus Ato was better than Ato alone for the control of DD.

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FOOT CARE—LOWER EXTREMITIES

Table: Per protocol analysis ZYH1 2mg, ZYH1 4mg and Placebo in addition to Ato 10mg in DD (*P < 0.05)Laboratory Parameter Visit ZYH1 2 mg(N=80)

Mean ± SD (% change)

ZYH1 4 mg(N=78)Mean ± SD (%

change)

Placebo(N=83)Mean ± SD (%

change)Triglyceride (mg/dL)

Baseline 273.8 ± 79.18 284.9 ± 87.80 284.7 ± 80.15

Week 12

149.7 ± 70.07(-44.9*)

142.1 ± 67.26(-47.2*)

204.0 ± 97.13(-25)

HDL-Cholesterol (mg/dL)

Baseline 36.2 ± 7.87 38.9 ± 11.28 38.6 ± 12.32

Week 12

40.0 ± 11.48(9.9*)

39.9 ± 8.80(8.1*)

37.0 ± 7.88(0.5)

Apolipoproteins A1 (mg/dL)

Baseline 134.3 ± 33.77 135.1 ± 29.31 138.7 ± 28.40

Week 12

155.6 ± 47.39(22.4*)

142.4 ± 48.57(9.2)

145.1 ± 49.32(9.2)

LDL-Cholesterol-Direct (mg/dL)

Baseline 131.5 ± 30.67 140.2 ± 29.38 140.2 ± 33.50

Week 12

97.0 ± 29.93(-26.2)

94.0 ± 27.60(-30.7*)

103.3 ± 30.53(-22.8)

Apolipoproteins B (mg/dL)

Baseline 97.6 ± 22.27 102.2 ± 21.40 103.4 ± 23.39

Week 12

71.0 ± 22.64(-26.6)

67.3 ± 19.29(-32.1*)

77.0 ± 18.79(-22.4)

Total Cholesterol (mg/dL)

Baseline 198.6 ± 38.30 209.9 ± 36.89 208.8 ± 39.35

Week 12

155.3 ± 36.10(-22.1)

151.9 ± 33.55(-25.8*)

168.4 ± 35.23(-17.4*)

Non-HDL Cholesterol (mg/dL)

Baseline 162.4 ± 37.47 171.0 ± 37.26 170.2 ± 41.51

Week 12

115.3 ± 35.91(-28.8*)

112.0 ± 33.52(-32.4*)

131.4 ± 36.49(-20)

Fasting Plasma Glucose (mg/dL)

Baseline 177.0 ± 66.29 175.1 ± 64.01 180.2 ± 68.47

Week 12

150.9 ± 75.28(-10.5*)

147.8 ± 60.75(-10.5*)

181.6 ± 94.38(7.4)


Guided Audio Tour: Walking on the Neuropathic Diabetic Foot (Posters 638-P to 645-P), see page 17.

& 638-PEarly Detection of Peripheral Neuropathy Using Virtual Obstacle CrossingBIJAN NAJAFI, GURTEJ GREWAL, RASHAD SAYEED, STEVE YASCHECK, YASER KHAN, ROBERT A. MENZIES, TALAL K. TALAL, Chicago, IL, Tucson, AZ, Doha, Qa-tar

This is a pilot study observing the effect of virtual reality technique on the early detection of diabetic peripheral neuropathy (DPN). We predicted that DPN patients would show a misjudgment of foot position during obstacle crossing, an altered reaction time, and an impaired balance.An innovative virtual obstacle crossing (VOC) paradigm using wearable sensors was de-veloped in attempts to detect lower extremity nerve damage due to DPN. Sixty-eight participants including diabetes with no, moderate and severe neuropathy and aged matched healthy controls were recruited. Severity of neuropathy was quantifi ed using vibratory perception threshold (VPT) values .The ability of perception of lower extremity was quantifi ed by measuring the rate of obstacle crossing success (OCS), reaction time (TR), and foot position while crossing a series of virtual obstacles with heights ranging from 5% to 20% of the subject’s leg length. Additionally, balance was as-sessed pre, during and post VOC trials.No signifi cant difference was found between groups for age and BMI (p>0.05). Results suggest VOC test allows separating between groups. The rate of OCS was signifi cantly reduced with increasing neuropathy severity (p<0.05). Results also suggest a signifi cant correlation between TR and VPT values (r=0.5, p<10-5). Finally, results sug-gest a signifi cant deterioration in balance due to diabetes, irrespective of

neuropathy severity (p<0.05).The results proposed the benefi t of virtual ob-stacle crossing as an objective method for detecting peripheral neuropathy at early stage. This is based on the reasoning that lower extremity proprio-ception decreases with increasing nerve damage. The increased reaction time, decreased OCS, and increased sway of the DPN patients in this study suggests their decreased proprioception, and, therefore, increased periph-eral nerve damage. Further studies should be addressed to compare VOC with other standard methods to confi rm whether VOC can detect DPN earlier than current methods.

Supported by: QNRF (NPRP08-499-3-109) and NIH (T35DK074390)

& 639-PNaltrexone Targets Angiogenesis Pathways to Accelerate Wound ClosurePATRICIA J. MCLAUGHLIN, JESSICA A. IMMONEN, IAN S. ZAGON, Hershey, PA

Delayed wound closure is a major complication of diabetes. These studies investigated an underlying biological axis, the opioid growth factor (OGF) - opioid growth factor receptor (OGFr) that has been shown to modulate epi-thelial cell replication. Blockade of opioid receptors from endogenous opioid peptides such as OGF (chemical name, [Met5]-enkephalin) which inhibit cell replication, by topical application of opioid antagonists such as naltrex-one (NTX), reversed delays in closure of 6 mm diameter full-thickness skin wounds in streptozotocin-induced type 1 diabetic (T1D) rats. Application 3 times daily of NTX decreased wound size over a 9 day period by more than 50% relative to residual wound area in T1D rats receiving vehicle. Histo-pathological evaluation of skin samples from T1D rats treated with NTX or vehicle, as well as normal tissue, has indicated that markers of angiogenesis were signifi cantly repressed in T1D rats, but were markedly elevated in T1D rats receiving NTX, relative to normal levels. Granulation tissue was evalu-ated periodically between 3-10 days following wounding using antibodies specifi c for fi broblast growth factor (FGF), vascular endothelial growth factor (VEGF), and alpha smooth muscle actin ( -SMA). FGF, a marker for angiogen-esis initiation, was reduced 64% in T1D rats on day 3, but elevated to normal levels in T1D rats receiving NTX. The number of VEGF- positive vessels was signifi cantly increased more than 2-fold in T1D rats receiving NTX relative to T1D-vehicle rats on days 3, 5, and 8. Moreover, the number of -SMA positive cells in T1D skin was markedly (p<0.05) elevated in tissue topically treated with NTX on days 3, 5, 8, and 10 relative to T1D-vehicle specimens. These data suggest that blockade of the OGF-OGFr axis with opioid antago-nists alters angiogenesis, an underlying physiological pathway associated with wound healing in diabetes, and provides additional support for the use of NTX as a non-toxic, effective, topical therapy for wound closure.

& 640-PH2S Improves Wound Healing via Restoring EPCs Functions in Type 2 Diabetic MiceFANG LIU, DANDAN CHEN, JIEMEI WANG, FUWANG LI, MINGWEI ZHANG, WEIPING JIA, ALEX F. CHEN, Shanghai, China, Pittsburgh, PA

Angiogenesis is crucial to wound healing in diabetes. Hydrogen sulfi de’s (H2S) pro-angiogenic effects have been recently reported. This study inves-tigated the effectiveness and possible mechanism of H2S donor in improving wound healing in db/db diabetic mice.A 6mm dorsal wound was made on male db/db and their matched control mice. The db/db mice were treated with sodium hydrosulfi de (NaHS), 4-hydro- xythiobenzamide group (HTB), or saline; The control mice were treated with DL-PAG or saline for 18 days. Wound size was recorded every other day; skin tissue in wound area and the plasma were harvested, and endothelial progenitor cells (EPCs) were cul-tured from bone marrow cells. Plasma H2S concentrations were measured. EPC tube formation and adhesive function after in vivo H2S therapy and invitro treatment with NaHS, PAG, or si-CSE were determined. The expres-sion of CSE, CBS,VEGF, and angiopoitien-1 (Ang-1) in skin tissue and EPCs were detected by Western blotting.The plasma H2S levels of db/db mice was signifi cantly lower than controls (P<0.01), and signifi cantly increased in the NaHS and HTB groups compared to db/db controls(both P<0.01). The wounds in NaHS and HTB groups healed signifi cantly faster than db/db group (both P<0.01) from day 4 to 16. PAG delayed the wound closure rate in the db/+ mice(P<0.05). The tube lengths of EPCs and the adhesive EPC numbers in-creased in NaHS and HTB groups, but decreased in PAG group. The tube formation and adhesion of EPCs from db/+ mice reduced signifi cantly after in vitro PAG intervention (both P<0.01) or CSE silencing (both P<0.01). Con-trarily, the tube length of EPCs increased signifi cantly in EPCs modifi ed by NaHS than in db/db EPCs (all P<0.01). The expression of Ang-1 and VEGF in wound skin tissue and in EPCs were up-regulated in the NaHS and HTB

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groups compared to db/db controls (all P<0.05).We concluded that H2S ac-celerates wound healing in diabetic mice; its effects are related to restore the EPC angiogenic function.

& 641-PFluctuating Sensorimotor Responses in Routine Foot Screening As-sessments—Results from the West of Ireland Diabetes Foot StudyADAM GARROW, LORNA HURLEY, LAURA KELLY, LIAM GLYNN, CAROLINE MCINTOSH, SEAN DINNEEN, Salford, United Kingdom, Galway, Ireland

Sensorimotor testing is an established part of diabetic foot screening. This analysis examined the variability of common screening tests over time.563 primary care patients attended a standardised foot ulcer risk assessment including light touch (10g monofi lament); sharp/blunt; temperature; vibration perception (VPT) and ankle refl ex response. 377/563 (67%) patients attended a follow-up assessment 19 (sd 2.1) months after the baseline visit. All as-sessments were carried out by health care professionals trained in senso-rimotor screening. The analysis examined changes in response in each of the clinical tests between baseline and follow-up. Kappa (k) statistics show the level of agreement between the two time points over and above what would be expected by chance. K values <0.2 indicate Poor; 0.2-0.4 Fair & 0.4-0.6 Moderate agreement. The proportion of patients with ‘normal’ sensation at baseline and also at 19 months was sharp/blunt 87.3%; monofi lament 82.3%; VPT 80.3%; temperature 72.5%; and ankle refl ex 69.8%. In contrast, a large proportion of patients with sensorimotor dysfunction at baseline were re-corded as having “normal” sensation at follow-up: sharp/blunt 43.9%; mono-fi lament 41.8%; VPT 43.9%; temperature 36.9%; and ankle refl ex 49.6%. The large variability was refl ected in Kappa values which suggested only fair to moderate levels of agreement between the baseline and follow-up assess-ments. (sharp/blunt 0.42; monofi lament 0.50; VPT 0.43; temperature 0.36 & ankle refl ex 0.28).Whilst instrument measurement error and inter-observer variability may partly explain differences between baseline and follow-up measures, the observed improvements in sensorimotor function were un-expected and perhaps counter-intuitive. The results may refl ect fl uctuations in peripheral nerve function in patients with partial rather than complete sensory loss. Caution is required when interpreting the results of clinical sensorimotor tests in diabetes.

Supported by: Diabetes Federation of Ireland, The Health Research Board

642-P

& 643-P25-Hydroxyvitamiv D [25(OH)D] Levels and Diabetic Foot Ulcer: Is There any Relationship?MOHAMMAD ZUBAIR, JAMAL AHMAD, ABIDA MALIK, Aligarh, India

In recent years, there has been an effort to understand possible roles of 25(OH)D, including its role in the immune system particularly on T cell-medicated immunity, pancreatic insulin secretion and insulin action. 25(OH)D stimulates the cell differentiation and reduces cell proliferation, which is essential for cell growth and wound healing. However, data on the associa-tion between low level of plasma 25(OH) D and diabetic foot syndrome are scarce. Circulating plasma 25(OH) D levels were measured in diabetic pa-tients with ulcer (n=162) and without ulcer (n=162) in a case control study. Of these patients, 85.1% had type 2 diabetes. Subjects with diabetic foot ulcer showed lower median plasma level of 25(OH)D [6.3(4.2-11.1) vs. 28.0(21.4-37.0)]ng/ml after adjusting the age and BMI. Regardless of the low levels of 25(OH) D in cases and controls, it was associated with neuropathy, sex (female), duration of ulcer healing, and smoking status and independent of confounding factors, including BMI (kg/m2), A1c (%), hypertension, neph-ropathy, foot ulcer, retinopathy, CAD, PAD, HDL-C (mg/dl) and LDL-C (mg/dl). The factors which predict the risk of developing ulcer independent of 25(OH)D status were BMI (>25kg/m2) [OR 20.18; RR 1.45], A1c (>6.9%) [OR 4.37; RR 1.77], HDL-C(<40mg/dl) [OR 1.16; RR 1.07], LDL-C (>100mg/dl) [OR 1.07; RR 1.03], triglycerides(>200mg/dl) [OR 1.40; RR 1.19], neuropathy [OR 6.88; RR 3.12], retinopathy [OR 3.34; RR 1.91], hypertension [OR 1.64; RR 1.28], nephropathy [OR 3.12; RR 1.87] & smoking [OR 4.53; RR 2.99] using odds and risk ratios. In conclusion, it is not clear whether the suppression of delayed wound healing seen during 25(OH)D defi ciency is due to the secondary ef-fect or is a direct action of vitamin D on certain components of the immune system. Long-term randomized trials are needed to see the impact of vitamin D supplementation on the outcome of diabetic foot patients

& 644-PSerum Magnesium Levels Are Signifi cantly Correlated With the Se-verity of Infl ammation in Patients With Diabetic FootSALIM OZENC, SIRZAT YESILKAYA, MUSTAFA CAKAR, EROL ARSLAN, SEREF DEMIRBAS, BAYRAM KOÇ, KENAN SAGLAM, Ankara, Turkey

Hypomagnesemia is not an uncommon fi nding in patients with uncon-trolled diabetes mellitus. It is associated with abnormal platelet action and the development of neuropathy, both of which are risk factors for the evolution of ulcers of the feet. A lately suggested data from animal studies shows that during chronic hypomagnesemia, loss of neutral endopeptidase activity contributes to neutrophil activation. In another, high CRP levels were found associted with lower magnesium levels in healthy groups. We aimed to investigate the correlation between serum magnesium levels and indica-tors of infl ammation in diabetic foot patients.Our study was made on the total of 88 patients; 57 diabetic foot patients and 31 controls. Median age of patient and control groups was 64 and 51, respectively. 25.8 % (n = 7) of controls and 17.5 % (n=10) of patient group were female. Wagner grade of the patient’s foot ulcers were grade 2 on 18 patients and grade 1, 3 and 4 on each 13 patients. Serum magnesium levels of diabetic foot patients (me-dian:1,92 mmol/l) were signifi cantly lower than controls (median:2.1 mmol/l) (p<0.05). Sedimentation levels of diabetic foot ulcer patients (median: 38 mm/hr) were signifi cantly higher than controls (median: 14 mm/hr)(p<0.05). Serum CRP levels of diabetic foot patients (median: 18.0 mg/l) were signifi -cantly higher than controls (median: 13.8 mg/l)(p<0.05). On Pearson correla-tion analysis, there was a signifi cant negative correlation between serum magnesium levels and serum CRP levels of the patient group (p<0.05).Serum magnesium depletion is present and shows a relationship with develop-ment of ulcers in subjects with type 2 diabetes. It has lately been proposed that severe hypomagnesemia is associated with low grade infl ammation in metabolic syndrome and cardiovascular infl ammation. In the literature, to our knowledge, our study is the fi rst to say that serum magnesium levels are signifi cantly associated with serum CRP levels in diabetic foot patients.

& 645-PPsychological Stress and Diabetic Foot Ulcer Healing: Preliminary FindingsLORETTA VILEIKYTE, BIING JIUN-SHEN, LAURA CAMPBELL, ANDREW J. BOUL-TON, MATTHEW J. HARDMAN, Manchester, United Kingdom, Athens, OH

Research indicates that psychological stress (PS)-induced immunomodu-lation delays acute wound repair. Here we explored the potential role of PS in the more complex diabetic foot ulcer (DFU)-healing paradigm. Ninety three type 2 DM patients (84% male; mean age 57yrs) with plantar neuropathic

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DFU (University of Texas Classifi cation: 69% grade 1A; 11% 1B; 16% 2A; and 4% 2B) completed baseline self-report measures of generalized (Perceived Stress Scale, PSS; Hospital Anxiety and Depression Scale, HADS; and State-Trait Anger Expression Inventory, STAXI) and DFU-specifi c PS (NeuroQoL-In-terpersonal Burden (NeuroQoL-IP) and Patient Interpretation of Neuropathy (PIN) Scales: PIN-Amputation Worry and PIN-Anger at Docs). DFU-specifi c biomarkers (IL6, IL1-beta, MMP2 and MMP9) were determined via quanti-fi cation of immunohistochemical tissue localization and normalized biopsy gene expression. Systemic biomarkers (IL6 and IL1-beta) were measured from patient serum via ELISA. Bivariate analyses revealed multiple mea-sures of increased generalized and DFU-specifi c PS were associated with: a) decreased local IL1-beta at baseline: HADS-Depression (r=-.27; p<.001) and NeuroQoL-IP (r=-.38; p<.01); b) decreased MMP9: HADS-Depression (r=-.29; p<.05); PIN-Worry (r=-.34; p<.05) and PIN-Anger (r=-.37; p<.01); c) in-creased MMP2: PIN-Worry (r=.32; p<.05) and PIN-Anger (r=.31; p<.05). STAXI was associated with higher levels of baseline systemic IL6 (r=.32; p<.01). Intriguingly, greater than 80% DFU area reduction at 6 weeks was less likely in patients reporting more severe PIN-Worry (r=-.36; p<.01) and PIN-Anger (r=-.30; p<.01) and associated with higher baseline levels of systemic IL6 (r=-.29; p<.05) and local MMP2 (r=-.32; p<.05). These preliminary data iden-tify potential psychological stress-induced biomarkers linking stress to DFU chronicity.

Supported by: 5-R01-DK07-1066-05

646-PCathepsin D, Adiponectin, TNF- , IL-6 and hsCRP Plasma Levels in Subjects With Diabetic Foot and Possible Correlation With Clinical Variables: A Multicentric StudyJAMAL AHMAD, MOHAMMAD ZUBAIR, ABIDA MALIK, MOHAMMAD A. SID-DIQUI, SUBHASH K. WANGNOO, Aligarh, India, New Delhi, India

Diabetic foot, characterized by a pronounced infl ammatory reaction, de-creased collagen content and biosynthesis and accelerated degradation are crucial in wound healing. Cathepsin D has been implicated in cell growth, apoptosis and its inhibitor to reverse the inhibition of collagen biosynthesis in wounded rat skin with diabetes. To date, the increased proteolytic activity of cathepsin D in diabetic foot has not been evaluated and the pathogenic signifi cance of this infl ammation has received little attention. Circulating levels of cathepsin D, acute-phase reactant and cytokines were measured in diabetic foot ulcer (DFU) (n=211) and without ulcer (n=208). Of the patients, 89.73% had type 2 diabetes. Subjects with DFU showed higher median plasma level of Cathepsin D [556.3(312.6-587.3) vs 306.3(92.6-337.3)] RFC/ml, TNF- [96.6(79.9-121.5) vs 8.4(7.1-9.20)] ng/ml, IL-6 [32.2(8.52-48.4) vs 4.9(4.5-5.6)] ng/ml, hsCRP [12.6(11.2-14.1) vs 3.90(3.50-4.60)] mg/ml and lower median plasma levels of adiponectin [8.50(7.10-9.5) vs 13.3(12.1-14.2)] ng/ml. A positive correlation was found between grades of ulcer, BMI, A1c and retinopathy for cathepsin D, for adiponectin, between grades of ulcer, BMI, retinopathy, nephropathy & smoking, for IL-6, between grades of ulcer, BMI, nephropathy, CAD & smoking, for hsCRP, grades of ulcer, BMI, LDL-C, nephropathy & smoking while total cholesterol, nephropathy, PAD, smoking and neuropathy for TNF- . Diabetic subjects with various grades of DFU showed a higher plasma cathepsin D, IL-6, hsCRP, TNF- and lower adi-ponectin levels in comparison with diabetes without foot ulcer independent of the concomitant infections. It would be interesting to fi nd out whether an activation of immune system precedes the development of foot ulcer and whether cathepsin D inhibitor & anti-infl ammatory therapies will be effec-tive in improving the outcome in such patients

647-PAll Negative Pressure Dressings Are Not Equal: A Comparison of Negative Pressure Therapies in Porcine Wound Healing ModelNICHOLAS J. TANNOUS, DEBORAH NOBLE, EDWARD ROY, JIYING HUANG, GEORGETTE ONI, IMELDA DELGADO, GAURAV THAKRAL, LAWRENCE LAVERY, KATHRYN DAVIS, Dallas, TX

Negative pressure dressings are now a common modality in both inpa-tient and offi ce based wound care. There are, however, several available options with varying economic implications. Variations in dressing type and suction pressure may have effects on the wound both on macroscopic and microscopic levels. This study looks at the outcome on wound healing in a porcine model when comparing the lower pressure Convatech negative pres-sure dressing to the higher pressure KCI system.Five animals were included in this IACUC approved study. For each animal, three circular full thickness wounds, 5 cm in diameter, were surgically created down to fascia on their dorsum. One wound was covered using the Engenex® Bio-dome™ technol-ogy from Convatec, which consists of negative pressure wound therapy at

75 mm Hg. The second was covered using V.A.C.® therapy from KCI at a negative pressure of 125 mm Hg. The third was a control wound that was covered with a thin layer of Medline Skin Integrity® Hydrogel, followed by a non-occlusive gauze dressing. The area of each wound was recorded twice weekly over a 21 day period.KCI wounds were signifi cantly smaller on day 3 than the control (p= 0.036) and Convatech (p=0.048). By day 10 there were no signifi cant differences between control wounds and negative pressure dressings (p= 0.7455). By day 21 control wounds were signifi cantly small-er than wounds treated with negative pressure systems (p= 0.0434). KCI wounds were smaller than Convatech wounds by day 21 but not signifi cantly so (p=0.23).This study demonstrates that there are signifi cant differences between negative pressure systems. This difference may be attributable to the suction pressure. After 10 days the positive effect of these dressings appears to be reduced. This has implications for clinical practice and further studies are warranted to investigate the optimum timing and pressure for negative pressure wound therapy.

648-PObstructive Sleep Apnea is Associated With Oxidative Stress and Microvascular and Endothelial Dysfunction in Patients With Type 2 DiabetesABD A. TAHRANI, KIRAN DUBB, ASAD ALI, MANJIT SINGH, ANTHONY H. BARNETT, MARTIN J. STEVENS, Birmingham, United Kingdom, Coventry, United Kingdom

We aimed to assess the impact of OSA on oxidative stress (OS) and mi-crovascular function in adults with T2D. Patients were recruited randomly from the diabetes clinic of a UK hospital. OSA (apnoea hypopnea index (AHI)

5 events/h (OSA+))was assessed using home based multi channel device (Alice PDX, Philips Respironics, USA). Microvascular skin fl ow was assessed using laser speckle contrast imaging (Moor Instruments Ltd, UK) at the mid thigh level. OS was assessed by measuring serum 3-nitrotyrosine (NT) (nM) and plasma lipid peroxide (LP) (μM/ml).Patients (n=102) were included (73 OSA+). NT (23.5 (16.7-36.1) vs. 15.5 (11.5-24.3), p=0.007) and LP (18.4 (8.3-37.4) vs. 7.9 (0.8-22.8), p=0.01) levels were higher in OSA+ patients and cor-related with OSA severity (Table 1).After adjustment for a wide range of variables, OSA remained an independent predictor of NT (B=0.16, p=0.01) and LP (B=1.09, p=0.03) levels, OSA+ patients had lower blood fl ow and im-paired endothelium responses (Table 2).OSA increases OS and is associated with impaired microvascular function which might contribute to the devel-opment of microvascular complications. Trials examining the impact of OSA treatment on these parameters are needed.

Table 1: The correlation between oxidative stress measures and OSA. ODI: Oxygen desaturation Index

AHI ODI Time spent with oxygen

saturations < 80%

Nadir nocturnal oxygen

saturationsSerum nitrotyrosine r=0.38,

p<0.001r=0.36,p<0.001

r=0.25,p=0.01

r=-0.23,p=0.02

Plasma lipid peroxide r= 0.19, p=0.06

r=0.22,p=0.03

r=0.25,p=0.02

r= -0.24,p=0.02

Table 2: Data presented as median (IQR). P values adjusted for age, BMI and diabetes duration

OSA-(n=24)

OSA+(n=47)

P value-unadjusted

P valueadjusted

Baseline fl ux 35.60(26.75-42.37)

30.80(15.70-20.70)

< 0.001 < 0.001

Heating induced fl ux 168.90(133.20-209.05)

154.60(129.30-192.80)

0.405 0.726

Acetylcholineinduced fl ux

130.95(99.07-177.52)

99.30(68.90-120.20)

0.01 0.029

Na nitroprusside induced fl ux

146.50(117.05-204.62)

103.00(62.30-135.10)

0.001 < 0.001

Baseline conductance (measured as fl ux/Mean arterial BP)

0.40(0.28-0.48)

0.20(0.16-0.31)

< 0.001 < 0.001

Acetylcholineconductance

1.43(1.09-1.83)

1.07(0.75-1.29)

0.002 0.023

nitroprussideconductance

1.61(1.15-2.14)

1.16(0.62-1.41)

0.001 < 0.001

Supported by: NIHR (UK)

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649-PStudy of Heat Shock Protein Hsp47 Expression in Chronic Wound of Controlled and Uncontrolled Type 2 DiabetesMellitusDEEPA POKHARIA, HEMANT KUMAR, MANISH MISHRA, AWADHESH K. ARYA, RICHIK TRIPATHI, RAKESH K. SINGH, MOHAN KUMAR, KAMLAKAR TRIPATHI, Varanasi, India, Kingstown, Saint Vincent and the Grenadines

HSP47, a collagen binding stress proteinplays a vital role in procollagen processing during collagen synthesis. This studyreveals the possible ef-fect of type 2 diabetes mellitus (T2DM) on Hsp47 expression in fi broblast cells. Forthis 50 T2DM subjects having chronic wound and 20 non diabetic subjects havingwound had been registered. They were divided into three groups 20 subjects werenon diabetic having wound (G1), 25 subjects were controlled diabetic having wound (G2)(HbA1c %< 7.0, FBS<7.7mmol/l) and 25 subjects were uncontrolled diabetic havingwound (G3) (HbA1c %> 7.0, FBS>7.7mmol/l). They were evaluated forglycemic control (FBS, PPBS, HbA1c %) and expression of Hsp47 at wound area ofthe subjects. Biochemical tests were analyzed by Syncron CX5 autoanalyzer. Expression ofHsp47 were as-sayed by immunohistochemistry method and the intensity of immunoreac-tivity of Hsp47 was evaluatedaccording to a scale of zero no expression, 1faint,2 moderate and 3 strong expression. Group G3 shows the poor expres-sionof Hsp47 (score 1+), Hsp47 expressed moderately in group G2 score 2+ and groupG1scored 3+ for Hsp47. Hsp47 expressed strongly in dermal fi bro-blastsof the granulation tissue of G1. G2 express Hsp47 moderately while G3 express minimally.Our studies indicate that Hsp47 mediates aberrant col-lagen homeostasis inuncontrolled T2DM wound; this may be due to dysfunc-tion of fi broblast cells. Suchimpairments could contribute to delay wound healing. Several pharmacologicaland genetic approaches may be considered to address Hsp47 directed therapies fortreating non healing wound.

Supported by: ICMR-SRF

650-PHbA1c and Long-Term Mortality in Patients With Diabetic Foot UlcerMAGNUS LÖNDAHL, KATARINA FAGHER, ANDERS NILSSON, Lund, Sweden, An-gelholm, Sweden

Recommendations for glycemic control in patients with cardiovascular disease have been debated. The aim of this study was to evaluate the im-pact of HbA1c on long-term mortality in the high-risk group of patients with diabetic foot ulcers. 8-year mortality was evaluated in a consecutive and prospectively registered group of type 2 diabetes patients, younger than 80 years, visiting a multidisciplinary diabetic foot clinic with an ulcer that did not heal within a period of 4 weeks treatment. Of the 223 eligible patients nine were excluded due to lack of HbA1c and the remaining 214 patients (37.9% females, age 69.1 (63-76) years) grouped according to their baseline HbA1c (<7.5% (58mmol/mol) (n=81, group 1), 7.5-8.9% (n=70, group 2) and 9.0%- (n=63, group 3)). There were no differences between groups in sex distribution or prevalence of myocardial infarction, heart failure, hyperten-sion, dyslipidemia, end-stage renal disease or peripheral vascular disease, but patients in group 3 were younger than those in group 1. Eight-year mor-tality is given in fi gure 1 and age adjusted HR for mortality are given in table 1. Despite similar baseline cardiovascular co-morbidities, HbA1c below 7.5% was associated with increased mortality in this study population of patients with diabetic foot ulcer.

Table 1HR (95% CI)

HbA1c <7.5% vs HbA1c 7.5-8.9

p-value HR (95% CI)HbA1c <7.5% vs

HbA1c 9.0%-

p-value HR (95% CI)HbA1c <7.5% vs

HbA1c 7.5%-

p-value

2-year mortality 2.20(1.09-4.46) 0.03 1.83(0.89-3.70) 0.10 1.91(1.08-3.39) 0.0264-year mortality 1.84(1.12-4.46) 0.02 1.96(1.14-3.40) 0.02 1.83(1.19-2.80) 0.0066-year mortality 1.46(0.95-2.25) 0.08 1.64(1.02-2.63) 0.04 1.49(1.02-2.16) 0.048-year mortality 1.43(0.96-2.13) 0.08 1.51(0.98-2.32) 0.06 1.42(1.01-2.02) 0.047

Supported by: Thelma Zoégas Foundation, Swedish Diabetes Foundation, R&D County of Skane

651-PUse of Biomarkers of Oxidative Stress as Indicators of Risk Factor for Diabetic FootCAIO J. TEIXEIRA, ANA CARLA P. OLIVEIRA, TALITHA F. STEFANELLO, MÁRCIA A. CARRARA, ANACHARIS B. SÁ-NAKANISHI, JURANDIR F. COMAR, CARLOS A. SANTOS, ROBERTO B. BAZOTTE, MÁRCIA R. BATISTA, Maringá, Brazil

We investigated the role of oxidative stress markers as indicators of risk of injury of the lower extremities in patients with type 2 diabetes mellitus. In 29 patients were assessed the following parameters: 1. Anthropometric measures for calculating the Body Mass Index (BMI); 2. Glycated hemoglo-bin (HbA1c); 3. Blood markers of oxidative status (total antioxidant capacity - TAC, reduced protein thiols and levels of carbonylated proteins), and 4. Test of sensation in the feet (National Hansen’s Disease Program - U.S.). Patients were divided into two groups according to the index of sensation: group 1 (low risk - 18 patients) and group 2 (high risk of injury - 11 patients). The metabolic evaluations were performed at two moments in each group, baseline and later (110 days). The results were: 1. The group 1 had a mean age 56.9±8.7 years and the mean duration of disease of 8.8±6.9 years, while group 2 had a mean age of 63.4±7.6 years and the mean of disease duration of 18.8±10.4 years (P<.001); 2. According to BMI, the group 2 was framed as obese (BMI=32.4±6.7 kg/m²) and insulin users, while group 1 was classifi ed as overweight (BMI=29.54±4.3 kg/m²) and users of oral antidiabetics; 3. The group 1 presented an increase of HbA1c (%) during the study period (from 5.8 to 7.4, P=.0002), while group 2 showed only a trend to increase (from 8.1 to 8.9, P=.1748); 4. The TAC (mg/plasma albumin) in group 1 ranged from 21.9 to 17.3 (P<.0001) and group 2 from 20.8 to 16.7 (P=.0020). For thiols groups (mg/ mol albumin), in group 1 ranged from 11.4 to 10.3 (P=.0013) and group 2 from 10.2 to 10 (P=.3652). Concerning the levels of protein carbonyls (mg/

mol albumin), in group 1 ranged from 4.4 to 7.3, (P<.0001) and group 2 from 4.7 to 7.5 (P=.0029). The results suggest that the evaluation of the plasma levels of carbonylated proteins, TAC and reduced protein thiols could furnish additional information to prevent the risk of diabetic amputation considering that the alteration of these biomarkers was associated with loss of sensitiv-ity and foot ulcerations.

652-PInitial Vascular Response to CLEAR Cleat Cycling in Patients at Risk for DFURYAN T. CREWS, STEVEN R. SMITH, SHANNON B. LIU, Chicago, IL

Exercise has been linked to improved wound healing. Potential mecha-nisms for this improvement are increases in skin blood fl ow and skin oxy-gen tension. While diabetic foot ulcers (DFU) are extremely diffi cult to heal, typical wound locations prohibit most types of exercise. The CLEAR Cleat was designed to allow DFU patients to safely participate in aerobic exercise by minimizing physical stress to the wound (offl oading).This study sought to determine whether CLEAR Cleat cycling increased blood fl ow to the forefoot.Ten subjects with diabetic peripheral neuropathy, with or without addition DFU risk factors, were recruited to participate (aged 51±8 y; BMI 32±6). Individuals with active DFU were excluded. Each subject completed two 5-minute stationary cycling sessions at the same self-selected cadence and resistance level. Standard athletic shoes and pedals were used during one session, and the CLEAR Cleat was used with one foot during the other

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session. Perfusion levels were examined at the hallux by surface laser Dop-pler probe (tpu - tissue perfusion units) and the forefoot by plantar surface temperature (infrared thermography) both pre and post each cycling bout.The cycling signifi cantly increased microcirculation at the hallux during both cycling conditions (pre 3.15±2.9 vs. post 6.8±6.3 tpu). Mean forefoot temper-ature did not differ between groups or pre (28.2±.7°C) vs. post (28.2±.8°C) exercise. With the exception of mild leg cramping and muscle tightness in two subjects, no adverse events occurred. The increased microcirculation in these high risk subjects indicates that the cycling may improve blood fl ow to forefoot DFU and therefore aid healing. The lack of change in the gross assessment of surface temperature may be due to the limited duration of cycling or the relatively low exertion level by the subjects. Future studies utilizing subjects with active DFU should look at the use of the cleat in a rou-tine exercise regimen. They may determine if the thermal response changes over multiple cleat sessions.

Supported by: NIH (T35DK074390)

653-PIntravenous Antimicrobial Therapy of Diabetic Foot Ulcers: An Ef-fective Outpatient Program in TanzaniaZULFIQARALI G. ABBAS, JANET LUTALE, LENNOX K. ARCHIBALD, Dar es Salaam, United Republic of Tanzania, Gainesville, FL

Diabetic foot ulcer infection cause substantial morbidity and mortality, prolonged hospital stays, and increased hospital costs in Tanzania. Thus, we initiated an outpatient program for (IV) antimicrobial therapy in two out-patient clinics in Dar es Salaam, Tanzania. During February 2005 -December 2011 (study period), diabetes patients with infected foot ulcers were evalu-ated then started on IV antimicrobial therapy administered twice daily for 10-15 days depending on severity. Treatment included various combinations of second and third-generation cephalosporins, penicillins, macrolides, qui-nolones, and anti-anaerobic agents. Logistic regression analysis was carried out and adjusted odds ratio (AOR) and 95% confi dence intervals (CI) calcu-lated. Of 909 patients treated during the study period, 595 (65%) were male; median age was 55 years; 900 patients received an extended-spectrum third-generation cephalosporin and complete ulcer healing was attained in 760 (84%). Independent factors associated with complete healing were ulcer area <1,000 mm2 (AOR: 2.1, CI: 1.4-3.1) or concomitant receipt of an anti-anaerobic agent (AOR: 4.5, CI: 3.0-6.8). Independent factors associated with delayed ulcer healing included macrovascular disease (AOR: 2.1, CI: 1.4-3.2); hypertension (AOR: 1.6, CI: 1.04-2.4); and addition of amoxicillin/clavulanic acid (AOR: 2.7, CI: 1.5-4.7) or quinolones (AOR: 5.1, CI: 3.3-7.9) to the thera-peutic regimen. In conclusion, we established the feasibility of affordable, outpatient IV antimicrobial therapy for diabetic foot ulcer infection in Tan-zania. Best outcomes were documented for patients who received a single extended-spectrum third-generation cephalosporin with an anti-anaerobic agent. Augmentation of this regimen with penicillins, macrolides, or quino-lones, did not enhance ulcer healing rates. These fi ndings potentially have cost saving implications for the management of infected diabetic foot ulcers in Tanzania.

654-PDaily Foot Care Adherence and Patient EducationJINSUP SONG, GARY FOSTER, GUENTHER BODEN, DANA TANGO, REAGAN KANE, ALEXANDRA HANLON, JAMES FURMATO, Philadelphia, PAWhile studies have shown that diabetic education can improve foot care, many diabetic patients are experiencing non-traumatic lower limb amputa-tion. This study examines if a novel diabetic foot care education using per-sonal plantar pressures improves adherence to daily foot care. Study partici-pants (N=96) were at-risk type 2 DM patients (63.5% male; mean age 53.9 years; diabetic foot risk score: 36.5% 1; 47.9% 2A; 8.3% 2B; 2.1% 3A; 5.2% 3B). Study participants were randomly assigned to the control (45) or the in-tervention group (51). All participants received standard foot care brochures, but only the subjects in the intervention group were shown their barefoot plantar pressures and received an explanation as to why they need to in-spect their feet daily and wear protective shoes. All subjects received rou-tine foot care at baseline, 1, 3, 6, 9, and 12 months. Adherence to daily foot care behaviors was assessed using Footcare Behavior Questionnaire. While the subjects in the intervention group had greater adherence to daily foot inspection than the control group throughout the study period, a signifi cant difference was noted only at 3 month evaluation. No signifi cant difference was noted in barefoot indoor walking. Additional studies and independent measures of foot care are needed to evaluate if the proposed diabetic foot education based on personal plantar pressures yields improved adherence and clinical outcomes.

Table 1. Percentages of adherence by item, group, and visit. Item responses were compared by group according to the percent of responses that im-proved from baseline using Fisher’s Exact tests.

During the past week

Control Intervention

Visit Adherence(%)

N Adherence(%)

N p value

examinedfeet

baseline 64.4 45 70.6 51

daily or month 1 76.7 43 87.5 48 0.3376twice per day

month 3 71.1 38 88.9 45 0.0422

month 6 70.6 34 83.3 42 0.3715 month 9 65.5 29 83.8 37 0.1507 month 12 77.8 18 86.4 22 0.3047never walk baseline 46.7 45 49.0 51 barefootindoor

month 1 53.5 43 66.7 48 0.3048

month 3 52.6 38 71.1 45 0.1293 month 6 58.8 34 66.7 42 0.4219 month 9 69.0 29 81.1 37 0.1304 month 12 61.1 18 63.6 22 0.4117

655-PImplications of Plantar Loading in Gait on Microvascular Function in Diabetes and Pre-DiabetesJAMES A. FURMATO, JINSUP SONG, DANA N. TANGO, REAGAN KANE, Phila-delphia, PA

The relationship of walking to neuropathic foot ulcer (DNU) genesis is un-known. We hypothesize soft tissue property changes around the sub-papillary venous plexus delays onset of postocclusive hyperemic response (POHR) in in-dividuals with a history of DNU compared to healthy subjects. If similar chang-es are found in obese individuals (where pre-diabetes is prevalent), subclinical glycation may occur before it is recognized and addressed by a physician. We measured POHR to loads applied under the hallux in weightbearing subjects. Nine diabetic individuals with a history of DNU and 9 non-diabetic, healthy controls enrolled in a pilot study. The fi ndings were compared to 18 nondiabet-ic subjects in a weight loss study. We titrated each subject for the compres-sion needed to blanch and applied a 90s continuous load and 90 cycles pulsed load then followed each with 180s of refractory time. Laser Doppler signals were analyzed to fi nd the latency time between the end of occlusion and the initiation of POHR (TL) for the three groups. The mean TL for pulsed compres-sion was statistically greater in the diabetic group than in the control group (100.0±24.7 vs 68.71±-22.2 s; p=0.010). The TL for continuous compression was higher in the diabetic individuals as well (0.061±0.02 vs 0.047±0.020s; p=0.17). The obesity subjects showed mean pulsed TL=78.5±35.5s, longer than healthy controls, and continuous compression TL=0.060±0.031, closer to that of diabetic subjects suggesting deviation from normal. Prolonged TL in walking may lead to hypoxia in sensate individuals and DNUs in diabetic individuals. We suggest temporal sequencing of gait and a physiological test be used to indicate changes at the tissue and cellular level to mark progression of glyca-tion in diabetic and pre-diabetic feet.

DIABETES EDUCATION

Guided Audio Tour: Innovative Approaches that Foster Self-Management (Posters 656-P to 663-P), see page 17.

& 656-PSpecialty Trained Certifi ed Diabetes Educator (CDE) With Endocri-nologist Oversight Providing Metabolic Management (MM) With Diabetes Self-Management Training (DSMT) to Increase Rural Health Access in the Adult Patient With Uncontrolled Type 2 Diabe-tes Mellitus Improves HgA1c and Patient SatisfactionMARY A. JOHNSON, JOHN W. KENNEDY, H.L. KIRCHNER, JEFFERY M. BARRETT, Danville, PA

The increase in uncontrolled Type 2 DM, combined with a shortage of endocrinologists, led a rural health system in Pennsylvania to pilot the use

complications—hypoglycemia ada.pdf

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