insulin action—adipocyte biology category · insulin action/ molecular metabolism posters insulin...

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INSULIN ACTION—ADIPOCYTE BIOLOGY

lysis. Islet transplantation has emerged as a promising treatment for T1D, though its clinical application is constrained due to limited islet availability, adverse effects of immunosuppression on islet function, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to help preserve islet function and prevent immune-mediated rejection after transplantation into T1D patients. We have developed a novel cytoprotec-tive nanothin coating for islet encapsulation consisting of tannic acid, an immunomodulatory antioxidant, and hypothesize that islet encapsulation in tannic acid-containing multilayers will restore euglycemia in diabetic mice and delay immune-mediated rejection of islet transplants. Tannic acid-con-taining nanothin coatings signifi cantly reduced the synthesis of noxious in-nate immune-derived ROS and pro-infl ammatory cytokines, including TNF-α, IL-1β, and IL-12p70 from LPS-stimulated bone marrow-derived macrophages. More importantly, tannic acid-coatings resulted in blunted diabetogenic T cell activation with decreased TNF-α (2-fold, p<0.005) and IFN-γ (5-fold, p<0.001), hallmark Th1 cytokines that induce pancreatic β-cell destruction in T1D. Murine islets encapsulated in tannic acid-containing multilayers did not affect glucose-stimulated insulin secretion or compromise islet viability. These results highlight the exciting possibilities that tannic acid-encapsu-lated islets possess great promise in mediating islet allo- and xenograft ac-ceptance to restore euglycemia in T1D patients. Future studies will defi ne the fundamental roles of immune responses and polymer chemistry in islet transplantation to cure T1D.

Supported By: American Diabetes Association (7-12-CD-11 to H.M.T.); National Institute of Diabetes and Digestive and Kidney Diseases (R01DK099550)

& 1845-PBody Mass Index, Diabetes Type, and Mortality after Cardiac Trans-plantationRODOLFO J. GALINDO, YAEL T. HARRIS, AMISHA WALLIA, JOANNA STEIN, PRI-YATHAMA VELLANKI, GUILLERMO E. UMPIERREZ, Great Neck, NY, Chicago, IL, Atlanta, GA

Cardiac transplantation is a successful therapy with 81% 1-year survival. The impact of BMI and pre-transplant diabetes (PreTxDM) or new-onset dia-betes after transplantation (NODAT) on mortality after cardiac transplanta-tion is not known.

The United Network for Organ Sharing (UNOS) database was searched for fi rst-time adult cardiac transplant recipients with >1 year of follow-up from 07/01/2004 to 03/31/2014. Primary outcome was mortality after 1-year post transplantation.

Of the 8,673 cardiac recipients, 25.7% had PreTxDM, 13.4% had NODAT, and 60.9% had no diabetes (No-DM). According to BMI, group I (15- <18.5- underweight), II (18.5-<25- normal), III (25-<30- overweight), IV (30-35- obe-sity), V (>35-45- morbid obesity) represented 2.5%, 34.5%, 38.2%, 19.1%, and 5.7%, respectively. Eighty-eight percent were alive after 1 year, with a mean follow up time of 4 ±1.5 years (± SD, range: 1.0-9.4 years). Mortal-ity in patients with PreTxDM, NODAT and NoDM was 9.1%, 12.5%, 16.9%, respectively. Mortality in groups I to V was 16.8%, 10.6%, 10.7%, 13.5% and 14.4%, respectively. In multivariate analysis, mortality was higher in under-weight patients compared to normal BMI (odds ratio [OR]: 1.8, CI 1.2-2.6). Higher mortality rates were also seen in obese and morbid obese subjects, compared to normal weight and overweight, but differences were not signif-icant. Compared to No-DM, mortality was higher in patients with PreTxDM (OR 2.8, CI 2.3-3.3) and NODAT (OR 1.6, CI 1.3-2). Risk of mortality in patients with PreTxDM was higher than those with NODAT (OR 1.7, CI 1.4 - 2.2).

In summary, diabetes and BMI independently affect mortality in cardiac transplant patients. Underweight is associated with higher mortality, but the mortality impact of obesity is not signifi cant. Both PreTxDM and NODAT are associated with increased mortality after 1-year post-transplantation, with the highest odds in PreTxDM. More research is needed to determine weight and glycemic control management goals among cardiac transplant subjects.

1846-PPreservation of Beta Cell Function following Total Pancreatectomy and Islet AutotransplantationZEHRA TEKIN, OMID SAVARI, KAROLINA GOLAB, SABARINATHAN RAMACHAN-DRAN, LINDSAY SCHENCK, MARTIN TIBUDAN, W. JAMES CHON, MICHEAL J. MIL-LIS, JEFFREY B. MATTHEWS, ANDRES GELRUD, PIOTR WITKOWSKI, Chicago, IL

The aim of the study was to assess the number of patients remaining insulin free after total pancreatectomy and islet autotransplantation in our center.

Total pancreatectomy followed by islet autotransplantation was per-formed in 18 patients in age of 34.8 (11-60) and with BMI of 24.8 (18-35). Six-teen had chronic pancreatitis with intractable pain, remaining 2 benign pan-creas tumor or small ampullary cancer. Genetic mutations (PRSS1, SPINK1

or CFTR) were present in 10 (55%) patients. Two month exogenous insulin therapy was implemented after procedure to support islet graft recovery. Follow up was 23.5 (1-70) months.

The islet tissue pellet volume was 9.68 ml (2-30). Viability was 95% (81-98). Four recently transplanted patients are still under routine supportive insulin therapy. Other 7 (50%) are currently off insulin with excellent glucose control and HbA1c below 6. Remaining 7 individuals still require insulin in-jections, however none of them experiences “brittle” form of diabetes; no severe hypoglycemic episodes were reported. Transplanted beta cell mass was signifi cantly higher- in insulin free patients comparing to those with insulin therapy, 228 kIEQ (145k-330 kIEQ) vs. 140 kIEQ (40-292 kIEQ), respec-tively. Islet mass per patient body weight was also substantially higher in the same group- 3,444 IEQ/kg (1,611-4,824) vs. 2,346 IEQ/kg (556-4,375 IEQ/kg), respectively. Islet gradient purifi cation was applied in 4 cases and resulted in insulin independence in 3 individuals. BMI as well as time of chronic pan-creatitis prior to operation did not differ in patients who became insulin free and insulin dependent. None of the patients developed long-term complica-tions related to the islet transplant procedure.

Islet autotransplantation effi ciently preserved islet endocrine function in patients after total pancreatectomy allowing for insulin independence in half of them and stable glucose control in remaining. The success was cor-related with higher islet mass transplanted.


Guided Audio Tour: Fat in the Flames—Adipose Tissue Infl ammation (Posters: 1847-P to 1853-P), see page 13.

& 1847-PHyperinsulinemia Promotes Obesity-associated Adipose Tissue Infl ammationADILSON GUILHERME, DAVID J. PEDERSEN, LAURA V. DANAI, LAUREN HEYDA, JESSICA L. COHEN, JUERG STRAUBHAAR, SARAH M. NICOLORO, MICHAEL P. CZECH, Worcester, MA

In obesity, adipose tissue (AT) dysfunction is associated with infl ammation, systemic insulin resistance and hyperinsulinemia. However, the mechanisms that drive AT infl ammation in obesity and disrupt adipocyte metabolic func-tions are incompletely understood. Here we show that reducing circulating insulin levels about 50% in obese mice by streptozotocin or diazoxide treat-ments surprisingly suppressed the elevated adipose tissue infl ammation, as measured by a reduction of AT crown-like structures and by a decrease in macrophage markers and pro-infl ammatory cytokine expression.

This decreased cytokine expression in the AT of streptozotocin treated obese mice was associated with partial restoration of expression of en-zymes in the de novo lipogenesis (DNL) pathway and of 14C-glucose con-version into triglyceride-fatty acids. Lowering insulin levels in obese mice also enhanced insulin stimulated Akt protein kinase phosphorylation and restored insulin responsiveness of AT DNL. Moreover, responsiveness of blood glucose to injected insulin was improved by streptozotocin and diaz-oxide treatments without changes in body weight and blood glucose levels. Additionally, chronic insulin treatment in mature 3T3-L1 adipocytes led to a 10-fold increase in CCL2 mRNA level within 6 h. Interestingly, pre-treatment of 3T3-L1 adipocytes with the ERK inhibitor PD98059 ablated the insulin-induced increase in CCL2 expression, indicating that chronically elevated in-sulin promotes CCL2 expression in mature adipocytes in an ERK-dependent manner. Taken together, these results indicate that obesity-associated hy-perinsulinemia unexpectedly drives AT infl ammation in obese mice, which may in turn contribute to factors that suppress DNL and systemic insulin sensitivity.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases (DK085753, DK030898)

& 1848-PMedium from Human Monocyte-derived Macrophage Conditioned in High, but Not Normal, Glucose Inhibits Triacylglycerol Accumu-lation and Peroxisome Proliferator-Activated Receptor γ Expres-sion in Differentiating Human PreadipocytesVIAN PESHDARY, ANNEMARIE GAGNON, ALEXANDER SORISKY, Ottawa, ON, Canada

Adipose tissue accumulates via differentiation of preadipocytes into adi-pocytes (adipogenesis) and/or adipocyte hypertrophy. Low adipogenic ca-pacity promotes adipocyte hypertrophy, causing infl ammatory macrophage

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accumulation and insulin resistance. High glucose (HG) leads to macrophage infl ammation, but it is unknown if it infl uences their anti-adipogenic effect. Our aim was to test if medium conditioned by macrophages in HG inhibits human adipogenesis (Research Ethics Board-approved). Human monocyte-derived macrophages (MDM) were prepared from healthy volunteers. MDMs were exposed to 5 mM (NG) or 25 mM (HG) glucose for 24 hr. Condi-tioned medium was collected and its effect on differentiation, compared to glucose-matched control medium, was evaluated using human subcutane-ous abdominal preadipocytes from patients undergoing elective abdominal surgery. When preadipocytes were induced to differentiate in macrophage-conditioned medium (MacCM) generated in HG (HG-MacCM), triacylglycerol (TG) accumulation and protein expression of peroxisome proliferator-acti-vated receptor γ (PPARγ) were inhibited by 32±5% and 46±11%, respectively, (mean±SE; n=6, p<0.01 for both) vs. control. There were no changes in these markers in response to NG-MacCM (n=6). Infl ammatory markers were also evaluated in the differentiating preadipocytes. In HG-MacCM, but not in NG-MacCM, mRNA expression of interleukin 6 (IL-6) rose by 3.4±0.7 fold (mean±SE; n=5; p<0.05) vs. control. In both HG-MacCM and NG-MacCM, monocyte chemotactic protein 1 (MCP1) increased by 4.6±0.8 fold (p<0.01) and 3.2±0.4 fold (p<0.05) vs. control, respectively (mean±SE; n=5), however, the HG-MacCM increase was more potent (p<0.05). Our data suggest expo-sure of macrophages to HG may infl uence adipose tissue function by inhibit-ing adipogenesis and promoting infl ammation.

Supported By: Heart and Stroke Foundation of Canada

& 1849-PDose-dependent Effects of Vitamin D on Insulin Sensitivity and Adi-pose Tissue Infl ammationKEHAO ZHANG, AKANKASHA TIWARI, MICHELLE CAREY, OANA A. SANDU, PREETI KISHORE, MEREDITH HAWKINS, Bronx, NY

Vitamin D (VitD) defi ciency is associated with impaired glucose tolerance and diabetes. Recent literature suggests individuals with 25-hydroxyvitamin D (25(OH)D) levels ≥50ng/ml have 60% lower risk of diabetes compared with <12ng/ml. To defi ne metabolically optimal levels, we studied the effects of progressive VitD repletion on insulin action, adipose tissue infl ammation and fi brosis.

N=20 obese, insulin resistant subjects (age 45±4 y, BMI 34±1 kg/m2, HOMA 5±1, 25(OH)D 14±1ng/ml) received oral VitD3 or placebo for 6 months. Stepped euglycemic, hyperinsulinemic clamps were performed at baseline (B), following VitD repletion to Level II (LII, ~30ng/ml) and then to Level III (LIII,~ 50ng/ml) to measure endogenous glucose production (EGP) and periph-eral glucose disappearance (Rd).

EGP decreased by 29% at LII and by 36% at LIII versus B (B=1.4±0.2, LII=1.0±0.1,LIII=0.9±0.2 mg/kg/min, both p<0.05 vs. B). Rd was unchanged. Following initial VitD repletion, there were signifi cant reductions in adipose gene expression of TNF-α, IL-6, iNOS, TGF-β1, and Collagen VI (all p<0.05). Collagen VI and CD68 immunofl uorescence were also reduced (Fig 1). No additional benefi t was noted in any parameter upon increasing VitD levels to ~50ng/ml. Thus, repleting VitD improves hepatic insulin resistance in con-cert with decreased adipose infl ammation and fi brosis, with maximal benefi t observed upon normalizing VitD levels.

Supported By: American Diabetes Association (1-12-CT-41 to P.K.)

& 1850-PCHOP Mediates Insulin Resistance via Adipose Tissue Macrophage PolarizationJUNHONG GAO, YASUSHI ISHIGAKI, TORU SUZUKI, KEIICHI KONDO, JUNTA IMAI, TESTUYA YAMADA, HIDEKI KATAGIRI, Sendai, Japan, Morioka, Japan

Obesity represents chronic infl ammatory states augmented by infi ltration of pro-infl ammatory M1 macrophages into white adipose tissue (WAT), lead-ing to insulin resistance. C/EBP homologous protein CHOP is well known to play important roles in ER stress-related cellular apoptosis and several other metabolic disorders. We examined the role of CHOP in the development of obesity-induced insulin resistance. By ameliorating peripheral insulin resis-tance, CHOP defi ciency prevented high fat diet (HFD)-induced glucose in-tolerance. In wild-type (WT) mice, quantitative RT-PCR revealed that HFD up-regulated CHOP expression and promoted adipose tissue macrophage (ATM) M1 polarization. In contrast, in CHOP-/- mice, M2 was the predomi-nant ATM phenotype, despite similar amounts of infi ltrating macrophages. CD11c+ cells were decreased and CD206+ cells were increased in WAT of CHOP-/- mice. Flow cytometric analyses of the stromal vascular fraction of WAT revealed that M1 (F4/80+CD11c+CD206-)/M2 (F4/80+CD11c-CD206+) macrophage ratios were signifi cantly decreased in CHOP-/- mice, indicating that CHOP defi ciency promotes ATM M2 polarization. Adipose expressions of PPARγ and Th2 cytokines were preserved in CHOP-/- mice. In naive mac-rophages from the spleens of WT and CHOP-/- mice, stimulation with IFN-γ and IL-13 similarly up-regulated TNF-α and IL-10, respectively. In contrast, in cultured 3T3-L1 adipocytes, ER stress decreased PPARγ and IL-13 expres-sions, but CHOP knockdown suppressed these down-regulations, suggesting the importance of adipose CHOP in this mechanism. These fi ndings indicate that HFD-induced ER stress plays important roles in M1 polarization involv-ing adipose CHOP up-regulation and Th2 cytokine down-regulation, leading to insulin resistance.

& 1851-PMedium Conditioned by Human Peripheral Blood Mononuclear Cells Inhibits Human Adipogenesis: Role of Oral Fat LoadANNEMARIE GAGNON, MARION COUSINS, COLETTE FAVREAU, KATHY HENRY, TEIK CHYE OOI, ALEXANDER SORISKY, Ottawa, ON, Canada

In response to chronic positive caloric pressure, the ability of preadipo-cytes to differentiate permits hyperplastic expansion of functional adipo-cytes, maintaining insulin sensitivity. Immune cell infi ltration of adipose tissue is associated with adipogenic defi cit leading to infl amed, insulin-re-sistant hypertrophied adipocytes. The purpose of this study was to evaluate the role of an oral fat load on the ability of medium conditioned by periph-eral blood mononuclear cells (PBMCs) to inhibit human adipogenesis. Blood samples were collected from fasting human volunteers (n=21) prior to and 4h (post-prandial; PP) after ingestion of a fat load (research ethics board-approved). Plasma triacylglycerol (TG) levels rose from 1.6±0.6 to 3.1±1.4mM (mean±SD; p<0.001), post prandially. PBMCs were isolated and either lysed for RNA quantifi cation, or placed in culture for 24h to generate conditioned medium (fasting or PP PBMC-CM). These test media, and control medium not exposed to cells, were evaluated for their effect on human adipocyte differentiation. Fasting PBMC-CM inhibited the adipogenesis of human preadipocytes, by 42% vs. control medium (p<0.01), as assessed by intracel-lular TG accumulation and levels of adipogenic markers aP2 and peroxisome proliferator activator γ (PPARγ). With PP PBMC-CM, inhibition of intracellular TG accumulation and aP2 expression was more pronounced, at 65% (p<0.01 vs. control medium and p<0.05 vs. fasting PBMC-CM). A similar trend was observed for PPARγ levels, but did not reach statistical signifi cance (PP vs. fasting PBMC-CM). In PBMCs, mRNA expression of MCP-1 and ICAM-1 was increased in response to the fat load (1.7- and 1.4-fold vs. fasting PBMC, p<0.05). The increase in MCP-1 mRNA levels was positively correlated with the increase in plasma TG observed post-prandially (r=0.49, p=0.04). Our data suggest acute nutrient stress causes PBMCs to become infl amed and more anti-adipogenic, which could infl uence adipose tissue remodeling.


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& 1852-PLinagliptin-mediated DPP-4 Inhibition Attenuates Obesity-induced Insulin Resistance through an M2-dominant Shift in Adipose Tissue MacrophageTSUGUHITO OTA, FEN ZHUGE, YINHUA NI, LIANG XU, MAYUMI NAGASHIMADA, NAOTO NAGATA, SHUICHI KANEKO, Kanazawa, Japan

Dipeptidyl peptidase-4 (DPP4) is widely expressed, including immune cells. However, the role of DPP4 in macrophage-mediated adipose tissue in-fl ammation remains unclear. Here, we show that linagliptin, a DPP4 inhibitor, ameliorates adipose tissue infl ammation and whole-body insulin resistance in high-fat diet (HFD)-induced obese (DIO) mice by regulating both adipose tissue macrophage (ATM) recruitment and M1/M2 status. C57BL/6J mice were fed a HFD or a HFD containing linaglitptin (HFD+Lina; 3 mg/kg body weight) for 8 weeks. Expression of mRNA for DPP4 expression was signifi -cantly increased in stromal vascular fraction compared to adipocyte fraction of epididymal white adipose tissue (eWAT) of DIO mice. DIO mice had 1.7-fold increase in DPP4 activity in eWAT compared to wild type (WT) mice (p < 0.01), whereas linagliptin markedly reduced eWAT DPP4 activity by 89% (p < 0.01) in DIO mice. Linagliptin administration improved HFD-induced glucose intolerance, and hyperinsulinemia (HFD 1.07±0.25 vs. HFD+Lina 0.52±0.08 ng/ml, p < 0.05; fasting state) and also enhanced insulin signaling assessed by IRβ and Akt phosphorylation in eWAT of DIO mice. HFD+Lina mice had de-creased macrophage infi ltration and crown-like structure formation in eWAT compared with HFD mice even though weight and adiposity were similar. Furthermore, fl ow cytometry analysis revealed that HFD+Lina mice had 35% fewer CD11c+CD206-(M1) ATMs whereas 25% more CD11c-CD206+(M2) ATMs than HFD mice, resulting in predominance of M2 over M1 ATM popu-lation. In parallel, linagliptin (50-200 nM) suppressed LPS/IFNγ-induced M1 markers mRNA expression (TNFα and MCP-1) in peritoneal macrophages whereas it augmented IL4-induced M2 markers mRNA expression (Arg1, Chi3l3, and Mgl1) in a dose-dependent manner. In conclusion, linagliptin-mediated DPP4 inhibition causes a dynamic M2 dominant shift of ATM, and thereby attenuates obesity-induced infl ammation and insulin resistance.

Supported By: Japan Ministry of Education, Culture, Sports, Science and Tech-nology

& 1853-PLack of IL-6-type Cytokine Signaling in Adipocytes Relieves Obesi-ty-induced Hepatic Insulin ResistanceSTEPHAN WUEEST, FLURIN ITEM, FABRIZIO C. LUCCHINI, WERNER MÜLLER, EUGEN J. SCHOENLE, DANIEL KONRAD, Zürich, Switzerland, Manchester, United Kingdom

IL-6-type cytokines, which all signal via the common signal transducer glycoprotein 130 (gp130), were shown to affect differentiation, insulin sensitivity as well as lipolysis of adipocytes rendering gp130 a potential therapeutic target to combat obesity and its associated diseases. However, effects of individual IL-6 family members on adipocytes may be opposing. Thus, we aimed to elaborate on how merged IL-6-type cytokine signalling in adipocytes impacts on obesity and insulin sensitivity using newly generated adipocyte-specifi c gp130 knockout mice. Lack of IL-6-type cytokine signal-ling had no signifi cant effect on adipocyte differentiation as judged by fat pad weights, adipocyte size as well as PPARγ protein levels neither in lean nor in high fat diet-induced obese mice. In contrast, the ability of insulin to suppress free fatty acid release was improved and basal lipolysis was reduced in mesenteric adipocytes isolated from obese knockout compared to control littermates. Such blunted lipolysis was paralleled by reduced phosphorylation of the lipolytic regulator ERK1/2, a kinase activated down-stream of IL-6-type cytokine signalling. Consistently, free fatty acid levels in the portal vein were reduced in high fat diet-fed knockout compared to control mice. Of note, lack of gp130 in adipocytes partly protected mice from obesity-induced hepatic insulin resistance, resulting in signifi cantly blunted high fat diet-induced whole body insulin resistance as determined during hyperinsulinaemic-euglycaemic clamp studies. Taken together, lack of IL-6-type cytokine signalling in mesenteric adipocytes reduces basal and insulin-inhibited lipolysis and relieves obesity-induced hepatic insulin resistance. Consequently, blocking gp130 dependent signaling in adipocytes may be a novel approach to blunt detrimental fat-liver crosstalk as observed under obesity.

Supported By: Olga Mayenfi sch Foundation

1854-PAdipocyte Autophagy Is Required for Normal Insulin-Glucose Ho-meostasis and Suppression of Lipid Hydroperoxides at the Whole Body LevelJINJIN CAI, TANYA FOROSTYAN, LUIS FANDINO, YONG HWAN HAN, KARLA MARIA PIRES, SHAOBO PEI, STEFAN OFFERMANNS, SIHEM BOUDINA, TIMOTHY E. GRAHAM, Salt Lake City, UT, Bad Nauheim, Germany

The role of autophagy in adipocyte biology is incompletely understood. Prior studies in mice evaluated effects of inhibiting adipocyte autophagy during early embryogenesis: resultant phenotypes were lean and obesity-resistant, with BAT-like WAT depots rich in mitochondria, increased energy expenditure and lipid beta-oxidation, and improved insulin-sensitivity and glucose-tolerance. However, abnormal development in these models ob-scures insight into the functions of autophagy in normal, mature adipocytes. To address this, we generated mice in which Atg3, a requisite mediator of autophagy, is deleted in adulthood via a tamoxifen (TAM)-inducible Cre transgene. TAM treatment at 8 wks of age deleted Atg3 and suppressed autophagy solely in adipocytes, producing adult adipocyte Atg3-knockout (AdAdAtg3KO) mice. Mitochondria increased 2-fold in AdAdAtg3KO adipo-cytes, possibly due to loss of mitophagy; oxidative phosphorylation and ATP production were unchanged, but ROS and lipid hydroperoxide (LPO) produc-tion increased. AdAdAtg3KO mice had normal growth, weight gain, food intake, and energy expenditure on chow and high fat diets, but exhibited insulin-resistance and glucose-intolerance on chow, which worsened with high fat feeding. Insulin signaling was impaired in AdAdAtg3KO adipocytes, and also in liver and skeletal muscle, suggesting autophagy-impaired adipo-cytes cause systemic insulin-resistance. Multiple adipokines and cytokines were measured and showed no changes in AdAdAtg3KO mice. However, LPO levels were increased >6-fold in serum and 3-fold in liver of AdAdAt-g3KO mice; in parallel, liver glutathione was reduced >50% and Nrf2 and NF-kappa-B were robustly activated, indicating oxidative stress. Therefore, adipocyte autophagy is necessary for maintaining insulin-glucose homeo-stasis, potentially through suppression of circulating LPOs that can induce oxidative stress and insulin resistance in other tissues.

Supported By: American Diabetes Association (7-13-BS-056 to T.E.G.); U.S. De-partment of Veterans Affairs (I01BX000937); National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (R01DK100826)

1855-PJNK2 in Myeloid Cells Impairs Vasodilator Properties of Perivas-cular Adipose Tissue in Muscle during Short-Term Western Diet ExposureFEMKE P. HOEVENAARS, RICK I. MEIJER, VICTOR W. VAN HINSBERGH, JOHN S. YUDKIN, ERIK H. SERNE, YVO M. SMULDERS, ETTO C. ERINGA, Amsterdam, Netherlands, London, United Kingdom

Obesity is associated with accumulation of perivascular adipose tissue (PVAT) in muscle, which contributes to impaired microvascular delivery of insulin and glucose to skeletal muscle. PVAT has anticontractile properties that are lost in obesity, thereby blunting insulin induced vasodilation.

We hypothesized that a short term western diet (WD) impairs insulin-in-duced vasodilatation via PVAT, and that infi ltration of JNK2 expressing cells from bone marrow is involved in this impairment.

Male C57BL/6J mice were exposed to a two week Western diet or chow after transplantation of JNK2+/+ or JNK2-/- bone marrow. Gracilis muscle resistance arteries were isolated for analysis of insulin-induced vasore-activity ex-vivo using pressure myography. In vivo, insulin sensitivity was assessed using a euglycemic hyperinsulinemic clamp in combination with determination of muscle perfusion via contrast ultrasonography.

Ex-vivo vasoreactivity experiments showed that control PVAT induces insulin-induced vasodilation. However, after two weeks exposure to a WD the ability of PVAT to induce insulin-induced vasodilation was blunted. In contrast, when chow resistance arteries were exposed to PVAT from WD fed JNK2-/- chimeric mice, this disruption was corrected.

In vivo, insulin sensitivity and insulin-induced microvascular recruitment in muscle were reduced by the WD. Insulin sensitivity showed partial re-covery to control level in WD fed JNK2-/- chimaeric mice. In addition, WD alone disturbs muscle perfusion compared to chow but a partial improve-ment was showed when JNK2 is deleted in bone marrow of WD fed mice (+30%, n=3).

In conclusion, impaired insulin-induced muscle perfusion is an early phe-nomenon after short term western diet exposure, which is caused by en-dothelial as well as PVAT dysfunction. The latter is caused by JNK2 cells which infi ltrated from the bone marrow into PVAT.

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1856-PFat Transplantation Ameliorates High-Fat Diet-induced Hypergly-cemia in InsrP1195L/+ MiceEUN YOUNG LEE, KENICHI SAKURAI, XILIN ZHANG, YASUHIKO MINOKOSHI, TAKASHI MIKI, Chiba, Japan, Okazaki, Japan

Increase in dietary fat intake is considered attributable to the recent in-crease in the patients with type 2 diabetes mellitus (T2DM), but its mecha-nism is not fully clarifi ed. In the present study, we examined the effect of high fat diet (HFD) feeding on developing hyperglycemia in insulin receptor mutant (InsrP1195L/+) mice, which exhibited normoglycemia under normal diet. We found that mRNA expression of glucose-6-phosphatase (G6pc) in liver was signifi cantly increased on refeeding, while that of phospho-enolpyruvate carboxykinase 1 (Pck1) was not altered in InsrP1195L/+/HFD mice. Interestingly, we found that gluconeogenesis from glycerol but not from pyruvate was increased in InsrP1195L/+/HFD mice. Lipolysis as as-sessed by phosphorylated hormone sensitive lipase (phospho-HSL) was increased in the white adipose tissue (WAT) of InsrP1195L/+/HFD mice (3.14 folds in InsrP1195L/+/HFD mice vs. wild-type (WT)/HFD mice). In addition, lipolysis of InsrP1195L/+/HFD mice was signifi cantly increased also in vitro. Treatment of the mice with a PPARγ agonist pioglitazone revealed that PPARγ acts as a critical determinant of lipolysis in InsrP1195L/+ mice but not in WT mice. Taken together with these results, we hypothesized that the increased glycerol release from WAT triggers the increased gluconeo-genesis from glycerol in liver, leading to the development of hyperglycemia in InsrP1195L/+/HFD mice. To test this possibility, we transplanted wild-type subcutaneous fat to InsrP1195L/+/HFD mice and found that the transplanta-tion signifi cantly ameliorated hyperglycemia and abnormal G6pc expression in liver of InsrP1195L/+/HFD mice. Serum adiponectin levels of InsrP1195L/+/HFD mice were signifi cantly lower than those of WT/HFD mice, but the fat transplantation did not improve the decreased serum adiponectin. Accord-ingly, the restoration of normal function in the subcutaneous fat could be a novel therapeutic target of T2DM in humans.

1857-PLipocalin 2 Defi ciency Disrupts Retinoid Metabolism in Adipose Tissue and Thermogenic Effect of All-trans Retinoic Acid in High-Fat Diet-induced ObesityHONG GUO, ROCIO FONCEA, SHEILA M. O’BYRNE, HONGFENG JIANG, YUANYU-AN ZHANG, WILLIAM S. BLANER, DAVID A. BERNLOHR, XIAOLI CHEN, Minne-apolis, MN, New York, NY

Lipocalin 2 (Lcn2) has been recently characterized as an adipose-derived cytokine belonging to the lipocalin subfamily of hydrophobic ligand binding proteins. Our previous studies have shown that Lcn2 defi ciency impairs adap-tive thermogenesis via a non-adrenergic pathway. However, the mechanism for Lcn2 role in energy metabolism and non-adrenergic stimulation of ther-mogenesis is unclear. Herein, we investigated the role of Lcn2 in the regu-lation of retinoid metabolism and retinoic acid (RA) action in high-fat diet (HFD)-induced obesity and thermogenesis. We showed that LCN2 is a high affi nity RA binding protein. Lcn2 defi ciency disrupted retinoid metabolism as evidenced by decreased RA and retinol levels in adipose tissue. Upon 12 weeks of HFD feeding, Lcn2 KO mice showed a signifi cant down-regulation of genes involved in RA signaling pathway, intracellular RA transport, and RA biosynthesis in epididymal adipose tissue compared to WT mice. More-over, the gene and protein levels of RBP4 and STRA6, the key regulator of retinol transport and uptake, were also signifi cantly altered in adipose tissue of Lcn2 KO mice. More interestingly, Lcn2 KO mice exhibited an attenuated effect of all-trans retinoic acid (ATRA) on reducing body weight and fat mass as well as on increasing body temperature compared with WT mice under the HFD feeding condition. The effect of ATRA was also abolished on the mRNA expression of genes involved in lipogenesis and RA signaling path-way as well as RA responsive genes in adipose tissue of HFD-fed Lcn2 KO mice. We further demonstrated that Lcn2 defi ciency diminished RA induction of PGC-1α and UCP1 expression in primary differentiated brown adipocytes. Lcn2 is required for the full action of RA on p38MAPK and HSL phosphoryla-tion in brown adipocytes and adipose tissue. We conclude that Lcn2 plays a role as an important regulator in retinoid metabolism and thermogenic effect of RA in adipose tissue.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases (R01DK080743)

1858-PFAK Signaling Controls Insulin Sensitivity through Adipocyte SurvivalCYNTHIA T. LUK, SALLY Y. SHI, ERICA P. CAI, THARINI SIVASUBRAMANIYAM, MANSA KRISHNAMURTHY, JARA J. BRUNT, STEPHANIE A. SCHROER, DANIEL A. WINER, MINNA WOO, Toronto, ON, Canada

Obesity-induced insulin resistance is associated with adipose tissue ex-pansion and adipocyte death, but the complex pathways regulating these processes remain unclear. Focal adhesion kinase (FAK) is an intracellular ki-nase central to integrin signaling and known to be important in development, growth, migration and survival. While FAK is overexpressed in many human tumors, its role in tissue growth in other contexts is largely unknown, and adipose tissue is uniquely capable of non-neoplastic expansion with energy excess. We previously found that in both mice and humans, FAK increased in adipose tissue with obesity and diabetes. To further study adipocyte FAK in diabetes, we used a Cre-loxP system to generate adipose tissue-specifi c FAK knockout mice. We also knocked down FAK in 3T3-L1 adipocytes in vitro with siRNA. Disruption of FAK resulted in increased adipocyte death in mice under basal and high fat diet (HFD)-fed conditions, as well as in adipocytes in vitro, but with no changes in differentiation or proliferation. Mice had divergent adipose tissue remodelling, with increased adiposity under basal conditions or short term HFD feeding. However, ultimately adipose tissue expansion and weight gain was impaired with prolonged caloric excess from HFD or genetic obesity, and mice were insulin resistant under both basal and stress conditions. Inhibition of apoptosis in knockout mice, either by ge-netic defi ciency of caspase 3 or by pharmacological inhibition of apoptosis attenuated adipocyte death and restored adiposity, improving insulin sensi-tivity. Together these results demonstrate that FAK is required for adipocyte survival and maintenance of insulin sensitivity, particularly in the setting of obesity and metabolic stress.

Supported By: Canadian Institutes of Health Research; Canadian Diabetes Association

1859-P

1860-PSerum Retinol Binding Protein (RBP4) Is Absent from Circulation of Mice with Hepatocyte-specifi c Genetic Deletion of RBP4SPENCER J. THOMPSON, JINJIN CAI, ASHOT SARGSYAN, WILLIAM S. BLANER, TIMOTHY E. GRAHAM, Salt Lake City, UT, New York, NY

Serum retinol binding protein (RBP4), the Vitamin A transport protein, is implicated in the pathogenesis of type 2 diabetes and cardiovascular dis-ease. Serum RBP4 levels are increased in obesity and other insulin-resis-

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tant states; in addition, a genetic polymorphism within the RBP4 promoter increases serum RBP4 and confers risk for diabetes and heart disease in humans. RBP4 acts on liver, muscle, and both adipocytes and immune cells within adipose tissue to cause insulin resistance and glucose intolerance. It has been proposed that under normal conditions, serum RBP4 is produced primarily by liver, whereas extrahepatic sources -adipose tissue in particu-lar- are the cause of elevated serum RBP4 in the setting of insulin-resistance. However, the relative contributions of specifi c tissues to serum RBP4 levels in vivo have not been formally determined. To assess this, we studied mice with hepatocyte-specifi c genetic deletion of RBP4 (liver RBP4-KO, or LRKO mice). RBP4 could not be detected in serum of LRKO mice, nor in urine, the principal route of RBP4 excretion. Retinol, measured by HPLC, was reduced 95% in serum of LRKO mice. However, RBP4 mRNA was expressed in LRKO adipose tissue, and adipose tissue explants secreted RBP4 protein at levels equal to WT mice. Feeding high fat diet (HFD) increased adiposity and caused insulin-resistance in LRKO and WT mice; liver RBP4 mRNA was not altered, but adipose tissue RBP4 mRNA and explant RBP4 secretion increased in WT and LRKO mice fed HFD. Despite increased adipose tissue RBP4 production, RBP4 remained undetectable in serum or urine of LRKO mice fed HFD. We conclude that adipocyte-produced RBP4 does not contribute to circulating RBP4 concentrations in vivo, and extrahepatic RBP4 from any source most likely exerts autocrine or paracrine actions confi ned to the tissues that pro-duce it. By extension, potential effects of novel RBP4 ligands postulated by others most likely exert their activities locally rather than systemically.


1861-PA Novel Role of AMPK in the Regulation of Adipose Tissue Remodel-ing in Obesity-induced Insulin ResistanceTING LUO, ALLISON NOCON, XIAOLIANG RUI, MENGWEI ZANG, Boston, MA

Adipose tissue fi brosis and remodeling have been implicated on the pathogenesis of obesity, but little is known about how adipose remodeling is regulated in response to overnutrition. Here we identify that anti-diabetic drug metformin inhibits extracellular matrix (ECM) accumulation, a hallmark of adipose tissue fi brosis, and increases adaptive thermogenesis of white adipose tissue (WAT). Human studies showed aberrant adipose expansion and excessive ECM in omental white fat pad of obese subjects, compared to that of healthy subjects. Metformin (250mg/kg/day) administration to ob/ob mice led to decreased fat mass and improved insulin sensitivity and glucose homeostasis. Masson’s trichrome staining showed that adipose ex-pansion and extensive interstitial fi brosis in epididymal fat depots of ob/ob mice were reduced, and Sirius Red staining confi rmed that collagen deposi-tion caused by obesity was decreased upon metformin treatment. Improved adipose dysfunction by metformin was associated with activation of AMPK, suppression of aberrant elevation of HIF-1α, α-smooth muscle actin, TGFβ1 and Smad3 phosphorylation, the downstream effector of TGFβ1, and a re-duction in macrophage infi ltration and crown-like structure in WAT of ob/ob mice. In vitro studies with primary preadipocytes showed that the ability of TGFβ1 to induce expression of fi brogenic genes (Col1α1 and Col3α1) and key regulators of ECM (MMP-2, MMP-9, MMP-14, and TIMP-1) was inhib-ited by metformin. The fi brogenic response to TGFβ1 was also repressed by overexpressing constitutive active AMPK and its upstream kinase LKB1. Fur-thermore, metformin acted on white fat depots in vivo and in vitro to stimu-late expression of UCP1 and other brown-like thermogenic genes. Taken together, adipose AMPK activation ameliorates obesity-induced fi brosis and increases browning of white fat, leading to maintained adipose architecture and function and improved metabolic dysregulation.

Supported By: National Institutes of Health

1862-PInsulin Inhibits Autophagy Independently of mTORC1 Signaling in 3T3L1 AdipocytesJINJIN CAI, ASHOT SARGSYAN, SPENCER J. THOMPSON, TIMOTHY E. GRA-HAM, Salt Lake City, UT

Autophagy is an evolutionarily conserved catabolic process required for cellular homeostasis during nutrient deprivation and other forms of stress. Insulin inhibits autophagy in several cell types, including adipocytes. How-ever, specifi c signaling mechanisms by which insulin inhibits adipocyte au-tophagy are not well-defi ned. We studied regulation of autophagy by insulin in 3T3L1 adipocytes. Amino acid-deprivation induced autophagy 1.8-fold, and insulin treatment (100 nM) completely suppressed this response. In contrast, autophagy induced by the mitochondrial uncoupling agent, CCCP, was not in-

hibited by insulin at any concentration. Insulin inhibits autophagy in several cell types by activating mTOR complex 1 (mTORC1), which inhibits activation of the Atg1-related kinase Ulk1, a positive regulator of autophagy. Surpris-ingly, treatment of 3T3L1 adipocytes with mTORC1-inhibitor, rapamycin, completely blocked insulin-stimulated p70 S6 kinase (S6K) phosphorylation in basal and nutrient-deprivation conditions, but did not affect inhibition of autophagy by insulin. However, treatment with mTOR kinase active site-in-hibitors completely prevented inhibition of autophagy by insulin, suggesting insulin acts through a mTOR-dependent signaling mechanism(s) distinct from mTORc1 to inhibit autophagy. Consistent with this, siRNA-mediated silencing of mTORc2-regulator, Rictor, or inhibition of Akt, the downstream target of mTORC2 signaling, completely prevented inhibition of autophagy by insulin. In contrast, siRNA silencing of mTORc1-regulator, Raptor, mimicked effects of rapamycin, blocking phosphorylation of S6K without affecting inhibition of autophagy by insulin. These data suggest that insulin inhibits autophagy through a unique rapamycin-insensitive, Raptor/mTORC1-independent path-way. Further studies are needed to defi ne the specifi c points of regulation in the autophagy pathway targeted by insulin signaling via the Rictor/mTORC2/Akt cassette in 3T3L1 cells.


1863-PAdipose Nicotinamide N-methyltransferase Expression Is Inversely Correlated with Peripheral Insulin Sensitivity and Increases after a Hypercaloric High-Fat-High-Sugar Diet in Lean MenMURAT KILICARSLAN, BARBARA B. KAHN, KARIN E. KOOPMAN, JOHANNES A. ROMIJN, MARIETTE T. ACKERMANS, UNGA A. UNMEHOPA, AART J. NEDER-VEEN, MATTHAN W. CAAN, SUSANNE E. LA FLEUR, MIREILLE J. SERLIE, Amster-dam, Netherlands, Boston, MA

Nicotinamide N-methyltransferase (NNMT) is increased in white adipose tissue in obesity and type 2 diabetes mellitus (T2DM). Adipose-specifi c NNMT knockdown prevents weight gain and insulin resistance on a high fat diet by increasing energy expenditure in mice, making it a potential thera-peutic modality for obesity and T2DM. However, the association between adipose NNMT expression and insulin sensitivityand the direct effects of caloric excess on NNMT expression in humans are unknown. To study the effects of caloric excess on subcutaneous adipose tissue (SAT) expression of NNMT, 36 healthy men (BMI 22.2 [19.6-24.9] kg/m2) consumed a hyper-caloric, high-sugar (HS) or high-fat-high-sugar (HFHS) diet (40% caloric sur-plus) or a control (eucaloric) diet for 6 weeks. Before and after the diets, we performed a 2-step hyperinsulinemic-euglycemic clamp to measure both he-patic (insulin-mediated suppression of endogenous glucose production(EGP)) and peripheral (rate of disappearance [Rd]) insulin sensitivity, assessed total abdominal fat and liver fat by MRS and measured SAT NNMT expression using qPCR in SAT biopsies. At baseline, SAT NNMT expression correlated positively with abdominal adipose tissue volume (rs = 0.379, P = 0.033) and inversely with Rd (rs = -0.342, P = 0.045). SAT NNMT expression did not cor-relate with basal EGP or EGP suppression. Insulin sensitivity did not change after the diets and.SAT NNMT expression remained unchanged after the HS and control diets while after the HFHS diet SAT NNMT expression signifi -cantly increased (P = 0.023). To conclude, SAT NNMT expression is inversely associated with insulin sensitivity and increases after a hypercaloric HFHS diet in lean men. These results suggest that SAT NNMT may play a role in whole-body glucose metabolism and is modulated by macronutrient compo-sition during hypercaloric conditions in healthy subjects.

1864-PCoordinated Increases in Estrogen Receptor Alpha and Fat Brown-ing Genes Can Explain Metabolic Improvement due to CB1 Antago-nism in the Fat Fed DogMORVARID KABIR, MALINI S. IYER, JOYCE M. RICHEY, QIANG WU, ORISON O. WOOLCOTT, DARKO STEFANOVSKI, STELLA P. KIM, CATHRYN M. KOLKA, VIORI-CA IONUT, RICHARD N. BERGMAN, Los Angeles, CA

We have recently demonstrated that CB-1 receptor antagonism increased expression of genes involved in beiging/browning of adipose tissues, spe-cifi cally in the subcutaneous (SC) and visceral (VIS) depots from the fat-fed canine model. The mechanism(s) by which the CB1 antagonist Rimonabant (RIM) promotes adipocyte beiging/browning is unknown. PGC1α, which is a co-factor and transcriptionally regulates the expression and activity of estrogen receptor α (ERα), was signifi cantly elevated following RIM treat-ment. Here we demonstrate for the fi rst time that following RIM treatment there is a signifi cant increase in ERα gene expression in the subcutaneous

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(SC) and visceral (VIS) fat depots, the same adipose depots in which we demonstrated enhanced beiging/browning. Animals were fed a HFD (52% fat) for 6 weeks followed by a continued 16 weeks of fat feeding with either HFD + placebo (PL) (n=9) or HFD + RIM (1.25 mg/kg per day; n=11). Biopsies from SC and VIS depots were obtained for gene expression: before HFD (Pre-fat), after 6 weeks of fat (HFD) and 16 weeks of HFD +/- RIM. RIM increased ERα, expression in both SC and VIS depots by 3 fold (P<0.001) compared to pre-fat and HFD groups. Positive correlations were found between PGC1α and UCP1 with ERα in the VIS depot (PGC1α r=0.52, P<0.05; UCP1 r=0.66, P<0.001). In the SC depot, we found positive correlation between UCP1 and ERα r=0.62, P<0.005). Thus, our data suggest one mechanism by which the CB1-R antagonist increases beiging/browning of adipose tissue, through up-regulation of the key factors, such as PGC1α, UCP1 and ERα thought to be critical for adipose tissue transformation. Increased browning of fat appears to be an important mechanism by which the cannabinoid system regulates body fat, improves glucose homeostasis and enhances overall metabolism.

1865-PAdiporedoxin Negatively Regulates Insulin Signaling in AdipocytesALESSANDRO PESCHECHERA, COLLETTE J. LAFLAMME, TOVA MESHULAM, PAUL F. PILCH, Boston, MA

Adiporedoxin (Adrx) is a novel adipocyte protein, which is resident in the endoplasmic reticulum (ER) and has an active site -CXXC- motif characteris-tic of the thioredoxin/peroxiredoin super family of cellular redox regulators. Adrx acts to affect the secretion and/or the plasma membrane amounts of disulfi de bond-containing proteins such as adiponectin and the insulin recep-tor (IR). Thus we hypothesized that Adrx may regulate the insulin signaling pathway as a result of altered cell-surface IR, and indeed, 3T3-L1 adipocytes over expressing (OE) and under expressing (KD) Adrx show corresponding increases and decrease in the IR. These cells were then subjected, or not, to increasing doses of insulin (0, 0.3, 1, 10 nM), and the levels of total and phos-phorylated Akt and AS160 were determined along with [3H]-2-deoxyglucose (2-DOG) uptake. In Adrx OE cells, the insulin-induced phosphorylation levels of Akt and AS160 were at the same as in control cells, and no signifi cant differences were observed in 2-DOG uptake. In contrast, in KD adipocytes, insulin-dependent phosphorylation of Akt and AS160 was diminished as compared to control cells, as was insulin-dependent glucose uptake. Thus the data support the notion of spare IRs in the OE cells, but limiting IR after down regulation of Adrx. Furthermore, the Adrx gene is regulated by tu-mor necrosis factor alpha (TNFa) in cultured murine adipocytes. Overnight incubation of these cells with TNFa results in a down regulation of Adrx mRNA and protein consistent with TNFa’s known actions as a mediator of cellular insulin resistance. These fi ndings have important implications for Adrx amounts in pathological states in vivo such as insulin resistance and its associated complications.

1866-PAdipose Tissue FGF21 Resistance Contributes to Hypoadiponectine-mia and Insulin Resistance in Obesity: Role of miR-34aKAREN S.L. LAM, CYRUS Y.C. CHAN, ALEXIA STILLITANO, CHI-MING WONG, YONG PANG, AIMIN XU, Hong Kong, China

Fibroblast Growth Factor 21 (FGF21) is a hormone with benefi cial effects on glucose and lipid homeostasis. We have previously reported its stimula-tory effect on adiponectin secretion. However, serum FGF21 is paradoxically elevated in obesity and high FGF21 levels predict incident type 2 diabetes, suggesting that FGF21 resistance enhances the development of obesity-related type 2 diabetes. Here we sought to confi rm the presence of FGF21 resistance in the adipose tissues of obese/overweight humans and inves-tigate for the underlying mechanism. The expression levels of FGFR1, the receptor for FGF21, and β-klotho, its co-receptor protein, and miR-34a, were measured in visceral adipose tissues (VAT) collected during surgery from 24 overweight/obese (BMI > 23) Chinese women and 29 age- and sex-matched lean controls. To elucidate the effect of increased miR-34a on the expres-sion of β-klotho, FGFR1 and adiponectin, 3T3-L1 pre-adipocytes were in-fected with lentiviral vector expressing miR-34a, before differentiation into mature adipocytes. Obese/overweight subjects had raised serum FGF21, reduced serum adiponectin, reduced VAT expressions of FGFR1 (all p<0.01) and β-klotho (p<0.05), but increased VAT expression of miR-34a (p<0.05), by RT-PCR. VAT FGFR1 and β-klotho expressions correlated inversely with the HOMA-IR insulin resistance index (both p<0.05), but positively with serum adiponectin (both p<0.01). On the other hand, VAT miR-34a expres-sion correlated inversely with that of β-klotho, FGFR1 and adiponectin (all p<0.01), but positively with HOMA-IR (p<0.05). Lentivirus-mediated 5-fold increase in adipocyte miR-34a expression was accompanied by reduced

β-klotho (p<0.01), FGFR1 (p<0.01) and adiponectin (p<0.001) expressions. We conclude that MiR-34a mediated FGF21 resistance is present in the adipose tissues of obese/overweight subjects and may contribute to obesity-related insulin resistance, in part via inducing hypoadiponectinaemia.

Supported By: Research Grants Council of Hong Kong (HKU2/CRF/12R)

1867-PTwo Divergent Signals from the Gs-Coupled T1R3 Homomeric Sweet Taste Receptor Converge at FoxO1 to Inhibit Adipogenesis in 3T3-L1 CellsYOUSUKE MASUBUCHI, ITARU KOJIMA, HIROSHI SHIBATA, Maebashi, Japan

3T3-L1 cells express a non-canonical T1R3 homomeric sweet taste recep-tor, the stimulation of which elicits Gs-alpha-mediated activation of the tu-bulin GTPase and microtubules disassembly leading to activation of the Rho/ROCK pathway and dephosphorylation of Akt and FoxO1, with consequent repression of pro-adipogenic transcription factors. Here, we report another anti-adipogenic signaling pathway downstream of this receptor. The addi-tion of sucralose prevented the decline of SIRT2 at 24 hours after the induc-tion of adipogenesis in 3T3-L1 cells, which coincided with deacetylation of FoxO1 as well as tubulin. The effect of sucralose on FoxO1 was attenuated with sirtinol, a sirtuin inhibitor or with overexpression of the SIRT2 deacety-lase mutant (SIRT2-N168A). Additionally, overexpression of the FoxO1 mu-tant mimicking the acetylated form (FoxO1-3KA (K242A, K245A and K262A)) attenuated the anti-adipogenic effect of sucralose. Thus, the T1R3 homo-meric sweet taste receptor generates two divergent signals in response to sweet agonist, which converge at FoxO1 causing its dephosphorylation and deacetylation to inhibit adipogenesis.

1868-PCDK5 Regulatory Subunit-associated Protein 1-like 1 (Cdkal1) Neg-atively Regulates Adipocyte Differentiation through Activation of the Wnt PathwayKAZUMI TAKE, WEI SUN, HIRONORI WAKI, YUTA HIRAIKE, JING YU, MASATO IWABU, MIKI OKADA-IWABU, KOHJIRO UEKI, TOSHIMASA YAMAUCHI, TAKASHI KADOWAKI, Tokyo, Japan

Cdkal1 was identifi ed as a susceptibility gene for both type 2 diabetes and obesity. Although Cdkal1 in pancreatic β cells was shown to modulate insu-lin secretion, little is known about its role in adipocytes. Cdkal1 expression is increased during 3T3-L1 adipocyte differentiation but eventually decreases when they are cultured over 3 weeks. Retroviral overexpression of Cdkal1 in 3T3-L1 cells strongly suppresses adipocyte differentiation, which is res-cued by forced expression of PPARγ but not by PPARγ agonist treatment, implying the critical role of downregulation of PPARγ expression by Cdkal1. Conversely, knockdown of Cdkal1 by shRNA promotes adipocyte differentia-tion. Experiments using deletion and site-directed mutagenesis of Cdkal1 constructs indicate that two CXXXCXXC motifs located in the N-terminal radical SAM domain are critical for the anti-adipogenic effect. Investigation on the pathways that regulate PPARγ expression pinpoints activation of the Wnt pathway as a possible mechanism. Our data indicated that Cdkal1 acts as a negative regulator of adipocyte differentiation.

1869-PAdipose Tissue Fatty Acid Composition Is Modifi ed by Lipolytic ActivityJESPER J. LUNDBOM, ANTTI HAKKARAINEN, ALESSANDRA BIERWAGEN, JAAKKO KAPRIO, AILA RISSANEN, NINA LUNDBOM, MICHAEL RODEN, KIRSI H. PIETILÄINEN, Düsseldorf, Germany, Helsinki, Finland

How non-dietary factors modify adipose tissue FA composition is largely unknown. Previous studies on adipose tissue FA composition and triglycer-ide turnover in humans have not taken into account the more metabolically active deep subcutaneous adipose depot. Also, most studies on adipose tissue FA composition have used invasive sampling procedures. This study aimed at analysis of adipose tissue FA composition and intramyocellular lipid (IMCL) content by non-invasive magnetic resonance spectroscopy (MRS) and body fat distribution by magnetic resonance imaging (MRI) in 16 pairs of healthy monozygotic twins (MZ) discordant for BMI and 8 healthy volunteers before and after one-hour bout of exercising. Unsaturation of FA in deep subcutaneous adipose tissue (DSAT) inversely associated with IMCL content (R=-0.5184, P=0.0056, N=28), suggesting a common mechanism responsible for the slow turnover adipose and fast turnover IMCL depots. Adipose tis-sue lipolytic activity could be the common mechanism, which selectively releases unsaturated FA, subsequently increasing systemic non-esterifi ed FA (NEFA) and IMCL content. Analysis of gene expression of adipose tissue lipolytic showed an inverse association between hormone-sensitive lipase

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(HSL) expression and DSAT unsaturation (R=-0.4292, P=0.0179, N=31). A single one-hour bout of exercising already decreased DSAT unsaturation by 4.0% (P=0.0014, N=8). In conclusion, adipose tissue lipolytic activity modi-fi es adipose tissue FA composition on an intraday basis through selective FA mobilization. These fi ndings may help to better understand of the role adipose tissue FA composition in insulin resistance.

Supported By: Novo Nordisk Foundation; Diabetes Research Foundation; Jalmari and Rauha Ahokas Foundation; Finnish Foundation for Cardiovascular Research; Academy of Finland; German Federal Ministry of Health; Ministry of Innovation


1870-PA Mouse Model of SHORT Syndrome Exhibits Impaired PI3K SignalingMARIE H. SOLHEIM, JONATHON WINNAY, ANDERS MOLVEN, PÅL R. NJØLSTAD, C. RONALD KAHN, Boston, MA, Bergen, Norway

The phosphatidylinositol 3-kinase (PI3K) pathway regulates a spectrum of cellular functions including glucose metabolism, cell growth/differentiation and apoptosis. The p85α regulatory subunit of PI3K, encoded by PIK3R1, is a critical mediator regulating activation of the pathway. Recently, we identi-fi ed a heterozygous missense mutation in PIK3R1 resulting in an Arg649Trp substitution in patients with SHORT syndrome characterized by short stat-ure, partial lipodystrophy and insulin resistance. To determine whether the Arg649Trp mutation is suffi cient to recapitulate characteristic features of SHORT syndrome in vivo and investigate the function of the mutant protein at a mechanistic level, we generated knock-in mice harboring this muta-tion. While homozygous mice were fetal lethal, heterozygous knock-in mice exhibited multiple features of SHORT syndrome including reduced body weight, reduced body length and a selective reduction in subcutaneous, not epididymal, adipose tissue. By 8 weeks of age, knock-in mice were hyperg-lycemic in the fed state when compared to controls (318±25 vs. 213±16 mg/dL). A parallel assessment of insulin levels at this age revealed marked hy-perinsulinemia in mutant animals (fed insulin 8.38±1.0 vs. 0.69±0.09 ng/dL). Glucose and insulin tolerance tests revealed a markedly impaired glucose tolerance and reduced response to exogenous insulin in mutant mice. At a molecular level, this was due to reduced activation of the PI 3-kinase path-way in adipose tissue and liver as assessed by the phosphorylation of AKT. Brown preadipocyte cell lines from control and heterozygous mutant mice also displayed a marked reduction in the insulin-dependent phosphorylation of AKT. This decrease in PI3K activity was due to a defect in the ability of the mutant p85α to interact with phosphorylated IRS-1. Thus, the Arg649Trp mutation in p85α acts as a dominant negative allele, blocking PI 3-kinase ac-tivity and creating a model of SHORT syndrome with lipodystrophy, growth retardation, and insulin resistance.

Supported By: National Institutes of Health (DK045935); University of Bergen; Research Council of Norway; Kristian Gerhard Jebsen Foundation

1871-PDifferential Roles of Insulin Signaling in Neurons and Astroglia to Modulate Energy Homeostasis and MoodWEIKANG CAI, MASAJI SAKAGUCHI, C. RONALD KAHN, Boston, MA

Insulin is well known to play critical roles in the central nervous system on both regulation of systemic energy homeostasis and neural functions includ-ing cognition and mood. Insulin receptors (IR) are expressed in both neurons and astroglial cells, and both are ablated in previous brain specifi c IR knockout mice created using Nestin-Cre. To better understand the differential roles of insulin in neurons and astrocytes, we crossed IR-fl ox mice with GFAP-Cre and CamKIIa-Cre mice to generate astroglial (GIRKO) and neuronal (CamKIRKO) specifi c insulin receptor defi cient mice. Both mice were born at the expected Mendelian ratio and showed no developmental abnormality through adult-hood. CamKIRKO mice showed specifi c and effi cient IR deletion in neurons in the cerebrum, while GIRKO mice showed IR deletion in astroglia, as well as a portion of cortical, hippocampal and cerebellar neurons. Both mice had im-paired insulin signaling in selective regions in the brain in response to insulin injection. Both mice have normal body weights and early growth and develop-ment. CamKIRKO mice showed an age-dependent increase in fasting glucose level and impaired glucose tolerance. CamKIRKO mice also had less white fat mass and elevated free fatty acid level in the serum. In contrast, GIRKO mice showed normal fasting glucose, glucose tolerance, insulin tolerance, and normal body composition and normal serum free fatty acid level. By 3-month

of age, GIRKO mice developed anxiety and depression phenotypes, while CamKIRKO mice appeared to have normal mood regulation. Taken together, our present study revealed differential roles of insulin signaling in neuron and astroglia. Insulin receptor deletion in CamKIIa-expressing neurons leads to im-paired systemic energy homeostasis. In contrast, insulin signaling in astroglial cells appears to have a more predominant role in mood regulation.

1872-PmTORC2 Regulates Skeletal Muscle Insulin Sensitivity by Control-ling Glycolytic Rate and Intramyocellular Triacylglycerol StorageMAXIMILIAN KLEINERT, MARKUS A. RUEGG, DAVID E. JAMES, ERIK A. RICHTER, Copenhagen, Denmark, Basel, Switzerland, Sydney, Australia

In skeletal muscle, mTORC2 is emerging as an important regulator of glu-cose metabolism. To understand the underlying molecular mechanisms of how mTORC2 regulates glucose metabolism in skeletal muscle, we performed un-biased, global quantitative phospho- and total proteomic analyses of mouse muscles lacking the critical mTORC2 subunit Rictor (Ric mKO). This revealed that mTORC2 controls skeletal muscle glycolysis and lipid storage signaling pathways. In agreement, incubated Ric mKO muscle exhibited a 45% reduction (p<0.05) in insulin-stimulated muscle glycolytic fl ux; and Ric mKO mice were insulin resistant in vivo. Ric mKO also exhibited greater reliance on fat as an energy substrate, re-partitioning of lean to fat mass and 60% higher (p<0.05) intramyocellular triacylglycerol (IMTG) content. The increase in IMTG was ac-companied by 4-fold elevated (p<0.05) protein expression of the lipid droplet coating protein, Perilipin 3 (PLIN3) due to increased AMPK activity and nuclear FoxO1 protein content in Ric mKO muscle. In contrast, mice overexpressing a dominant negative α2 AMPK construct displayed 56% decreased PLIN3 protein expression, 48% decreased (p<0.05) IMTG content and 51% decreased (p<0.05) nuclear FoxO1 protein content. This demonstrates that mTORC2 regulates skel-etal muscle insulin sensitivity by controlling muscle glycolytic fl ux and lipid stor-age in the form of IMTG via a novel AMPK-FoxO1-PLIN3 signaling axis.

1873-PAPPL2 Negatively Regulates Adiponectin and Insulin Sensitivity In VivoJIYOON RYU, XIAOBAN XIN, KUN DONG, XIAONING GUO, FENG LIU, LILY Q. DONG, San Antonio, TX

Adiponectin exerts its benefi cial functions by binding to its membrane receptors, AdipoR1 and AdipoR2. Previous studies indicate that adaptor pro-teins APPL1 and APPL2 act as an integrated “Yin-Yang” machinery to regulate adiponectin signaling in muscle cells. To investigate the role of APPL2 in vivo, we generated liver-specifi c APPL2 knockout mice (L-APPL2 KO). Liver-specifi c disrupting APPL2 expression has no effects on mouse development, body weight, and food intake. However, the KO mice showed increased adiponec-tin signaling, reduced gluconeogenesis, and improved glucose tolerance and insulin sensitivity compared to their control littermates under high fat diet conditions. Interestingly, liver FGF21 levels were signifi cantly enhanced in the L-APPL2 KO mice compared to their control mice. Consistent with this fi nding, adiponectin induced FGF21 expression in primary hepatocytes and the stimulatory effect of adiponectin was further potentiated by suppress-ing APPL2 expression. Our study identifi es APPL2 as a key negative regulator of hepatic adiponectin signaling and insulin sensitivity and uncovers a link between hepatic adiponectin signaling and FGF21 expression.

Supported By: American Diabetes Association (7-13-BS-043 to L.Q.D.); National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Dis-eases (R01DK080344-01)

1874-PMicroRNA-194 Modulates Glucose Metabolism and Its Skeletal Muscle Expression Is Reduced in DiabetesCELINE LATOUCHE, ALAINA K. NATOLI, MEDINI REDDY-LUTHMOODOO, SARAH E. HEYWOOD, BRONWYN A. KINGWELL, Melbourne, Australia

MicroRNAs (miRs) have recently been linked to the regulation of glucose metabolism and type 2 diabetes (T2D).

This study aimed to identify miRs associated with insulin resistance and T2D. Skeletal muscle biopsies were taken from patients with unmedicated type 2 diabetes (n=5) and age matched healthy controls (n=5), as well as from insulin resistant offspring of rat dams fed a high fat diet during pregnancy. Microarrays were used to assess miR expression in muscle samples. The role of differentially expressed miRs in regulating glucose metabolism was subsequently investigated in the L6 skeletal muscle cell line using specifi c miRNA inhibitors/mimics to examine effects on basal and insulin-stimulated glucose transport, incorporation into glycogen and oxidation.

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Twenty-three miRs were differentially expressed in the skeletal muscle of patients with T2D compared to healthy controls. In the skeletal muscle of the insulin resistant rat offspring, there were 7 differentially expressed miRs.compared to control offspring. Among these, we identifi ed miR-194 as signifi cantly reduced by 30 to 50% in both models. Computational prediction of miR-194 target genes indicated several targets linked to T2D, insulin and AMPK signaling pathways. Knockdown of miR-194 in skeletal muscle cells induced an increase in basal glucose uptake (+42% vs. non transfected cells, p<0.001) and glycogen synthesis (+30% vs. non transfected cells, p<0.05), and potentiated insulin-stimulated glucose uptake (+30% vs. non transfected cells, p<0.05). Glucose oxidation was not altered when silencing miR-194.

Type 2 diabetes is associated with regulation of several miRs in skeletal muscle including miR-194 which is involved in glucose uptake into muscle cells and glycogenesis. MiR-194 could be down-regulated in patients with T2D as a compensatory response to facilitate glucose uptake into tissue in the face of insulin resistance.

1875-PInsulin Resistance in Human iPS Cells Reduces Mitochondrial Oxi-dative Metabolism and Metabolic FlexibilityALISON M. BURKART, KELLY TAN, JOHN F. O’SULLIVAN, SALVATORE IOVINO, LAURA WARREN, ROBERT E. GERSZTEN, C. RONALD KAHN, MARY-ELIZABETH PATTI, Boston, MA

Insulin resistance, a critical component of type 2 diabetes (T2D), precedes and predicts T2D onset and is associated with mitochondrial dysfunction. To defi ne the precise cause-effect relationship between insulin resistance and mitochondrial dysfunction in disease development, we analyzed induced pluripotent stem cells (iPSC) from 5 healthy individuals and 4 patients with genetic insulin resistance due to insulin receptor mutations. Insulin-resistant iPSC had a 22% increase in mitochondrial number, but 16% decrease in area (p<0.05). Mitochondrial oxidative function was impaired, with a 40% de-crease in citrate synthase activity and 55% decrease in spare respiratory capacity (p<0.05). Simultaneously, multiple glycolytic enzymes were up-regulated, lactate production increased 80% (p<0.05), and expression of the upstream inhibitor of PDH (PDK4) was increased, leading to decreased PDH activity - all consistent with a shift towards anaerobic metabolism. Global metabolomic analysis using mass spectrometry revealed that insulin-resis-tant iPSC were unable to appropriately modulate metabolism in response to increased glucose or lipid load, including dysregulation of 2-amidoadipic acid and keto-methylvalerate, which were both previously associated with T2D risk. Branched chain amino acid, lysine, and alanine metabolism were also altered in response to high glucose (q<0.05), while amino acid metabo-lism, glycolysis, and CoA biosynthesis were altered by lipid (q<0.05). These perturbations were accompanied by a 34% increase in ADP/ATP ratio and 3-fold increase in AMPK activity (p<0.05), indicating energetic stress. Thus, genetically-determined insulin resistance can alter mitochondrial size and oxidative activity and reduce metabolic fl exibility. These alterations in me-tabolism can modify function not only of undifferentiated stem cells, but also of differentiated insulin-target tissues that arise from them.

Supported By: American Diabetes Association (7-12-MN66 to M-E.P.); T32DK007260 (to A.M.B.); Harold Whitworth Pierce Charitable Trust (to A.M.B.); Novo Nordisk Foundation (to M-E.P., C.R.K.)

1876-PThe Role of a Novel APPL1 Isoform in Regulating Hepatic Adiponec-tin Signaling and Glucose MetabolismAMANDA K. GALAN, MIN LU, FENG LIU, LILY Q. DONG, San Antonio, TX, Boston, MA

APPL1 (Adaptor protein containing Pleckstrin homology domain, Phos-photyrosine binding (PTB) domain and Leucine zipper motif) is an adiponec-tin receptor binding protein that mediates the insulin sensitizing effect of adiponectin. In the present study, we report an alternative-splicing variant of APPL1 (APPL1sv) that is highly expressed in the liver. Overexpression of APPL1sv inhibits and suppression of APPL1sv levels enhances, respectively, adiponectin-stimulated AMPK phosphorylation in mouse hepatocytes. AP-PL1sv binds to AdipoR1 and AdipoR2 under basal conditions, and the binding is suppressed by treating hepatocytes with adiponectin. Consistent with an inhibitory role of APPL1sv in regulating adiponectin signaling, adenovirus-mediated suppression of APPL1sv greatly enhanced insulin sensitivity and inhibited gluconeogenesis in mice. Our study identifi es a key negative regu-lator of hepatic adiponectin signaling and insulin sensitivity, which may shed light on the development of novel therapeutic treatment for insulin resis-tance and type 2 diabetes.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases (F31DK097939)

1877-PInsulin Receptor Regulation: A Role of the Last Enzyme of the De Novo Purine Synthesis Pathway (ATIC)MARTIAL BOUTCHUENG-DJIDJOU, GABRIEL COLLARD-SIMARD, SUZANNE FORTIER, SEBASTIEN HEBERT, ISABELLE KELLY, CHRISTIAN R. LANDRY, ROBERT FAURE, Quebec, QC, Canada

Insulin plays a primary role in managing circulating glucose levels. The mechanism by which the insulin receptor (IR) is coupled to ATP levels is not completely understood. We performed a bioinformatic screen of Golgi/endo-some (G/E) hepatic fractions and found that ATIC, which is a rate-limiting enzyme in the de novo purine biosynthesis pathway, and protein-tyrosine phosphatase-like A domaincontaining protein 1 (PTPLAD1) are associated with IR internalisation. The IR interactome (IRGEN) connects ATIC to AMPK within the G/E protein network (GEN). Common heritable variants associated with type 2 diabetes represent 45% of the IRGEN, including ATIC and AMPK. We show that PTPLAD1 and AMPK are rapidly compartmentalised within the plasma membrane (PM) and G/E fractions after insulin stimulation and that ATIC accumulates in the G/E fraction later. Using an in vitro reconstitu-tion system and siRNA-mediated partial knockdown of ATIC and PTPLAD1 in HEK293 cells, we show that both ATIC and PTPLAD1 affect IR tyrosine phosphorylation and endocytosis. We further show that insulin stimulation and ATIC knockdown readily increase the level of AMPKThr172 phospho-rylation in IR complexes. IR internalisation was markedly decreased after AMPKα2 knockdown, and treatment with the ATIC substrate AICAR, which is an allosteric activator of AMPK, increased IR endocytosis in cultured cells and in the liver. Therefore, these results suggest the presence of a signalling mechanism that senses adenylate synthesis, cell energy and IR activation states and that acts in regulating IR autophosphorylation and endocytosis.

1878-PSpecifi c Metabolic Profi les and Their Relationship to Insulin Resis-tance in Recent-Onset Type 1 and Type 2 Diabetes—Results from the German Diabetes Study (GDS)BIRGIT KNEBEL, KLAUS STRASSBURGER, JULIA SZENDRÖDI, JÖRG KOTZKA, MARSEL SCHEER, BETTINA NOWOTNY, KARSTEN MÜSSIG, AKIKO MÖLLER-HORIGOME, STEFAN LEHR, PETER SCHADEWALDT, PETER NOWOTNY, MAGRIET OUWENS, GIONANNI PACINI, HELMUT FINNER, BIRGIT KLÜPPELHOLZ, GUIDO GIANI, HADI AL-HASANI, MICHAEL RODEN, Düsseldorf, Germany, Padova, Italy

Insulin resistance (IR) is not only a feature of type 2 diabetes, but may also occur in long standing type 1 diabetes. As reliable biomarkers are yet unavailable, we hypothesized that plasma metabolome profi les allow the detection of common or unique biomarkers for IR in both diabetes types.

Patients of a prospective observational trial (German Diabetes Study) with type 2 or type 1 diabetes (T2D, n=196; T1D, n=96) and known disease dura-tion of less than one year underwent hyperinsulinemic-euglycemic clamp tests and mass spectrometry based fasting plasma metabolite analyses and were compared to glucose tolerant persons (CON, n=129).

Of 294 metabolites, 62 show a phenotype specifi c pattern using age, sex and BMI as covariates. These metabolites allowed group separation in prin-cipal component analyses and explained most of the variance between T1D, T2D and CON in partial least square discriminant analyses. Nevertheless, the metabolme patterns are comparable for T2D or T1D vs. CON with enrich-ment mainly in identical pathways e.g. protein biosynthesis. The direct com-parison of T1D vs. T2D shows diabetes type specifi c metabolomic alterations and enrichment of completely different pathways, e.g. ammonium recycling. To follow this point, we analyzed the associations of metabolites with IR as clamp derived M-value, as the essential predictor of disease progression independent to diabetes type. After adjustment for body mass and glycemic control, solely phosphatidylcholine PCaeC44:3 correlated with IR in T1D. In T2D, next to known alanine and tyrosine, lysine and alpha amino adipic acid were negatively related to IR.

In conclusion, metabolome analyses in recent onset diabetes patients confi rms altered metabolite patterns already in short diabetes duration, show diabetes-specifi c metabolic patterns, and identifi ed PCaeC44:3 in type 1 diabetes, and lysine and its degradation product in type 2 diabetes as novel predictors of IR.

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1879-PNovel Insulin Sensitizers Regulate Pyruvate Metabolism via a Di-rect Interaction with the Mitochondrial Pyruvate CarrierWILLIAM MCDONALD, ZHOUJI CHEN, KYLE MCCOMMIS, SERENA COLE, JEAN WHEELER, ANGELA BRIGHTWELL-CONRAD, DANIELLE HOLEWA, CINDY WOLFE, KRISTIN COULTER, PETER KILKUSKIE, ALFREDO TOMASSELLI, BRIAN FINCK, ROLF KLETZIEN, JERRY COLCA, Kalamazoo, MI, St. Louis, MO

We have previously shown with a photoaffi nity probe that the site of thiazolidinedione insulin sensitizers binding to the mitochondria involves a protein known as MPC2, a component of the mitochondrial pyruvate carrier (MPC), responsible for transport of pyruvate across the inner mitochondrial membrane. These new insulin sensitizers, as well as the fi rst generation insulin sensitizers, rosiglitazone and pioglitazone, specifi cally modulate pyruvate oxidation in various cell types. Herein, we have extended these observations using skeletal muscle, brain, and liver mitochondria and de-veloped an assay that can be used to evaluate many compounds for effects on pyruvate utilization. Using an Oroboros Oxygraph O2K system, the active TZDs, but not the inactive analog MSDC-1473, reduced pyruvate oxidation in the presence of malate (5mM) and ADP (1 mM) within 1 minute of the addi-tion of the active compounds. However, the effect of these compounds was lost in hepatic mitochondria from liver-specifi c MPC2 knockout mice. In order to obtain a more robust analysis of this activity, the assay was converted to 96 well plates using the O2 sensitive LuxCel reagent. Consistent with results obtained with the Oxygraph, active TZDs exerted a rapid, dose-dependent attenuation of pyruvate oxidation by isolated mitochondria. Maximal inhibi-tion was observed at 10 micromolar with half-maximal effects at less than 1 micromolar. There was no inhibition of succinate-mediated respiration. Moreover, when pyruvate concentrations were varied from 0.1-5 mM (leav-ing malate and ADP constant at 5 and 1 mM), TZDs competitively interacted with pyruvate in both muscle and brain mitochondria. These results provide additional evidence that insulin-sensitizing molecules specifi cally interact with the MPC and provide a system for further evaluation of this pharmacol-ogy, including a means to search for other agents with this mechanism of action.

1880-PNew Mouse Model of Diabetes/Tuberin-defi cient Showed Signifi -cant Increase in EMTSAMY L. HABIB, SITAI LIANG, San Antonio, TX

Progressive decline in renal function in diabetes is ultimately due to the increase in extracellular matrix proteins and alteration in epithelial-to-mes-enchymal transition (EMT). EMT is a process in which tubular epithelial cells undergo a phenotypic change into fi broblasts. Tubular epithelial cells could express fi broblast markers in diabetes, postulating of EMT. Major chang-es in cell number refl ect the balance between the cells proliferation and apoptosis. Our laboratory showed that hyperglycemia regulates renal cell apoptosis/fi brosis/proliferation but the target mechanism by which hyperg-lycemia contributes to EMT is not known. To test the role of tuberin in regu-lation of renal function and EMT proteins, TSC2+/- and TSC2+/+ (C57BL/6J background) mice were crossed with db/db mice (C57BL/6J background). The glucose levels in both TSC2+/-/dbdb mice and TSC2+/+/dbdb mice were around 180-240mg/dL. New strain of TSC2+/-/dbdb mice showed increase in proteinuria to almost 2-fold compared to TSC2+/+/dbdb mice (21.0 vs. 12.3mg protein/24h urine). The morphologic changes in kidney sections of TSC2+/-/dbdb showed signifi cant changes in tubular thickening, glomerular and tubu-lar hypertrophy compared to TSC2+/+/dbdb. Kidney hypertrophy in TSC2+/-/dbdb is increased 2-fold compared to TSC2+/+/dbdb mice. Creatinine clear-ance is increased to 2.5-fold in TSC2+/-/dbdb compared to TSC2+/+/dbdb mice. Signifi cant increase in vimentin and decrease in E-cadherin as mark-ers of mesenchymally and epithelial-derived cells undergoing an EMT were detected in kidney sections as well as in kidney homogenate of TSC2+/-/dbdb compared to TSC2+/-/dbdb mice. These data showed for the fi rst time that signifi cant decrease in tuberin under hyperglycemica condition resulted in increased vimentin and decreased in N-cadherin. These data suggests that decrease in tuberin led to increase epithelial-to-mesenchymal transi-tion and highlight the novel role of tuberin in enhance kidney tumorigensis in diabetes.

Supported By: U.S. Department of Veterans Affairs

1881-PInsulin Increases the Expression of the Genes Associated with Gly-coprotein 130 Signaling in Human Skeletal Muscle and This Effect Is Reversed by Free Fatty AcidsMAREK STRACZKOWSKI, MAGDALENA STEFANOWICZ, NATALIA MATULEWICZ, AGNIESZKA NIKOLAJUK, ELZBIETA OTZIOMEK, MONIKA KARCZEWSKA-KUPC-ZEWSKA, Białystok, Poland, Olsztyn, Poland

Glycoprotein 130 (gp130) cytokines, including interleukin 6 (IL-6), act through plasma membrane receptors consisting of 2 glycoproteins: a cytokine binding subunit (like IL-6R) and gp130, responsible for signal transduction. IL-6 may induce insulin resistance, however, in some conditions it may exert insulin-sensitizing effect in skeletal muscle. The aim of the present study was to assess the effect of insulin and free fatty acids on the expression of the genes associated with gp130 signaling in skeletal muscle of healthy humans and in cultured human myotubes. In experiment 1, we examined 20 healthy male subjects with normal glucose tolerance (mean age 25.2±3.2 years, mean BMI 26.5±4.6 kg/m2). The biopsy of vastus lateralis muscle was performed before and after 6-hour clamps without or with Intralipid/heparin infusion. In experiment 2, we performed muscle biopsy in 8 healthy male subjects with similar characteristics and then differentiated satellite cells into myotubes. We treated myotubes with insulin and palmitate separately and in combination. In both experiments, the expression of IL-6R, gp130 and associated genes was analyzed with Real Time PCR. In experiment 1, serum IL-6 increased and soluble forms of IL-6R and gp130 decreased similarly dur-ing both clamps (all p<0.05). Insulin infusion resulted in an increase in muscle expression of IL-6R (p<0.001) and JAK2 (p=0.041) in the entire group and in gp130 (p=0.043) in normal-weight individuals. Concurrent Intralipid/heparin infusion, which reduced insulin sensitivity by approx. 40%, abolished all these insulin’s effects. In experiment 2, incubation with insulin increased the expression of IL-6R, gp130 and SOCS3 (all p<0.05). Incubation with palmitate reversed all these changes and decreased JAK1 (p=0.041). Our data show that the regulation of genes associated with gp130 signaling in skeletal muscle may be important for lipid-induced insulin resistance.

Supported By: National Science Center of Poland (2011/01/B/NZ5/05380); Op-erational Program Innovative Economy (UDA-POIG.01.03.01-00-128/08)

1882-PInsulin Releases Brake on Autocrine VEGF Signaling in Endothelial Cells by Downregulating the Transcriptional Repressor CITED2SAMUEL LOCKHART, XUANCHUN WANG, DITTE SØRENSEN, LARS M. RASMUS-SEN, CHRISTIAN RASK-MADSEN, Boston, MA, Odense, Denmark

Impaired neovascularization of ischemic tissue in the myocardium and lower limb affects both recovery and survival from complications of diabe-tes. Since insulin can promote proangiogenic function in endothelial cells, poor angiogenesis in diabetes may be caused in part by insulin resistance in endothelial cells. We recently identifi ed CITED2 (CBP/p300-interacting transactivator 2) as a target of insulin signaling in this cell type. We now have data showing that CITED2 protein is upregulated in cardiac endothelial cells from mice with insulin receptor gene knockout targeted to endothelial cells. Adenoviral-mediated expression of wild-type FoxO1 or a constitutively active FoxO1 mutant increased CITED2 mRNA and insulin decreased CITED2 in cells expressing wild-type, but not mutant, FoxO1. Insulin-induced tube for-mation on Matrigel was increased by siRNA-mediated knockdown of CITED2 and decreased by overexpression of CITED2. Hypoxia increased endothelial cell CITED2 mRNA by up to 3-fold and this effect was completely prevented by insulin treatment. Autocrine vascular endothelial growth factor (VEGF) signaling has previously been shown to be essential for angiogenesis. When cultured endothelial cells were exposed to hypoxia, VEGF mRNA increased by up to 3-fold without insulin and up to 5-fold with insulin. siRNA-mediated knockdown of CITED2 prevented the additive effect of insulin on hypoxia-stimulated VEGF induction. Insulin did not regulate CITED2 in cultured mouse vascular smooth muscle cells. Furthermore, CITED2 expression was higher in endothelial cells than vascular smooth muscle cells in human artery biop-sies. We conclude that insulin may promote angiogenesis through inhibition of FoxO1, downregulation of CITED2, and augmentation of hypoxia-induced, autocrine VEGF signaling. Enhancing insulin sensitivity of this pathway or directly inhibiting CITED2 may improve angiogenesis in patients with type 2 diabetes.

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1883-PModulation of Myocardial Ketone Body Oxidation by Increased Glu-cose DeliveryMANOJA K. BRAHMA, MARK A. MCCRORY, ADAM R. WENDE, Birmingham, AL

An adult heart exhibits metabolic fl exibility by utilizing a variety of sub-strates including fatty acids, carbohydrates, and ketone bodies to meet its constant fuel demand. The loss of this fl exibility during overt diabetes is believed to be an underlying mechanism of diabetic cardiomyopathy. De-spite signifi cant levels of circulating ketone bodies, cardiac ketone bodies oxidation is attenuated in type 1 diabetic mice. However, the mechanism of this reduction and regulation of the ketolytic pathway in a diabetic heart is incompletely understood. Several reports suggest that metabolites from accessary pathways of glucose metabolism can critically regulate cellular function in multiple tissues including heart. We therefore sought to defi ne high glucose-induced molecular mechanisms leading to regulatory changes in the myocardial ketolytic machinery in a diabetic heart. Our fi ndings from the heart of streptozotocin (STZ)-induced diabetic mice confi rmed that diabetes promotes signifi cant (P < 0.05) transcriptional suppression of two important ketolytic genes for succinyl-CoA:3-oxoacid CoA transferase (SCOT; encoded by Oxct1) and 3-hydroxybutyrate dehydrogenase, type 1 (BDH1; encoded by Bdh1) and induction of a ketone synthesis gene 3-hydroxy-3-methylglu-taryl-Coenzyme A synthase 2 (HMGCS2; encoded by Hmgcs2). Accordingly, β-hydroxybutyrate, a major ketone accumulated in the diabetic myocardium indicating impaired cardiac ketone bodies utilization. Interestingly, cardio-myocyte-restricted expression of the insulin-regulated glucose transporter 4 (GLUT4) in transgenic mice (mG4H) alone was suffi cient to reduce cardiac Oxct1 expression at mRNA and protein levels. This transgene-specifi c sup-pression was maintained even in the diabetic myocardium suggesting that increased glucose delivery may modulate myocardial ketone body catabolic machinery by multiple mechanisms independent of other changes that occur during diabetes.

Supported By: JDRF; National Institutes of Health (K99R00HL111322)

1884-PGRP78-defi cient Macrophages Induce Muscle Expression of IL-13 and Prevent Diet-induced Insulin ResistanceJONG HUN KIM, EUNJUNG LEE, DAE YOUNG JUNG, RANDALL H. FRIEDLINE, SEZIN DAGDEVIREN, PAYAL R. PATEL, XIAODI HU, DUY A. TRAN, KEVIN S. HSU, KUNIKAZU INASHIMA, PANKHURI WALIA, ROGER J. DAVIS, KI WON LEE, AMY S. LEE, JASON K. KIM, Worcester, MA, Seoul, Republic of Korea, Los Angeles, CA

Skeletal muscle insulin resistance is associated with obesity and in-fl ammation, but the underlying mechanism is unclear. GRP78 is a major ER chaperone that controls the unfolded protein response (UPR), and mice with Grp78-defi cient macrophages (Lyz-Grp78-/-) are protected from diet-induced insulin resistance in skeletal muscle (Fig. 1). After 9 wks of high-fat diet (HFD), Lyz-Grp78-/- (KO) and Lyz-Cre (WT) mice (n=5~8/group) became obese, but plasma IL-13 levels were signifi cantly increased by more than 5-fold in KO mice (Fig. 2). Consistent with this, muscle expression of IL-13 and IL-13 re-ceptor (IL-13rα1) tended to increase in KO mice (Fig. 2). Bone marrow-derived macrophages (BMM) were isolated and showed a signifi cant upregulation of Grp94 and spliced XBP1, which are indicative of UPR, in KO mice (Fig. 3). BMM from KO mice also showed increased levels of IL-6 mRNA and protein levels (Fig. 4). Lastly, IL-6-treatment in mouse myoblast cell line caused a 2-fold increase in IL-13 and IL-13rα1 levels. Overall, our fi ndings indicate a novel pathway by which Grp78-defi cient macrophages, possibly via upregu-lation of UPR and IL-6, induce muscle expression of IL-13 that may increase glucose metabolism and prevent diet-induced insulin resistance in skeletal muscle.

Supported By: National Institutes of Health (R01DK080756)

1885-PProfi ling the Biosynthesis of Novel Insulin Sensitizing and Anti-infl ammatory LipidsMARK M. YORE, MATTHEW J. KOLAR, ISMAIL SYED, PRATIK ARYAL, ARCHANA VI-JAYAKUMAR, ALAN SAGHATELIAN, BARBARA B. KAHN, Boston, MA, La Jolla, CA

Branched Fatty Acid esters of Hydroxy-Fatty Acids (FAHFAs) are a newly discovered lipid class. A subgroup, Palmitic Acid Hydroxy Stearic Acids (PAH-SAs), are elevated ~18 fold in adipose tissue (AT) of mice that overexpress the Glut4 glucose transporter in AT (AG4OX). PAHSA levels are regulated by Carbohydrate-response element binding protein (ChREBP). In mice, PAHSA levels are regulated by fasting/refeeding and high fat diet (HFD). In insu-lin-resistant people, PAHSA levels in serum and SQ AT are lower than in insulin-sensitive people, and levels correlate highly with insulin sensitivity. We aimed to determine how tissue PAHSA levels are regulated. We devel-oped an in vitro assay which measures PAHSA biosynthetic capacity using palmitoyl-CoA and hydroxy-stearic acid substrates. We detected PAHSA biosynthetic activity in human SQ and omental AT and in all mouse tissues tested including perigonadal (PG) and SQ AT, BAT, liver, kidney, heart, brain, small/large intestine, muscle and spleen. Biosynthetic activity was 6-fold higher in 3T3-L1 adipocytes than in pre-adipocytes. In mouse PG AT, PAHSA biosynthesis decreases with fasting and increases with refeeding indicating that PAHSA biosynthetic enzymes undergo nutritional control. Surprisingly, PAHSA biosynthetic capacity does not correlate with relative PAHSA lev-els in these tissues. Paradoxically, PAHSA biosynthesis is decreased 50% in both PG and SQ AT and increased 100% in liver of AG4OX mice in spite of opposite changes in PAHSA levels. PAHSA biosynthetic capacity in SQ AT is unaffected by HFD feeding and ChREBP deletion while PAHSA levels are decreased. In summary, tissue biosynthetic capacities do not refl ect PAHSA levels. Also, nutritional and genetic manipulations which alter tissue PAHSA content in vivo may have no effect or opposing effects on in vitro biosyn-thetic capacity. Thus, substrate availability and/or degradation are likely to regulate PAHSA levels in vivo and these pathways may provide new thera-peutic targets for diabetes.

Supported By: National Institutes of Health (R01DK098002); JPB Foundation

1886-PRapid Parallel Measurements of Macroautophagy and Mitophagy Using a Single Fluorescent BiosensorASHOT SARGSYAN, MEGHAN LABASKY, LUIS FANDINO, TANYA FOROSTYAN, SPENCER J. THOMPSON, LUCY K. COLOSIMO, JINJIN CAI, TIMOTHY E. GRA-HAM, Salt Lake City, UT

Impairment of autophagy and autophagy-mediated mitochondrial turnover (mitophagy) are implicated in insulin-resistance, hepatic steatosis, and type 2 diabetes. Methods for measuring autophagy and mitophagy have relied on laborious single-cell microscopy. Here we report a fl ow cytometry (FCM)-based assay employing an organelle-targeted fl uorescent protein biosensor (Rosella) to measure autophagy and mitophagy in parallel. Rosella is a chi-mera of a pH-sensitive green fl uorescent protein (pHluorin) and a pH-stable red fl uorescent protein (dsRed.T3). Attaching Rosella to cellular “cargos” makes it possible to monitor delivery to acidic lysosomes. We sought to use autophagasome- and mitochondria-targeted Rosella constructs (Rosella-LC3 and mito-Rosella, respectively) to quantify autophagy and mitophagy in HeLa cells, a well-characterized model for studying CCCP-induced autophagy and mitophagy. Cells expressing Rosella-LC3 and mito-Rosella, with or without Parkin co-expression, were treated with CCCP or vehicle. In cells expressing Rosella-LC3, CCCP induced a reduction in green fl uorescence that identifi ed cells with increased autophagy. In cells expressing mito-Rosella, CCCP also induced a green-shift, but only in cells expressing Parkin. FCM analysis of green vs. red fl uorescence revealed a time- and dose-dependent induction of autophagy by CCCP that did not depend on Parkin expression; in contrast, induction of mitophagy by CCCP required Parkin, as previously reported. Au-tophagy and mitophagy were inhibited by lysosomal proton pump inhibitor,

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BafA1, indicating dependence on lysosomes. Responses were confi rmed by immunoblot analysis of LC3 and mitochondrial markers. Measuring 10,000 cells by FCM required 7 seconds of analysis time. Therefore, this parallel assay for autophagy/mitophagy has the capacity to make hundreds to thou-sands of measurements in minutes to hours, enabling the screening of large libraries of compounds or genetic manipulations.


1887-PEffects of Pioglitazone on Circulating MiRNAs Associated with Glu-cose Homeostasis in Type 2 DiabetesATTILA SEYHAN, CHRISTIAN MEYER, RICHARD E. PRATLEY, YURY NUNEZ-LO-PEZ, Orlando, FL

MicroRNAs (miRNAs) regulate a wide variety of cellular responses in type 2 diabetes (T2D). Pioglitazone (PIO), a PPARγ agonist used to treat T2D, has been shown to improve β-cell function and insulin sensitivity and reduce infl ammation. We hypothesized that PIO treatment would alter circulating miRNAs associated with β-cell injury/function, insulin sensitivity and infl am-mation. This was tested in a 3-month trial in which 24 patients with T2D who were well-controlled (HbA1c ≤ 7.0%) with diet/exercise or metformin were randomized to either PIO or placebo (PBO) treatment (NCT00656864). Plasma levels of miRNAs previously associated with diabetes were measured be-fore and after treatment. Levels of 7 miRNAs changed in response to PIO treatment relative to PBO (Table 1). Two characteristic miRNA signatures emerged: (I) miR-24.1 and miR-375 decreased in PIO treated patients while increasing in the PBO group and (II) miR-21, miR-29a, miR-126, miR-146a, and miR-148a increased in the PBO group while remaining stable in PIO-treated subjects. Changes in miR-24.1 and mIR-29a tended (p<0.1) to correlate with changes in fasting C-peptide, while changes in miR-375 tended to correlate with changes in fasting plasma glucose. These data suggest that circulating miRNAs have potential as novel biomarkers in T2D to monitor responses to treatment and could be useful to elucidate the mechanism of action of novel therapies.

1888-PMice Telomerase Positive Cells in the PVN Region of the Hypothala-mus Regulate Diabetes in Irs2 Null MiceYUE YU, KYLE COPPS, MORRIS F. WHITE, Boston, MA

The global incidence of diabetes mellitus has increased dramatically over the past few years and continues to rise. Stem cell-based therapy has the potential to transform the treatment of diabetes and other metabolic disor-ders. Functional beta cells can be produced from iPS cells, and some reports suggest that adult stem cells_including cells from bone marrow, adipose or the pancreas_are capable of regenerating tissues for diabetes treat-ment. To investigate the role of adult stem cells in metabolic homeostasis and explore the possibility of adult stem cell-based therapy for diabetes, we investigated Irs2 (Insulin Receptor Substrate 2) defi cient mice (Irs2-/-), a diabetes mouse model that is well established in our laboratory. Irs2-/- mice exhibit diabetes mellitus, marked by hyperglycemia, glucose intolerance and diabetic associated diseases including vision loss, heart failure and early mortality. Some adult stem cells are detected in tissues of Irs2-/- mice by tracing mouse telomerase (mTert), a widely accepted marker for stem cells or potential progenitor cells. We found more mTert positive (mTert+) cells in olfactory bulb and PVN region in hypothalamus of adult Irs2-/- mice, com-

pared to wild type littermate controls. To investigate the therapeutic roles of these mTert+ cells for diabetes in Irs2-/- mice, we deleted Foxo1lox/lox alleles using mTert-CreER transgene and tamoxifen treatment. Foxo1 dele-tion in mTert+ cells of Irs2-/- adult mice decreased fasting glucose, improved oral glucose tolerance and mortality, and decreased mTert+ cell quantities in olfactory and PVN regions. Immunofl uorescence results suggested that mTert+ cells proliferated in the PVN region in Irs2-/- mice, and that upon deletion of Foxo1 cells promoted neuronal progenitor cell differentiation into mature functional neurons, which can coordinate peripheral glucose regula-tion in Irs2-/- mice.

1889-PSensitive and Easy-to-Use Bioluminescent Assays for Measuring Glucose Dependent Metabolic PathwaysMICHAEL VALLEY, MARY SOBOL, DONNA LEIPPE, JOLANTA VIDUGIRIENE, Madi-son, WI

Maintaining blood glucose levels within a narrow range is a critical physi-ological function requiring multiple metabolic pathways and involving sev-eral cell types. This process is controlled by hormones such as insulin and glucagon and depending on the feeding or fasting conditions, the cells adjust their glucose metabolism to keep the blood glucose level under tight control. Impairments in insulin signal transduction pathways are the main contribu-tors to insulin resistance and type 2 diabetes. From a broad perspective, researchers are interested in monitoring glucose levels, but more specifi c measurements of a key set of glucose dependent metabolites can be much more informative. We have harnessed the many advantages of biolumines-cence to develop assays for such metabolic biomarkers. By combining the sensitive detection of our NAD(P)H-Glo technology with various specifi c de-hydrogenases, we can tailor assays to detect glucose, glutamate, lactate, glucose-6-phosphate, 2-deoxyglucose-6-phosphate (for glucose uptake), or other cellular metabolites. Using a glucose uptake assay, compounds alter-ing translocation of glucose transporters can be screened and characterized rapidly. Glucose detection assays provide a convenient and robust approach for studying activation or inhibition of gluconeogenesis or glycogenolysis, while lactate detection assays can refl ect perturbations in glycolysis. These bioluminescent assays are robust and sensitive, and the assay windows are larger than similar fl uorescent or colorimetric methods. They can be used in various formats (e.g. 96- and 384-well plates), applied to various sample types (e.g. mammalian cells, tissues, and 3D microtissues), and multiplexed with other assays (e.g. cell viability) to maximize information per well.

1890-PInsulin Receptor Isoform A Is Implicated in Podocyte Injury in Dia-betic Kidney DiseaseALLA MITROFANOVA, XIMENA A. MORALES, MAYRIN CORREA-MEDINA, CHRISTOPHER PEDIGO, FARAH LECLERCQ, GEORGE W. BRUKE, SANDRA MER-SCHER, ALESSIA FORNONI, Miami, FL

Disruption of physiological insulin signaling occurs in podocytes in ex-perimental diabetic kidney disease (DKD). Two isoforms of insulin receptor (IR) have been described that either lack (IRA) or contain (IRB) the 12 amino acids encoded by exon 11. IRA signals primary through p70S6 kinase and IRB signals primary through AKT. Podocytes are dependent on functional lipid rafts and associated protein caveolin-1 (Cav1) for proper signaling and survival. We have recently shown sphingomyelin-phosphodiesterase-acid-like-3b (SMPDL3b), which regulates the activity of acid-sphingomyelinase in podocyte lipid rafts, is upregulated in DKD and may contribute to podocyte injury. We tested the hypothesis that SMPDL3b overexpression suppresses IRB signaling and facilitates IRA signaling leading to podocyte hypertrophy and apoptosis.

Podocytes were treated with insulin (0.1 or 1 nM, 30 min) and samples were used for Western blot analysis. Co-immunoprecipitation experiments (Co-IP) were performed in HEK cells, transfected with 1ug of GFP- or FLAG-SMPDL3b, FLAG-IRA, FLAG-IRB, GFP-Cav1 plasmid. Apoptosis analysis was measured using Caspase-3 Fluorometric Assay kit. Cell surface area was measured by confocal microscopy (OPERA XL).

Podocytes overexpressing SMPDL3b (OE) showed signifi cant increase of IRB/IRA mRNA expression ratio compared to control podocytes (CT) (1.00±0.00 and 0.74±0.05, p<0.05). Co-IP demonstrated that both IRA and IRB interact with Cav1. SMPDL3b overexpression augmented IRA/Cav1 interac-tion and suppressed IRB/Cav1 interaction. OE podocytes showed bigger cell surface area compared to CT (2778.0±246.5 and 2171.0±54.7, p<0.05). Insulin stimulation had no effect on the caspase-3 activity in CT podocytes while it induced apoptosis in OE cells (1.19±0.08 and 2.14±0.32, p<0.05).

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Our data suggest SMPDL3b overexpression in human podocytes impairs IRB signaling leading to increased apoptosis and augments IRA signaling resulting in cell hypertrophy.

Supported By: National Institutes of Health (DK090316); National Institutes of Health-National Institute of Diabetes and Digestive and Kidney Diseases (5U24DX076169)

1891-PAcute Hyperinsulinemia Decreases Pregenual Anterior Cingulate Cortex (pgACC) Myo-Inositol Independent of Glucose Levels in Healthy Nondiabetic SubjectsNICOLAS R. BOLO, ALAN M. JACOBSON, BRANDON HAGER, GAIL MUSEN, MATCHERI KESHAVAN, DONALD C. SIMONSON, Boston, MA, Mineola, NY

Increased brain myo-inositol (MI) levels have been found in patients with metabolic syndrome and T2DM. However, the independent effects of glu-cose and insulin on MI and other brain metabolites are not clearly defi ned. To examine this question, we measured concentrations of MI, glutamate, N-acetylaspartate, choline and creatine using proton magnetic resonance spectroscopy (MRS) localized in 8 ml of the pgACC during acute hypergly-cemia and hyperinsulinemia in 10 healthy non-diabetic subjects (mean ± SD age = 28 ± 7 yrs, 6 M / 4 F, HbA1c = 5.5 ± 0.3%, FPG = 93 ± 6 mg/dl, fasting in-sulin = 4.0 ± 2.5 µU/ml). During visit 1, MRS was performed in the fasted eug-lycemic (EU1) state followed by a 60 minute hyperglycemic (HG) clamp (mean glucose = 205 ± 17 mg/dl, insulin = 36 ± 24 µU/ml). During visit 2, at least 15 days later, subjects were again studied in the basal state (EU2) followed by a hyperinsulinemic euglycemic clamp (HI-EU3) (mean glucose = 96 ± 7 mg/dl, insulin = 32 ± 21 µU/ml) designed to match the insulin levels achieved during the HG clamp. We analyzed MRS data using the LCModel software to obtain absolute concentrations by scaling metabolite signals to the water signal in the pgACC, and performed paired comparisons of concentrations during 1) EU1 vs. HG (Visit 1), 2) EU2 vs. HI-EU3 (Visit 2), and 3) HG (Visit 1) vs. HI-EU3 (Visit 2). MI was signifi cantly decreased during HG (17.1 ± 3.5 mM) relative to EU1 (19.6 ± 4.2 mM, p=0.006) and during HI-EU3 (16.1 ± 1.7 mM) relative to EU2 (18.0 ± 2.6 mM, p=0.013). No signifi cant difference was found between the HG (hyperglycemia) and HI-EU3 (matched hyperinsulinemic euglycemia) clamps. No differences were found for other metabolites. Thus, high plasma insulin decreases MI, while high plasma glucose has no signifi cant effect on MI in the pgACC. This suggests that elevated brain MI levels in patients with diabetes or metabolic syndrome may be due to reduced insulin levels or action in the brain, rather than hyperglycemia.

Supported By: National Institutes of Health (DK084202)

1892-PFunctional and Structural Studies on the Diabetes Risk Factor Mem-brane Protein ZnT8MARK J. DANIELS, MACIEJ JAGIELNICKI, MARK YEAGER, Charlottesville, VA

The human zinc transporter ZnT8 (SLC30A8), expressed predominantly in pancreatic β-cells, is key in maintaining the concentration of blood glucose through its role in insulin storage, maturation, and secretion. This transporter is associated with type 2 diabetes through a risk allele that encodes a major non-synonymous SNP at Arg325, and also with type 1 diabetes as a major autoantigen. Interestingly, non-functional mutants reduce diabetes risk, suggesting a therapeutic function for compounds that inhibit ZnT8 activity.

This ~40 kDa protein is known to form homodimers in β-cells, where it regulates transport of Zn2+ into insulin secretory granules. Despite its role in diabetes and its concomitant potential as a drug target, little is known about the structure or mechanism of ZnT8. The only structural information available is from studies on the homologous bacterial transporter YiiP. Our goal is to provide insight into the function of ZnT8 as a Zn2+/H+ antiporter as revealed by structural and functional studies.

We have successfully purifi ed ZnT8 constructs from Pichia pastoris and insect cell culture overexpression systems. With negative-stain electron microscopy (EM) and single particle image analysis, the molecular boundary of an EM density map at moderate resolution was suffi ciently well defi ned that we could dock a homology model of ZnT8. Our structure is consistent with a homodimer, and the bipartite appearance of each monomer is interpreted as the ~22 kDa alpha-heli-cal TM bundle and the ~10 kDa C-terminal domain. We have also expressed the N- and C-terminal domains of ZnT8 as protein fusions in E. coli and characterized their fold as mixed a-β by circular dichroism spectrometry. Complementary to our structural analyses, we developed in vivo functional assays in our P. pastoris and insect cell expression systems and found that ZnT8 enhances Zn2+ transport in both. The results of this research will be a starting point for drug design in targeting diabetes and its resulting complications.

Supported By: American Diabetes Association (7-13-BS-038 to M.J.D.)

1893-PIron Chelation Induces Hif1-alpha and Bnip3l/Nix-dependent Mito phagyASHOT SARGSYAN, MEGHAN LABASKY, LUIS FANDINO, TIMOTHY E. GRAHAM, Salt Lake City, UT

Increased iron stores are associated with type 2 diabetes, and phlebotomy to reduced iron stores in humans with high ferritin improves glycemia. Iron chelation therapy and low iron diets confer similar benefi ts in mouse mod-els of obesity and insulin resistance. Since mitochondrial function is highly dependent on iron availability, we hypothesized that iron might regulate mitochondrial quality control through mitophagy, the autophagy-mediated degradation of mitochondria in lysosomes. We employed a mitochondria-targeted lysosomal biosensor to measure effects of iron chelation on mi-tophagy in Hela cells, a well-characterized model for studying regulation of autophagy and mitophagy. Iron chelation with deferiprone (DFP) induced rapid and robust mitophagy; after 24 hrs, markers of mitochondrial content were reduced 50% vs. vehicle-treated control cells. Iron chelation stabilized Hif1-alpha, as previously reported. Silencing Hif1-alpha with siRNA inhibited DFP-induced mitophagy, indicating induction of mitophagy requires Hif1-alpha. Consistent with this, Echinomycin, a selective inhibitor of HIF1-alpha transcriptional activity, also inhibited DFP-induced mitophagy. Treatment of cells with IOX2, a prolyl-hydroxylase inhibitor that stabilizes Hif1-alpha, induced mitophagy independently of DFP. Hif1-alpha-mediated induction of Bnip3L/Nix, an autophagy adaptor protein present in the mitochondrial outer membrane, is a proposed mechanism for hypoxia-induced mitophagy. Silencing of Bnip3L with siRNA inhibited DFP-induced mitophagy, indicating Bnip3L plays a similar role in iron-regulated mitophagy. We conclude that iron chelation induces mitochondrial degradation via stabilization of Hif1-alpha, which in turn induces expression of Bnip3L, an autophagy adaptor protein that ultimately mediates mitophagy. We speculate that systemic iron status could infl uence mitochondrial quality and content through regulation of mitophagy by a similar mechanism in other cell types.


1894-PEndoplasmic Reticulum Stress Induced by Tunicamycin Increases Resistin mRNA through the PERK-ATF4-CHOP Pathway in THP-1 Hu-man MonocytesJUNPEI HAMADA, HIROSHI ONUMA, HIROKI HIRAI, KOJI TAKEMOTO, RYOICHI KAWAMURA, YASUNORI TAKATA, WATARU NISHIDA, SEIICHI HASHIDA, EIICHI ISHII, HARUHIKO OSAWA, Toon, Japan, Tokushima, Japan

Resistin, secreted from adipocytes, causes insulin resistance in mice. In humans, resistin gene is mainly expressed in monocytes and macrophages. Tunicamycin is known to induce endoplasmic reticulum (ER) stress, and re-duced resistin gene expression in 3T3-L1 mouse adipocytes. The aim of the present study was to examine whether ER stress affects resistin gene ex-pression in human monocytes.

The relation between resistin mRNA and ER stress markers mRNA was analyzed by RT-PCR in isolated monocytes of thirty healthy volunteers. The effect of lipopolysaccharides (LPS) or tunicamycin on resistin mRNA was analyzed in THP-1 human monocytes. Signaling pathways leading to resistin mRNA were assessed by the knockdown using siRNA or overexpression of key molecules involved in unfolded protein response.

Resistin mRNA was positively associated with either BiP or CHOP mRNA in human isolated monocytes (R=0.453, P<0.05, and R=0.420, P<0.05, re-spectively). In THP-1 cells, resistin mRNA was increased to ~ 1.5 fold in the presence of LPS. LPS also increased mRNA of BiP, PERK, and CHOP as well as resistin. Likewise, tunicamycin increased resistin mRNA. The mRNA of ER stress markers (BiP, PERK, ATF4, CHOP, IRE1, and XBP1), except for ATF6, was enhanced by tunicamycin. Tunicamycin-induced resistin mRNA and ER stress markers mRNA were inhibited by 4-phenylbutyric acid, a chemical chaper-one. The knockdown of either PERK, ATF4, or CHOP reduced tunicamycin-induced resistin mRNA. Resistin mRNA was not affected by the knockdown of ATF6, IRE1, or XBP1, whereas their own mRNA was reduced. Conversely, overexpression of ATF4 or CHOP gene increased resistin mRNA.

Therefore, ER stress induced by tunicamycin increased resistin mRNA through the PERK-ATF4-CHOP pathway in THP-1 human monocytes.

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INSULIN ACTION—GLUCOSE TRANSPORT AND INSULIN RESISTANCE IN VITRO


Guided Audio Tour: Glucose Transport—C’mon in, Sugar (Posters: 1895-P to 1900-P), see page 13.

& 1895-PInsulin Regulates Rab3/Noc2 Complex Dissociation to Promote Glut4 Translocation in Rat AdipocytesFRANCOISE KOUMANOV, VINIT J. PEREIRA, JUDITH D. RICHARDSON, SAMAN-THA L. SARGENT, DANIEL J. FAZAKERELY, GEOFFREY D. HOLMAN, Bath, United Kingdom, Sydney, Australia

In un-stimulated cells GLUT4 is moved through a series of intracellular compartments and becomes sequestered in a specialised intracellular vesi-cle compartment. Upon insulin stimulation it is translocated from this reser-voir compartment to the plasma membrane. The transit of GLUT4 between subcellular compartments is known to be dependent on a series of Rab pro-teins. However, the extents to which the activities of these Rabs are regu-lated by insulin action are still largely unknown. We have developed a new photoaffi nity reagent (Bio-ATB-GTP) which allows GTP binding proteomes to be explored. Using this reagent we have identifi ed Rab3B as a new candi-date insulin-stimulated G-protein that is linked to GLUT4 exocytosis in adi-pocytes. We confi rm Rab3 isoforms involvement in this process using con-stitutively active and dominant negative mutants and by Rab3 knockdown. We have identifi ed Noc2 as a negative regulator of GLUT4 translocation that interacts with a GDP-bound Rab3B. Insulin-stimulated GTP-loading of Rab3B disrupts the Noc2-Rab3B interaction, leading to displacement of Noc2 from the plasma membrane. We propose that this process relieves the inhibitory effect of Noc2 and consequently facilitates GLUT4 translocation.

Supported By: UK Medical Research Council

& 1896-PThe RAB10 Effector Protein SEC16A Regulates GLUT4 Traffi cking in AdipocytesJOANNE H. BRUNO, ALEXANDRIA BRUMFELD, DAVID IAEA, TIMOTHY E. MCGRAW, New York, NY

Adipocyte glucose uptake in response to insulin is essential for physi-ological glucose homeostasis. Understanding this response is of critical importance in gaining further insight into insulin action, as well as into the development of insulin resistance and type 2 diabetes. The process of in-creasing glucose transport into adipocytes in response to insulin requires the insertion of the glucose transporter GLUT4 into the plasma membrane, which mediates the ensuing increase in glucose uptake. Previous investiga-tions have characterized RAB10, a member of the highly conserved family of RAB GTPases, as being a major player in this process, although the precise mechanism through which it acts to promote GLUT4 storage vesicle deliv-ery to the plasma membrane remains unclear. Here we have used affi nity chromatography and subsequent mass spectrometry to identify adipocyte-specifi c RAB10 interacting proteins, and have characterized SEC16A as a novel RAB10 effector. Knockdown of SEC16A in 3T3-L1 adipocytes inhibits GLUT4 translocation to the plasma membrane in response to insulin, while having no effect on the traffi cking of non-insulin responsive cargo, such as the transferrin receptor. This inhibition appears to be due to a decrease in the insulin-stimulated GLUT4 exocytosis in SEC16A knockdown cells, which phenocopies that which has been observed for RAB10 knockdown. Further studies into the precise molecular mechanisms through which SEC16A is promoting GLUT4 traffi cking downstream of insulin and RAB10 could give us new insights into the mechanism of insulin action and the development of insulin resistance, as well as provide novel therapeutic targets for type 2 diabetes.

Supported By: Medical Scientist Training Program (GM007739)

& 1897-PAdipose Rab10 Knockout Induces Insulin IntoleranceREEMA P. VAZIRANI, LEIGH A. SADACCA, MELANIE BUCKMAN, TIMOTHY E. MCGRAW, New York, NY

Insulin regulation of glucose homeostasis is mediated by the traffi cking of the glucose transporter GLUT4 in muscle and adipose tissues, as well as the modulation of gluconeogenesis in the liver. Expression of adipose GLUT4 has been shown to be important for systemic insulin sensitivity since adipose GLUT4 knockout impairs insulin action not only in adipose, but also in the muscle and liver. Insulin-stimulated GLUT4 translocation involves activa-tion of Akt and subseqent inhibition of the Rab GAP AS160. In adipocytes,

AS160 inhibition results in activation of the GTPase Rab10. The physiological relevance of Rab10-mediated traffi cking of GLUT4 specifi cally in adipose is unknown. To probe this question, we created adipose-specifi c Rab10 knock-out (ARKO) mice. We found that adipocytes from ARKO mice have decreased insulin-stimulated glucose uptake, as well as impaired insulin-stimulated GLUT4 translocation to the plasma membrane. These results confi rm that adipocyte GLUT4 translocation machinery in primary adipocytes is similar to the signaling pathway elucidated in 3T3-L1 adipocytes. Insulin tolerance tests show that ARKO mice on both high and low fat diets are insulin intoler-ant. Furthermore, RNA sequencing analysis of white adipose tissue shows a signifi cant similarity in upregulated gene expression between adipose Rab10 knockout mice and adipose GLUT4 knockout mice. Unlike the adipose GLUT4 knockout, the defect in GLUT4 translocation in ARKO mice is moder-ate ~40%. This demonstrates that fully functional GLUT4 vesicle traffi cking is important for insulin sensitivity.

& 1898-PATBP, Adipocyte-specifi c Tubulin-binding Protein, Can Control Cy-toskeletal Structure and Insulin Stimulated Glucose UptakeMASASHI KURODA, RIE OKITSU, MARI KONDO, KASUMI NAKAGAWA, TOSHIO HOSAKA, NAGAKATSU HARADA, HIROSHI SAKAUE, Tokushima, Japan, Mitaka, Japan

The structure of cytoskeleton is reported to be involved in insulin-de-pendent glucose uptake. Upon insulin stimulation, GLUT4-containing vesi-cles move on microtubule to plasma membrane and GLUT4 is exposed to extracellular compartment, which allows glucose to enter to the cell. We have here identifi ed tubulin-associated protein, named Adipocyte-specifi c Tubulin-Binding Protein (ATBP), in white adipose tissue of C57BL/6 mice fed a high fat diet. Then, the purpose of this study is to cralify the role of ATBP in microtubule network structure and glucose transport in adipocytes. First, to check the association and collocalization of ATBP and tubulin, we performed immunoprecipitation and immunostaining against both molecules in 3T3-L1 adipocytes transfected with FLAG-tagged ATBP. The result showed the bind-ing and the collocalization of ATBP-FLAG and tubulin in 3T3-L1 adipocytes. Furthermore, inmmunostainning analysis showed enhanced fl uorescence accumulation of tubulin in ATBP-FLAG-overexpressed 3T3-L1 adipocytes, suggesting that ATBP can promote tubulin polymerization and bundling in adipocytes. We next carried out knockdown of ATBP in adipocytes with ad-enovirus mediated transfection to study the role of ATBP in triacylglycerol (TG) accumulation and insulin-dependent glucose uptake in 3T3-L1 adipo-cytes. The knockdown of ATBP caused impairment of insulin-induced GLUT4 translocation to plasma membrane and glucose transport, and decreased TG accumulation in adipocytes. Finally, we investigated body weight and the metabolism in ATBP knockout mice. ATBP knockout mice showed de-creased adiposity, reduced body weight and improved glucose tolerance when exposed to high-fat diet. These results indicated that ATBP has an important role in glucose homeostasis and lipid accumulation in adipocytes by affecting microtubule network. It might be possible that ATBP is a novel therapeutic target molecule against obesity.

& 1899-PTctex1d2, a Novel Syntaxin4 Interacting Protein That Negatively Regulates Insulin-Stimulated Glucose Transporter 4 Translocation and Glucose Uptake in 3T3-L1 AdipocytesYOKO SHIMODA, SHUICHI OKADA, JEFFREY PESSIN, MASANOBU YAMADA, Maebashi, Japan, Bronx, NY

Tctex1d2 is an open reading frame that encodes for a functionally un-known protein that contains a Tctex1 domain found in dynein light chain family members. Tctex1d2 expression in preadipose tissue was much less than in adipose tissue. Skeletal muscle, insulin sensitive tissue expressing glucose transporter 4, also expressed Tctex1d2 but tended to be less than adipose tissue. In adipose tissue Tctex1d2 expression was greater in visceral fat tissue than in subcutaneous fat tissue. Acute insulin stimulation in 3T3-L1 adipocytes overexpressing Tctex1d2 had no effect on the phosphorylation of Akt. However insulin-stimulated GLUT4 translocation and 2-deoxy glucose uptake was signifi cantly inhibited by Tctex1d2 overexpression but signifi -cantly increased by Tctex1d2 knock down. Tctex1d2 was found to associate with Syntaxin4 in an insulin dependent manner and inhibit Doc2b binding to Syntaxin4. Visceral adipose tissue from healthy human subject expressed Tctex1d2 much more than human diabetic subject. We hypothesized that Tc-tex1d2 is a negative regulator of Doc2b/Syntaxin4 interaction and thereby inhibits insulin-stimulated GLUT4 translocation and glucose uptake and might be involved in as one of the causes of diabetes mellitus.

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Importantly, sFRP4 is also expressed in adipose tissue. The current study in-vestigated whether the expression of sFRP4 in visceral adipose tissue (VAT) is altered in morbid obesity and type 2 diabetes, and whether sFRP4 interferes with metabolic control in primary human skeletal muscle cells (hSkMC).

Using real-time PCR analysis, sFRP4 gene expression levels were quanti-fi ed in VAT-biopsies from morbidly obese men (n=45, of whom 20 have type 2 diabetes) and control men (n=22) undergoing abdominal surgery. The effects of recombinant sFRP4 on insulin action and the expression of genes involved in glucose and lipid metabolism were studied in primary hSkMC from four different donors.

Expression of sFRP4 was 2.3-fold increased in VAT from patients with type 2 diabetes versus patients with morbid obesity or controls (P<0.002). In vitro, sFRP4 impaired insulin-stimulated Akt-phosphorylation by 50% in primary hSkMC (P<0.05). Furthermore, gene expression profi ling data sug-gest that sFRP4 disturbs energy substrate metabolism as illustrated by in-creased expression of CPT1B (1.8-fold), pyruvate dehydrogenase kinase 3 (PDK3; 2.5-fold), phosphorylase kinase gamma 1 (PHKG1; 2.7-fold), AMPKα2 (1.7-fold), and phosphoribosyl pyrophosphate synthetase 1 (PRPS1; 1.7-fold), and reduced levels of HSL (1.3-fold), AMPKβ (1.9-fold), and the fatty acid transporter solute carrier family 27 member 6 (SLC27A6; 2.3-fold) in primary hSkMC exposed to sFRP4 (n=4; all P< 0.05).

Collectively, these data show that the expression of the Wnt regulator sFRP4 is increased in VAT from patients with type 2 diabetes. In vitro, sFRP4 impairs insulin action and may affect glucose and lipid metabolism in skel-etal muscle.

1903-PCannabinoid Receptor 1 (CNR1) Expression in Human Adipose Tis-sue (AT) Correlates with Markers of Insulin Resistance (IR) and Obe-sity and May Be a Mediator of Glucocorticoid-induced LipolysisCHERNO O. SIDIBEH, MARIA J. PEREIRA, PRASAD KAMBLE, PETROS KATSOGI-ANNOS, MAGNUS SUNDBOM, MARIA K. SVENSSON, JAN W. ERIKSSON, Upp-sala, Sweden

Glucocorticoids and the endocannabinoid system are involved in the regu-lation of energy balance. We recently showed that CNR1 is upregulated in human AT by the synthetic glucocorticoid dexamethasone (dexa). Here, we study CNR1 involvement in glucocorticoid-induced IR in human AT.

Human subcutaneous (sc) and omental (om) AT, obtained from non-diabet-ic volunteers (13M/30F, 24-66 yr, BMI 20.7-55.5 kg/m2), was incubated with or without dexa (0.003-3 µM, 24h) in the presence or absence of the CNR1 antagonist AM281 for the last 4h of incubation. CNR1 expression as well as adipocyte lipolysis and glucose uptake were measured.

Dexa increased CNR1 expression dose-dependently in AT (sc: 14-fold, om: 29-fold; p<0.001). CNR1 gene expression was higher in sc than om AT in overweight-obese subjects, while levels were similar in normal-weight sub-jects. Furthermore, CNR1 gene expression in both depots correlated posi-tively with markers of IR and obesity, including insulin (sc: r=0.52, p<0.001; om: r=0.40, p<0.05), HOMA-IR (sc: r=0.56, p<0.001; om: r=0.38, p<0.05) and BMI (sc: r=0.75, p<0.001; om: r=0.45, p<0.01), and negatively with HDL-cho-lesterol (sc: r=-0.47, p<0.001; om: r=-0.39, p<0.05).

Dexa (0.3 µM) pre-treatment increased the rate of lipolysis by about 30%, compared with control (p<0.05), whereas the CNR1 antagonist AM281 prevented this effect (p<0.05). Dexa also reduced the basal rate of glucose uptake by about 35% (p<0.05) and showed a tendency in reducing insulin-stimulated glucose uptake by about 24% (p=0.069). However, treatment with the CNR1 antagonist AM281 did not prevent the effects of dexa on glucose uptake.

Our fi ndings suggest that CNR1 is upregulated in states of IR and obesity. Furthermore, CNR1 may be a mediator involved in glucocorticoid-regulated lipolysis in human adipocytes. This study gives further support for a role of the peripheral endocannabinoid system in obesity and IR.

Supported By: AstraZeneca; Sahlgrenska University Hospital; Portuguese Foun-dation for Science and Technology; Regional FoU-support Västra Götalandsre-gionen; Swedish Heart-Lung Foundation; Swedish Diabetes Association

1904-PFavorable In Vitro and In Vivo Actions of Insulin-like Growth Factor Binding Protein (IGFBP)-1 and Its RGD Domain in Insulin Sensitivity and Glucose RegulationNATALIE J. HAYWOOD, PAUL A. CORDELL, JESSICA SMITH, NADIRA Y. YULDA-SHEVA, MARK T. KEARNEY, STEPHEN B. WHEATCROFT, Leeds, United Kingdom

In prospective studies, low circulating levels of IGFBP1 predict the de-velopment of type 2 diabetes. We have previously shown that in vivo over expression of IGFBP1 improves insulin sensitivity and protects against ath-erosclerosis. IGFBP1 can impact on cellular functions via an RGD (α5β1 inte-

& 1900-PPKCµ Activation Inhibits AMPK through Ser485/491 Phosphoryla-tion and Inhibits Basal Glucose Uptake in C2C12 MyotubesKIMBERLY A. COUGHLAN, BELLA SUDIT, RUDY J. VALENTINE, NEIL B. RUDER-MAN, ASISH K. SAHA, Boston, MA

Our lab has previously shown that exposure to high glucose activates protein kinase C (PKC), inhibits AMP-activated protein kinase (AMPK), and causes insulin resistance in skeletal muscle. The inhibition of AMPK is medi-ated both by a decrease in phosphorylation at its activation site (Thr172) and by an increase in phosphorylation at its inhibitory site (Ser485/491). We re-cently showed that pharmacological PKC activation by the phorbol ester PMA stimulates phosphorylation of AMPK Ser485/491 and inhibits AMPK’s enzyme activity in the absence of changes in p-AMPK Thr172 in C2C12 myotubes. In the present study, we sought to determine which PKC isoform is responsible for the inhibitory phosphorylation of AMPK and whether these events affect basal glucose uptake in C2C12 myotubes. Since previous studies using broad PKC inhibitors showed that PKCµ activation correlated with phosphorylation of AMPK Ser485/491, we used the specifi c PKCµ inhibitor CRT0066101. Cells were pretreated for 1h with 10µM CRT0066101 prior to PMA treatment (50nM, 30min). As previously shown, PMA treatment signifi cantly increased phospho-rylation of AMPK at Ser485/491. Pretreatment with CRT0066101 prevented this phosphorylation. Phosphorylation of AMPK Thr172 was unchanged by treatment with PMA or the inhibitor. AMPK activity, as measured by the SAMS peptide assay, was signifi cantly diminished by PMA treatment, and this effect was completely prevented by pretreatment with the PKCµ inhibitor. Using 3H 2-deoxyglucose uptake, we found that PMA treatment signifi cantly decreased glucose uptake, which was prevented by pretreatment with the PKCµ inhibi-tor. In conclusion, PKC activation inhibits AMPK activity through Ser485/491 phosphorylation and inhibits basal glucose uptake in C2C12 myotubes, both of which can be prevented by the specifi c PKCµ inhibitor CRT0066101. Thus, PKCµ inhibition may be a novel strategy for the prevention or treatment of insulin resistance in skeletal muscle.

Supported By: T32HL07969

1901-PFree Fatty Acid-induced Insulin Resistance Is Associated with Dys-regulated Iron Metabolism in Human Skeletal Muscle CellsTAE HO KIM, SUNG-E CHOI, SANG-A LEE, JA YOUNG JEON, SO YOUNG OCK, SO-YEON AN, MIN SUK LEE, YUP KANG, SEUNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, KWAN-WOO LEE, Seoul, Republic of Korea, Suwon, Republic of Korea

Although iron is an important component of the respiratory system in mi-tochondria, the relationship between iron metabolism and insulin resistance (IR) in skeletal muscles has not been well studied. In this study, we inves-tigated the relationship between iron metabolism and palmitate-induced insulin resistance in human skeletal muscle cells. We measured cellular iron levels in palmitate-induced insulin resistant skeletal muscle cells using calcein-AM. In addition, we tested the effects of a variety of chemical iron chelators such as deferoxamine (DFO), deferasirox (DS), FeCl3, and FeSO4 in palmitate-induced insulin resistant skeletal muscle cells using the glucose uptake method. Because iron is a critical factor for respiratory metabolism, the current study was initiated to determine whether abnormal iron metabo-lism is involved in palmitate-induced insulin resistance. Palmitate treatment reduced insulin-induced Akt phosphorylation and glucose uptake but in-creased the intracellular iron content in skeletal muscle cells compared with control cells. When palmitate-treated skeletal muscle cells were treated with iron chelators, insulin-stimulated phosphorylated Akt levels and glu-cose uptake were recovered. However, iron supplementation by treatment with FeCl3 and FeSO4 augmented palmitate-induced insulin resistance. In-terestingly, iron supplementation in normal skeletal muscle cells reduced insulin-stimulated phosphorylated Akt and glucose uptake and evoked insu-lin resistance. Similarly, treating skeletal muscle cells with TNF-α elicited similar effects. The current study revealed that iron overload in skeletal muscle cells induced insulin resistance, whereas reduced intracellular iron levels protected palmitate-induced insulin resistance. Therefore, these data provide a therapeutic strategy for the treatment of insulin resistance.

1902-PSecreted Frizzled-Related Protein 4 Is increased in Type 2 Diabetes and Interferes with Insulin Action in Skeletal MuscleTINA HÖRBELT, PIA FAHLBUSCH, JOHANNES B. RUIGE, MARGRIET OUWENS, Düsseldorf, Germany, Ghent, Belgium

Secreted frizzled-related protein 4 (sFRP4) is a regulator of the activity of the Wnt signaling pathway. Circulating levels of sFRP4 are elevated in obesity even before the onset of type 2 diabetes, and associated with insulin resistance.

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INSULIN ACTION—SIGNAL TRANSDUCTION, INSULIN, AND OTHER HORMONES

grin binding) motif independent of IGF binding. However, whether IGFBP1 is causally implicated in insulin sensitivity and glucose regulation and could be exploited therapeutically remains unexplored.

C2C12 myotubes, 3T3 adipocytes, HepG2 hepatocytes, and INS823/13 pancreatic β-cells, were used to investigate the effects of acute treatment of rIGFBP1 and the synthetic hexapeptide GRGDTP (which binds α5β1-integrin) on insulin sensitivity and glucose regulation, through several complimentary in vitro assays. To examine whether the RGD motif of IGFBP1 is mechanistically implicated in glucose regulation, we administered GRGDTP peptide to both C57BL/6 mice with diet-induced obesity and KKAy diabetic Mice. Treatment of C2C12 myotubes with rIGFBP1 or GRGDTP, enhanced insulin stimulated IRS1, AKT phosphorylation and insulin stimulated glucose uptake (P≤0.05, ≤0.02 and P≤0.01 respectively). Co-incubation with glucose and IGFBP1 or GRGDTP, increased glucose stimulated insulin secretion in INS823/13 pancreatic β-cells (P≤0.01 and ≤0.05 respectively). Acute administration of GRGDTP signifi cant-lyimproved glucose clearance and insulin sensitivity in mice with diet-induced obesity and KKAy diabetic mice (P≤0.05). We have shown that the RGD integ-rin-binding domain of IGFBP1 may play an important role in insulin sensitivity and glucose regulation and represents a potential novel therapeutic agent in the fi eld of type 2 diabetes that requires further investigation.

1905-PMiR-26b Modulates Insulin Sensitivity in Adipocytes by Interrupt-ing PTEN/PI3K/AKT PathwayXIRONG GUO, CHENBO JI, CHUNMEI SHI, YAPING ZHAO, GUANGFENG XU, XI-ANWEI CUI, Najing, China

Background: MicroRNAs (miRNAs) have emerged as epigenetic regulators of metabolism and energy homeostasis. Resently, there is a growing body of evidence pointing to miRNAs that play important regulatory roles in insulin sensitivity.

Objective: To explore the mechanism of miR-26b involving in obesity-related insulin sensitivity in adipocytes.

Design: Quantitative real-time PCR were performed to determine miR-26b expression in obese rodent models, obesity subjects and insulin-resistant adipocytes. To analyze the roles of miR-26b overexpression and inhibition on glucose uptake in adipocytes. Western blotting was used to detect the pro-tein level of molecules involving in PI3K pathway. Bioinformatics and Dual Luciferase Assay were identifi ed the target gene of miR-26b. To comprehen-sively assess the regulative roles of miR-26b on PTEN/PI3K/AKT pathway and the relationship between miR-26b and metabolism characteristic of hu-man obese subjects.

Results: We demonstrated that levels of miR-26b are reduced in visceral adipose tissue in obese rodent models, human obesity subjects, and insulin-resistant adipocytes. We also observed that miR-26b promotes insulin-stimu-lated glucose uptake and increases insulin-stimulated GLUT4 translocation to the plasma membrane in human mature adipocytes. We presented evidence that miR-26b modulates insulin-stimulated AKT activation via inhibiting the target gene, PTEN. Moreover, we found that miR-26b have a signifi cant effect on improving insulin sensitivity via PTEN/PI3K/AKT pathway. Furthermore, we confi rmed that the expression level of miR-26b is negatively correlated with increasing BMI and HOMA-IR in human obese subjects.

Conclusion: Our results suggest that decreased miR-26b expression in vis-ceral adipose tissue may be involved in obesity-related insulin resistance by interrupting PTEN/PI3K/AKT pathway.

Supported By: National Key Basic Research Program of China (2013CB530604)

1906-PExposure to Insulin and High Glucose Inhibits Insulin Signaling in Human PreadipocytesJASON EL BILALI, ANNEMARIE GAGNON, ALEXANDER SORISKY, Ottawa, ON, Canada

Adipose tissue expansion (obesity) characterized by adipocyte hypertro-phy is associated with insulin-resistant adipocytes. In contrast, hyperplas-tic growth of adipose tissue, due to the differentiation of preadipocytes, is associated with preservation of adipocyte insulin sensitivity. High glucose in combination with insulin impairs insulin signaling and insulin-stimulated glucose uptake in human adipocytes, but the impact on human preadipo-cytes is not known. We hypothesized that exposure to insulin and high glu-cose would lead to attenuation of insulin signaling in human preadipocytes. Preadipocytes were isolated from the stromal vascular fraction of human subcutaneous adipose tissue obtained from 6 female patients undergoing elective surgery (Research Ethics Board-approved). Mean age was 57±6 years, and mean body mass index (BMI) was 27±4 kg/m2 (±SD). Cells were pre-incubated in 5 mM glucose (normal glucose; NG) or 25 mM glucose (high

glucose; HG) in the presence of 0.6 nM insulin for 48 hr, followed by acute 100 nM insulin treatment for 5 min. Immunoblot analysis revealed that in-sulin-stimulated Akt phosphorylation at serine (ser)-473 increased strongly with NG. Under high glucose + 0.6nM insulin conditions (HG+Ins), insulin-stimulated Akt phosphorylation levels were inhibited by 54±13% (mean ±SE, n=3, p<0.01) vs. NG. With acute insulin stimulation of preadipocytes in HG+Ins, tyrosine phosphorylation of insulin receptor-β subunit and insulin receptor substrate (IRS)-1 was inhibited by 77±8.8% and 66±17% (mean±SE, n=4, p<0.01 for both), respectively, vs. NG. Taken together, our data sug-gest that insulin signaling in human preadipocytes is inhibited by HG+Ins conditions. Since human preadipocytes are vital for healthy adipose tissue remodeling, it is possible that in vivo hyperglycemia and hyperinsulinemia that occurs in obese type 2 diabetic patients may exacerbate adipose tissue dysfunction.


1907-PPotential of Empaglifl ozin, a Specifi c SGLT2 Inhibitor, to Improve Glucose Homeostasis and Slow Down β-Cell Deterioration in Se-vere Preclinical Model of DiabetesERIC MAYOUX, BIRGIT STIERSTORFER, MICHAEL MARK, GERD LUIPPOLD, Bib-erach, Germany

Type 1 and type 2 diabetes are both characterized by progressive β-cell failure, associated with exhaustion of remaining β-cells to overcome, re-spectively, autoimmune assault or insulin resistance. Indeed the additional stress transfer to the remaining functional β-cells triggers a vicious circle leading to further β-cell death and disease progression. In this study we compared effi cacy of treatments, with different mode of action, to control blood glucose, maintain β-cells function, in rats with β-cells assault induced by streptozotozin (STZ).

STZ- rats were treated for 4 weeks with pioglitazone (10mg/kg p.o.), met-formin (300mg/kg p.o.), glimepiride (10mg/kg p.o.), empagliglozin (10mg/kg p.o.) or liraglutide (200mg/kg s.c.).

Prandial and fasting glucose, HbA1c, and C-peptide release were deter-mined in the plasma at end of treatment, while insulin content of pancreas was measured after sacrifi ce. In addition, oral glucose tolerance test (oGTT) was performed after 2 weeks of treatment.

Among those treatments, only empaglifl ozin was able to improve sig-nifi cantly the blood glucose parameters. Empaglifl ozin reduced also signifi -cantly the AUC glucose compared to the other groups during oGTT. While C-peptide secretion was dramatically decrease in all groups in comparison to none-diabetic animal, C-peptide secretion was signifi cantly more elevated (> 280%) with empaglifl ozin in comparison to other groups. At sacrifi ce, insulin content in pancreas was signifi cantly higher in empaglifl ozin group (> 450%) in comparison to other treatments.

These results demonstrate that the SGLT2 inhibitor, empaglifl ozin, with an insulin and β-cell independent mechanism exhibit signifi cant anti-diabetic properties and preserve β-cell function in severe model of β-cells failure. Therefore, SGLT2 inhibitors could have the potential to slow down the dis-ease progression in type 2 as well type 1 diabetic patients.


1908-PHepatic SIRT1 Stimulates Fibroblast Growth Factor 21 to Improve Hepatic Insulin Sensitivity and Control Glucose HomeostasisQI GONG, FEIFEI ZHANG, ZHIMIN HU, XUQING CHEN, YAMEI HAN, HAOYANG JIANG, JING GAO, YU LI, Shanghai, China, State College, PA

The hepatokine FGF21 has emerged as a novel metabolic regulator that has potential to treat obesity and diabetes. We recently demonstrated that SIRT1 is a key upstream regulator of FGF21 to regulate hepatic fatty acid metabolism. However, whether SIRT1 regulate hepatic insulin sensitivity and control glucose homeostasis through FGF21 is not known. We showed that hepatocyte-specifi c SIRT1 knockout mice (SIRT1 LKO) fed with high-fat high-sucrose diet exhibited a reduction of hepatic insulin sensitivity as mea-sured by decreased phosphorylation of Akt, GSK3β and GS, the key regula-tor of glycogen synthesis. The phenotype in SIRT1 LKO mice is associated with reduced hepatic and circulating levels of FGF21, and increased mTORC1 activity. Conversely, genetic or pharmacologically manipulation of SIRT1 by adenovirus-mediated overexpression of SIRT1 or by SIRT1 agonist resvera-trol respectively protected against insulin resistance in diet-induced obese mice. Moreover, administration with FGF21 inhibited phosphorylation of S6K

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and 4E-BP1, the downstream targets of mTORC1, and resulted in improved hepatic insulin sensitivity in diet-induced obese mice and in insulin-stimulat-ed hepatocytes. Importantly, SIRT1 defi ciency resulted in increased mTORC1 activity and decreased insulin signaling, which was rescued by overexpres-sion of FGF21 or treatment with mTORC1 inhibitor rapamycin. Consistently, treatment with FGF21 or rapamycin is suffi cient to alleviate aberrant glyco-gen storage in insulin-stimulated SIRT1-/- hepatocytes, suggesting improved hepatic insulin sensitivity. These results indicate that hepatic SIRT1 induces FGF21 and inhibits mTORC1 activity to improve hepatic insulin sensitivity. Our fi nding identifi es a novel mechanism of nutrient sensor SIRT1 on regulat-ing hepatic insulin signaling, and provides a mechanic insight into a role of SIRT1-FGF21 axis in regulating hepatic insulin sensitivity by inhibiting the mTORC1 complex.

Supported By: National Natural Science Foundation of China (81270930, 31471129)

1909-PHigh Fat Diet Impairs Receptor-mediated Insulin Uptake by the BrainSARAH M. GRAY, RICK I. MEIJER, EUGENE J. BARRETT, Charlottesville, VA

Insulin acts on the brain to regulate satiety, body weight, glucose and lipid metabolism, and may have a role in treating neurodegenerative disorders. Despite its importance, little is known of how insulin crosses the blood-brain barrier (BBB) to reach brain interstitial fl uid and act on target neurons. We hypothesized that the BBB endothelial cell (EC) has an insulin receptor-mediated vesicular transport system and that high fat diet (HFD)-induced insulin resistance could interfere with this process. To test whether insulin reaches brain tissue in vivo via the CSF circulation or by crossing the BBB, rats received 0.7 pmol 125I-insulin (125I-ins). Afterward, the vasculature was fl ushed for 5 min and brain tissue collected. 125I-ins appeared in cerebellum, cortex, and basal nuclei before reaching CSF. An insulin receptor selective antagonist (S-961) blocked brain 125I-ins clearance (p<0.01) as did 4 weeks of HFD (p<0.05, vs. chow fed rats). We also isolated brain ECs from HFD and chow fed rats and measured 125I-ins uptake (200 pM). Again HFD decreased EC 125I-ins uptake (p<0.01). To examine the pathway of insulin uptake by rat brain microvascular ECs (RBMVECs, Cell Applications), we measured the time course of insulin uptake and sensitivity to agents interfering with insu-lin signaling or lipid raft formation. 125I-ins uptake by RBMVECs reached a plateau after 15 min and was completely blocked by 10 nM S-961 (p<0.001), but not by an IGF-I receptor neutralizing antibody (Ab-3). Pharmacological inhibitors of either insulin signaling (wortmannin, genistein, and PD89059) or Src kinase (PP1) decreased 125I-ins uptake. Depleting cholesterol from lipid rafts with 10 mM methyl-β-cyclodextran or 5 ug/ml fi lipin signifi cantly decreased EC insulin uptake (p<0.05, each). We conclude that: a) insulin en-ters brain interstitial fl uid principally by crossing the BBB; b) HFD feeding compromises brain insulin transport; and c) insulin’s transport is mediated by its receptor, insulin signaling, and lipid rafts.

Supported By: American Heart Association (14PRE20100048); National Institutes of Health (T32HL007284, DK-057878)

1910-POvernight High-Fat Feeding Provokes Insulin Resistance and In-fl ammation in Vascular Endothelial CellsHONG WANG, KEVIN W. AYLOR, EUGENE J. BARRETT, Charlottesville, VA

Effective endothelial cell (EC) insulin signaling is required for insulin’s trans-endothelial transport (TET) to muscle and adipose tissue. High-fat diet (HFD) induces infl ammation and impairs insulin signaling in liver, muscle and adipose tissues within periods of 4-16 weeks. Intravenous lipid infusion for 2-6 hrs or a single high fat meal (within 6h) can provoke whole-body insulin resistance in lean healthy human subjects. Here we examined insulin signal-ing and transport and markers of cell infl ammation in freshly isolated aortic ECs of rats given one night of HFD (60% fat) vs. chow feeding. Compared to chow-fed rats (n=7 for each group), overnight HFD feeding increased serum insulin and triglyceride by 61% and 233%, respectively (p<0.05 for each). Serum glucose concentrations were not different. The HFD feeding also reduced both insulin-stimulated EC Akt phosphorylation at Ser473 and fl uor-oisothiocyanate (FITC) labeled-insulin uptake (the fi rst step of insulin TET) by 31% and 42%, respectively (p<0.0001 for each). Overnight HFD also induced EC infl ammatory responses as indicated by a decline in IκBα and greater nuclear NFκB localization (p<0.0001) and a ~7-fold increased expression of mRNA for inducible nitric oxide synthase (iNOS) compared to chow-fed con-trol. These HFD-induced abnormalities in EC insulin signaling and uptake and infl ammatory responses were effectively reversed by ex vivo treatment with nitric oxide donor, sodium nitroprusside (0.3 µM for 30 min). We conclude that the vascular EC is an early responder (within 20h) to a HFD insult as

manifested by rapid activation of IKKβ-NFκB pathway, increased oxidative stress, and impaired insulin signaling and transport. These data suggest that ECs may serve as a site for early effective therapeutic interventions target-ing nutrient-induced systemic insulin resistance.

Supported By: American Diabetes Association (1-11-BS-06 to E.J.B.); National Institutes of Health (DK057878, DK073059)

1911-PBile Acids Impair Hepatic Insulin Action In VivoTRAVIS CYPHERT, CHARLES FLYNN, OWEN P. MCGUINNESS, Nashville, TN

Alterations in bile acids and their signaling pathways have been linked to metabolic disease. In the liver bile acids can trigger the activation of the Akt pathway, which could enhance insulin action. However, a direct connection between insulin action and bile acid signaling in vivo has not been identifi ed. Our lab wanted to determine if an increase in plasma levels of bile acids could improve insulin action. We have identifi ed that different species of bile acids have different effects on insulin action. We examined the impact of physiologic (5-10uM) increases in taurocholic acid (TCA, a conjugated bile acid) and deoxycholic acid (DCA, a secondary bile acid) on insulin ac-tion in chronically catheterized conscious mice using a hyperinsulinemic (2 mU·kg-1·min-1)-euglycemic clamp. To our surprise, DCA (0.5 µmol·kg-1·min-1) decreased the glucose infusion rate (GIR) (control 22±3 vs. DCA 11±1 mg·kg-1·min-1) and this was due to an impaired suppression of hepatic glucose production (control 3±1 vs. DCA 9±1mg·kg-1·min-1). The infusion of DCA in-creased the hepatic gene expression of PEPCK (5.9±1.8 fold higher RNA ex-pression than control) and G6Pase (2.4±0.4 fold higher RNA expression than control). The increase in the expression of PEPCK and G6Pase RNA suggests that DCA impairs insulin’s ability to suppress gluconeogenesis. In contrast, TCA (0.5 µmol·kg-1·min-1) did not decrease GIR. TCA impaired insulin sup-pression of hepatic glucose production (control 3±1mg·kg-1·min-1 vs. TCA infusion 7±3mg·kg-1·min-1) but enhanced insulin action by increasing tissue glucose uptake into the soleus muscle and heart. Thus, bile acids exert a species-dependent effect on insulin action as TCA improved insulin action in some tissues while both DCA and TCA caused insulin resistance in the liver.

Supported By: National Institutes of Health (DK043748, DK078188)

1912-PSubstrate Specifi city of Insulin Degrading Enzyme and Its Role in the Regulation of Exogenous Insulin Clearance and Glycemic ControlHANNAH KEMPTON, MARGARET WU, GE DAI, JAMES MU, Kenilworth, NJ

Endogenous insulin, or insulin analog in the case of insulin-dependent patients, plays a central role in the regulation of glucose homeostasis. Insu-lin catabolism has been postulated, for over half a century, to be mediated primarily by insulin-degrading enzyme (IDE) without defi nitive in vivo confi r-mation. Recent studies employing genetic and pharmacological inhibition of IDE demonstrated its role in regulating endogenous insulin levels. However, additional metabolism relevant hormones, such as glucagon and amylin, were also shown to be IDE substrates. To better understand potential im-plications of IDE inhibition on insulin and glucose regulation, as well as IDE specifi city towards its commonly proposed substrates, in vitro studies using a FRET-based IDE activity assay were performed. Although IDE can cleave many of the peptides tested, including amylin, glucagon and beta-amyloid, it revealed a greater than 10-fold preference for insulin compared to other sub-strates, suggesting insulin is likely the primary IDE substrate in vivo espe-cially when considering their relevant physiological levels. Interestingly, IDE also showed differential activity towards modifi ed insulin analogs in vitro. For example, glargine and detemir have over 10-fold lower IC50s compared to native insulin. To study the effect of IDE on exogenous insulin clearance and its impact on insulin analogs’ in vivo profi le, their pharmacokinetic and pharmacodynamic profi les were compared in mice in the presence of an IDE inhibitor, in which studies enhanced glucose lowering effi cacy and delayed clearance of the analogs were clearly demonstrated. These results support IDE as a potential anti-diabetes target and suggest an IDE inhibitor could also be useful to enhance the therapeutic actions of insulin analogs.

1913-PFibulin5 Defi ciency Improved Insulin Resistance and Hepatic Ste-atosis Induced by High-Fat FeedingTOMOKO OKUYAMA, JUN SHIRAKAWA, HIROMI YANAGISAWA, YASUO TERAUCHI, Yokohama, Japan, Dallas, TX

Fibulin5 (Fbln5), a matricellular secretory protein, is essential for the de-velopment of elastic fi bers. We identifi ed that the expression of Fbln5 in pancreatic islets was signifi cantly increased in response to high glucose. To elucidate the roles of Fbln5 in glucose metabolism, we investigated Fbln5

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knockout (Fbln5KO) mice fed a standard chow (SC) or a high fat (HF) diet. The expression level of Fbln5 was increased in the liver of SC-fed wild-type (WT) mice after 24 hours of fasting, but such change in Fbln5 expression disap-peared under a HF diet. On either diet, Fbln5KO mice showed smaller body weight gain than WT mice. Although Fbln5KO mice had no signifi cant chang-es in glucose tolerance, serum insulin levels were signifi cantly decreased in HF diet-fed Fbln5KO mice compared with HF diet-fed WT mice. Insulin sensitivity was signifi cantly improved in HF diet-fed Fbln5KO mice compared with HF diet-fed WT mice. Pyruvate tolerance test showed decreased blood glucose levels in HF-fed Fbln5KO mice, suggesting that the loss of Fbln5 caused the reduction in hepatic gluconeogenesis in diet-induced obese mice. Fatty liver and hepatic triglyceride accumulation were ameliorated by the absence of Fbln5 on a HF diet. The phosphorylation level of Akt in the liver was enhanced in Fbln5KO mice after the injection of insulin. Unexpect-edly, however, the hepatic gene expression levels of PEPCK, G6Pase, PGC-1α were increased in Fbln5KO mice under fasting conditions. HF-fed Fbln5KO mice exhibited increased expression of PPARα in the liver under fasting con-ditions. Furthermore, the deletion of Fbln5 led to increment of hepatic FGF21 expressions during fasting in HF-fed mice. Although the phosphorylation ra-tio of JNK and eIF2α relative to total proteins in the liver were unchanged, the expression of total JNK was increased under fasting conditions in HF-fed Fbln5KO mice. Taken together, Fbln5 defi ciency improved insulin resistance and hepatic steatosis, possibly through the induction of FGF21.

1914-PTM-25659, a TAZ Modulator, Inhibits Palmitate-induced Insulin Re-sistance by Inducing FGF21KWAN-WOO LEE, TAE HO KIM, JA YOUNG JEON, SUNG-E CHOI, JONG GAB JUNG, EUN SUK HA, SO YOUNG OCK, SANG-A LEE, YUP KANG, MYUNG AE BAE, JIN HEE AHN, HANA JEONG, EUN SOOK HWANG, SEUNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, Suwon, Republic of Korea, Seoul, Republic of Korea

Transcriptional co-activator with PDZ binding motif (TAZ) plays a key role in regulating myogenic differentiation and muscle regeneration. In this study, we investigated the effects of TAZ on palmitate-induced insulin resistance.

The effects of TAZ on palmitate-induced insulin resistance were investi-gated using TM-25659 to modulate TAZ, C2 myotubes, and C57BL/6J mice. And, we investigated the relationship between the expressions of FGF21 and TM-25659 using immunoblotting and quantitative real-time PCR.

Palmitate reduced the phosphorylation of Akt, thereby impairing insulin signaling and eventually leading to reduced glucose uptake in C2 myotubes. However, treatment with TM-25659, which stimulates TAZ, blocked palmi-tate-inhibited glucose uptake and insulin signaling signifi cantly. To elucidate the mechanism of action of TM-25659, we measured the levels of the pro-infl ammatory cytokines TNF-α, IL-1-β, IL-6, and MCP-1, as well as FGF21. Different doses of TM-25659 inhibited the production of pro-infl ammatory cytokines and induced FGF21 mRNA and protein levels. We also measured the secretion of FGF21 from cells into the cytosol after treatment with TM-25659. To determine whether TM-25659 exerted the same effects in vivo, C57BL/6J mice were injected intraperitoneally with either vehicle or TM-25659 every other day for 1 week. TM-25659 increased FGF21 RNA and pro-tein levels signifi cantly in the skeletal muscle. Finally skeletal muscle cells were transfected with FGF21 siRNA, and FGF21 levels and the effects of TM-25659 on palmitate-induced insulin resistance were measured. TM-25659 could not rescue cells from palmitate-induced insulin resistance when FGF21 had been knocked down. Therefore, the TAZ activator TM25659 protected palmitate-induced insulin resistance via FGF21.

The present study suggests that TM-25659 might have therapeutic po-tential as a treatment for insulin resistance and diabetes by inducing FGF21 expression.

1915-PInsulin Glargine and (A21Gly, DiD-Arg) Insulin Do Not Promote Breast Cancer Growth in a Mouse ModelEMILY J. GALLAGHER, NORBERT TENNAGELS, ULRICH WERNER, DEREK LE-ROITH, New York, NY, Frankfurt, Germany

Since it was found that the insulin analog AspB10 led to mammary tumors in rats, there have been concerns over the potential mitogenicity of insulin analogs. As insulin analogs and delivery systems continue to be developed, there is a need to understand the mitogenic mechanisms of certain analogs. Previous studies have reported that insulin analogs that activate the insulin-like growth factor receptor (IGF-1R) may be more mitogenic, while others have suggested that prolonged activation of the insulin receptor (IR) may be responsible for the tumor promoting effects. Insulin glargine has greater affi nity for the IGF-1R than human insulin (HI) in vitro, but in vivo it is metabo-

lized to an M1 metabolite that has similar affi nity to the IR and IGF-1R as HI. (A21Gly, DiD-Arg) Insulin is a modifi ed form of insulin glargine that is resis-tant to in vivo metabolism and has higher affi nity for the IGF-1R than HI.

The aim of our study was to determine if insulin glargine and the non-metabolizable insulin (NMI) glargine analog (A21Gly, DiD-Arg) promote the growth and metastasis of breast cancer in a mouse model.

We used the MKR mouse for these studies. At 8 weeks of age, the 4th mammary fat pad of MKR mice was injected with murine breast cancer cells with different oncogenes (Met-1 cells, derived from polyoma virus middle T antigen breast cancer, or Mvt-1 cells, derived from a c-myc/vegf overex-pressing model). Tumor bearing mice were divided into 3 groups (n=8 per group) and injected with insulin glargine (12.5 IU/kg twice daily), NMI (12.5 IU/kg twice daily) or an equal volume of saline for 3 weeks. Tumors were measured for 3 weeks and at the end of the study tumors were weighed, and pulmonary macrometastases were quantifi ed. No difference in tumor size, weight or the number of metastases was found between the insulin glargine, NMI and control groups.

These data demonstrate the lack of mitogenic effect of insulin glargine and of a non-metabolizable form of glargine, in a mouse model of breast cancer.

Supported By: Sanofi

1916-PSerum Dipeptidyl Peptidase-4 Activity Is Associated with Meta-bolic Syndrome Prevalence in Type 1 Diabetic PatientsLEA DUVNJAK, KRISTINA BLASLOV, TOMISLAV BULUM, Zagreb, Croatia

The patophysiology of metabolic syndrome (MS) comprises a complex adipokine mediated cross-talk between visceral adipocytes and peripheral tissue. Dipeptidyl peptidase-4 (DPP4) was recently proposed as a novel adi-pokine linked to MS. We aimed to assess the relationship between fasting serum DPP4 activity, MS and its components in type 1 diabetic (T1DM) pa-tients.

Forty-four T1DM patients aged >18 and <65 years were enrolled. MS was defi ned according to International Diabetes Federation revised defi nition from 2009. Insulin sensitivity was calculated using the equation derived from euglycemic-hyperinsulinemic clamp studies-estimated glucose dispos-al rate (eGDR). DPP4 serum activity was determined spectrophotometrically as a rate of cleavage of 7-Amino-4-Methyl Coumarin (AMC) from H-Gly-Pro-AMC. Patients were divided into three groups according to DPP4 activity tertiles (<25.40; 32.4 and ≥36.54 U/L). The group from the third tertile group showed signifi cantly higher MS prevalence (92 vs. 53 vs. 0 %, p<0.001) and lower eGDR (5.79 vs. 6.93 vs. 8.48 mgkg-1min-1, p=0.004) compared to fi rst and second one. Systolic (136 vs. 131 vs. 119 mmHg, p=0.009), diastolic (83 vs. 82.5 vs. 77 mmHg, p=0.047) blood pressure and waist circumference (91 vs. 90 vs. 86 cm, p=0.037) were also higher in the third tertile group in comparison to other two groups. After adjustment for age, gender, disease duration, use of antihypertensives and statins, the linear regression analy-sis showed that eGDR decreases for 0.155 mgkg-1min-1 by each increase in serum DPP4 activity of 1 U/L (p<0.007) while in the multivariate logistic regression analysis DPP4 activity was positively associated with MS pres-ence (OR=1.785, p=0.005).

We showed that higher serum DPP4 activity is associated with lower in-sulin sensitivity and higher MS prevalence in T1DM patients. The potential role of DPP-4 acitivity in the complex pathophysiology of insulin resistance in T1DM needs to be established in future follow-up studies.

1917-PDelivery of Insulin to the Brain by Intranasal Administration In-creases Postprandial GLP-1 Secretion in HumansYOSHIKAZU HIRASAWA, HISAYO YOKOYAMA, NOOSHIN NAGHAVI, YOSHIHIRO YAMASHINA, RYOSUKE TAKEDA, AKEMI OTA, DAIKI IMAI, KAZUNOBU OKA-ZAKI, TOMOAKI MORIOKA, MASANORI EMOTO, MASAAKI INABA, TOSHIAKI MIYAGAWA, Osaka, Japan

Insulin in central nervous system regulates energy homeostasis in vivo. In humans, intranasal insulin administration increases insulin level in cere-brospinal fl uid without peripheral effect on glucose metabolism, which re-sults in reduced food intake and body weight, and increased postprandial thermogenesis. It remains to be unclear whether delivery of insulin to the brain affects incretin secretion through gut-brain axis or not. The purpose of the present study was to examine the effect of intranasal insulin on GLP-1 secretion, gastric emptying, and appetite in 9 normal-weight healthy young volunteers (age 21.9±3.4 (SD) years, 7 males and 2 females, BMI 19.8±1.6 kg/m2) in a randomized singe-blinded crossover design. On two separate days, at least one week apart, sixteen puffs of regular insulin (INS) 10U or normal saline as placebo (PLB) 0.1ml with total dose of 1.6mL (insulin 160 IU) in a

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random order was intranasally administered after an overnight fast and af-terwards, a fi xed meal test including 1.5 g paracetamol for gastric emptying test was performed in each participant. Blood glucose, insulin, active form of GLP-1 and paracetamol levels were measured at every 15-30 min during 180 min meal test and postprandial satiety during meal test by visual analog scale at every 30 min. There was no signifi cant differences in postprandial blood glucose and insulin levels during meal test between INS and PLB trials. Postprandial GLP-1 levels in INS trial during meal test showed a tendency to be higher than in PLB trial. The total increase of GLP-1 level in INS trial during meal test evaluated by area under the curve (AUC) was signifi cantly greater than in PLB trial (AUC, 862±372 vs. 604±389, p=0.046). Gastric emptying and postprandial satiety were not statistically different between the trials. In conclusion, delivery of insulin to the brain by intranasal administration increases postprandial GLP-1 secretion without modulation of gastric emp-tying and postprandial satiety in humans.

1918-PAn Adenoviral P53 Recombinant Improves Glucose Control in Insu-lin Resistant Diabetic Mouse ModelXUEMEI ZHANG, GARY XIAO, Dalian, China

The tumor suppressor transcriptional factor, p53, is known to regulate metabolic functions and control cell cycle progression and apoptosis. P53 has a wide range of effects on cellular glucose metabolism including glyco-lysis, pentose cycle, gluconeogenesis and glycogenolysis. In order to assess the impact of these p53 effects on glucose homeostasis in vivo, a recombi-nant adenoviral p53 vector (rAd-P53) was administered to insulin resistant diabetic mice. Changes in fasting blood glucose, serum triglycerides and C-peptide concentrations in treated and untreated mice were measured. Treatment with rAd-P53 improved glucose control in a dose and time de-pendent manner, and signifi cantly lowered fasting blood glucose and serum triglycerides as compared to those of viral vector control. Improved glucose control was associated with up-regulation of genes of the glycogenesis pathways (glucokinase (GK), glycogen synthase (Gs), and glucose transport-er 2 (Glut2), and down-regulation of genes of the gluconeogenesis pathways (fructose-1,6-bisphosphatase (Fbp1), glucose-6-phosphatase (G6pc) and gly-cogen phosphorylase (Gp) in the liver. Over-expression of Glut2, GK, PPAR-γ and insulin receptor precursor were also observed in pancreas tissues of the treated animals. Our data suggest that p53 improved glucose control by changing insulin sensitivity in this insulin resistant STZ/high-fat-diet diabe-tes mouse model.

Supported By: National Technology R&D Program for the 12th Five-Year Plan of China (2012BAK25B00)

1919-PErythropoietin via PPARγ-dependent PI3K/AKT Pathway Improves Hepatic Insulin ResistanceZHIJUAN GE, PENGZI ZHANG, TING HONG, SUNYINYAN TANG, RAN MENG, YAN BI, DALONG ZHU, Nanjing, China

Erythropoietin (EPO) has been recently shown to have benefi cial effects on glucose metabolism and insulin resistance (IR) in murine adipocytes and skeletal muscles. However, the precise mechanisms by which EPO improves hepatic IR remain unclear. Here, we reported that EPO administration in-creased gene and protein levels of peroxisome proliferator-activated recep-tor γ (PPARγ) and promoted the activation of phosphatidylinositol 3-kinase (PI3K)/AKT pathway, which were blocked by EPO receptor (EPOR) knock-down in palmitic acid-treated HepG2 cells. Importantly, PPARγ antagonist and PPARγ siRNA largely attenuated the ability of EPO to increase cellular glycogen levels and to restore AKT phosphorylation. Moreover, silencing of hepatic PPARγ in high-fat fed C57BL/6 mice and ob/ob mice impaired EPO-mediated increase in glucose tolerance and activation of hepatic PI3K/AKT pathway. Simultaneously, we found that EPO elevated SIRT1 expression as well as its activation through increasing the NAD+/NADH ratio, and SIRT1 depletion attenuated both expression and deacetylation of PPARγ and PPARγ-dependent activation of PI3K/AKT upon EPO intervention in HepG2 cells. In summary, these fi ndings suggest for the fi rst time that PPARγ func-tions as a novel downstream mediator in EPO/EPOR signaling and targeting SIRT1/PPARγ/PI3K/AKT pathway by EPO may have potential therapeutic im-plications for IR and type 2 diabetes.

Supported By: National Natural Science Foundation of China (81070636, 81370947, 81270906)

1920-PRegulation of Mitochondrial Localization and Activity by STARS, the Actin Cytoskeleton, and Small GTPase-Dependent SignalsAKIHISA MINO, MARY-ELIZABETH PATTI, Boston, MA

Expression of STARS (striated muscle activator of Rho signaling) and cy-toskeleton-related gene targets of the transcription factor SRF is increased in skeletal muscle from patients with type 2 diabetes, in parallel with insulin resistance. Experimental modulation of STARS regulates both glucose uptake and mitochondrial oxygen consumption. Since STARS regulates Rho signal-ing, we hypothesized that STARS effects on metabolism might be linked to cytoskeleton-dependent regulation of mitochondrial localization and activity. We fi rst assessed the effect of insulin on mitochondrial localization (Hsp60) and membrane potential (MitoTracker Red CMXRos) in C2C12 myocytes using confocal microscopy. After serum starvation, Hsp60 is predominantly cytoso-lic but acute insulin stimulation results in localization to the perinuclear area. Similarly, insulin increases MitoRed staining. We observed similar insulin-dependent effects in primary mouse myoblasts/tubes. These events were inhibited by inhibitors of actin polymerization (latrunculin B), microtubules (no-codazole) and the dynein motor protein (HPI-4). We also observed that insulin induces colocalization of mitochondria and sarcolipin, a modulator of calcium infl ux into ER for subsequent mitochondrial activation.

We next examined the impact of STARS on these phenotypes using wild type and STARS-KO primary myoblasts. In the basal state, both perinucle-ar mitochondrial localization (score: KO 3.0 vs. WT 1.8, n=21/19 cells) and membrane potential (score: KO 3.3 vs. WT 1.3, n=20/9 cells) were increased in myoblasts derived from STARS-KO mice. Similar effects were observed in myotubes and were reduced by Rho kinase inhibition (Y27632, 10 uM, 30min). These data suggest that STARS impact on metabolism may be medi-ated by RhoA-regulated cytoskeletal remodeling, which in turn modulates mitochondrial traffi cking to induce mitochondrial activation.

Supported By: American Diabetes Association (7-12-BS-145 to M-E.P.); Sanofi U.S.; National Institutes of Health (R41DK096772-01A1)

1921-PDeciphering the Effects of Insulin in the Mouse Gastrointestinal TractSTINA R. JENSEN, SARAH E. WHEELER, HENNING HVID, BO F. HANSEN, ERICA NISHIMURA, GRITH S. OLSEN, PATRICIA L. BRUBAKER, Toronto, ON, Canada, Måløv, Denmark

The metabolic functions of insulin have been well-described in liver, adipose and muscle tissue. However, despite insulin receptors being highly expressed throughout the gastrointestinal tract, little is known about the biological effect of insulin in this tissue. Here, we studied the effect of insu-lin at a supra-pharmacological dose on intestinal growth (due to the known mitogenic effects of insulin), barrier integrity and oral glucose tolerance in mice. For 12 days, C57BL/6J mice were subcutaneously administered saline or 300 nmol/kg human insulin daily; the absence of severe hypoglycemia was confi rmed at 1 and 5 hrs post-injection. Long-acting human Gly2-gluca-gon-like-peptide-2 (GLP2) was included as a positive control for small intes-tinal growth and barrier integrity. GLP2-treated mice had increased jejunal length and weight, and enlarged villi and crypts in jejunum; they also showed enhanced barrier integrity in the small intestine, reaching the level of the

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tight barrier in the colon; no changes in colon weight or length were de-tected. Mice treated with insulin did not show alteration in intestinal weight or length compared to vehicle. While villus height and crypt depth in jejunum showed no difference compared to vehicle, crypt depth in the large intestine was signifi cantly reduced in insulin-treated mice (p<0.05). Using chamber experiments demonstrated no changes in the resistance of jejunum and co-lon in response to insulin-treatment. In addition, 24 hrs before the last treat-ment, mice did not display altered glucose tolerance compared to vehicle. Finally, acute administration of insulin (t=-20 min) increased phosphorylated Akt in the colon compared to vehicle (p<0.0001), but not in jejunum. Together these fi ndings suggest insulin-treatment at a supra-pharmacological dose for 12 days does not cause enhanced growth in small and large intestine as opposed to GLP2, which is mitogenic in jejunum. Instead, our data suggest that insulin has biological effects specifi cally on the colon.

Supported By: Novo Nordisk

1922-PA Highly Sensitive and Specifi c Chemiluminescent ELISA to Mea-sure Active GLP-1 in Human and Rodent ModelsJULIE DONALDSON, PAM CRAVEN, NIRAJA PATEL, COLLIN SHAW, MARTIN BLANKFARD, Salem, NH

GLP-1 has been well characterized, through extensive research, as an in-cretin hormone that regulates both insulin and glucagon secretion. It is a highly sought after therapeutic target because of its benefi cial effects on glucose homeostasis, particularly through its regulation of beta cell mass, function, and viability. Currently available commercial ELISAs require large sample volumes or specialized instrumentation to achieve the desired sensi-tivity and to detect fasting levels of circulating GLP-1. Moreover, the speci-fi city and accuracy of these assays is variable, in some cases, leading to question the accuracy even between different lots of the same kit.

Keeping the aforementioned in mind, ALPCO has developed the STELLUX™ GLP-1 Active (7-36 amide) Chemiluminescence ELISA for the quantitative de-termination of GLP-1 (7-36 amide) in serum and plasma samples collected with DPP-IV inhibitor. Using a 25 µL sample size, this novel assay demon-strates excellent sensitivity of less than 1 pM (3.3 pg/mL), while specifi cally measuring GLP-1 (7-36 amide) without any cross reactivity to GLP-1 (1-36), (1-37), (7-37), or (9-36). The specifi city of detecting GLP-1 (7-36 amide) is im-portant, as it is the major circulating biologically active fragment. The con-servation of the GLP-1 sequence across species also makes a small sample volume size desirable for evaluating rodent models over multiple time points. The assay can discern fasted and fed levels in both rats and mice. Most importantly, lack of matrix interference evaluated via dilutional linearity and spike and recovery studies ensures that the assay is accurate and consistent across runs.

1923-PProgressive Alterations in Hepatic Insulin Resistance in the Human Obesity-Diabetes SpectrumMINI P. SAJAN, ROBERT IVEY, MACKENZIE LEE, JOSHUA SAJAN, ROBERT FA-RESE, Tampa, FL

How insulin resistance develops is obscure. In mouse obesity (O), there are initial increases in hepatic ceramide and activation of atypical PKC (aPKC) which selectively impairs Akt-dependent FoxO1 phosphorylation on the WD40/ProF scaffold, thus increasing gluconeogenesis, serum insu-lin, liver Akt and aPKC activity, and lipogenesis. In hepatocytes of humans with type 2 diabetes mellitus (T2DM), resistance is more advanced owing to impaired activation of IRS-1/PI3K and Akt, but aPKC activation remains elevated as IRS-2/PI3K activation is conserved and expression of aPKC-iota is increased by an insulin/aPKC-dependent mechanism. Importantly, inhibi-tion of hepatic aPKC improves these abnormalities in intact mice and human hepatocytes. Here, we examined livers of non-diabetic (ND) humans grouped (1-5) as per BMI (<25; 25-30; 31-35; 36-40; 41-45) along with a 6th T2DM group. In both T2DM and ND-O groups, there were increases in ceramide, aPKC activity, aPKC associated with WD40/ProF, and decreased Akt asso-ciation with WD40/ProF. Most notably in ND-O groups 2-5, there were (a) progressive decreases in IRS-1 levels, Akt activity and phosphorylation of Akt substrates, FoxO1 and GSK3β but not mTOR; and (b) progressive increas-es in; mRNA and protein levels of gluconeogenic enzymes (PEPCK, G6Pase), lipogenic enzymes (SREBP1c, FAS, ACC), and aPKC-iota but not aPKC-zeta. Further, in intact hepatocytes of T2DM humans, aPKC inhibitors reversed or diminished all abnormalities.

Conclusions: liver insulin resistance in humans is present throughout the O/T2DM spectrum, correlates with BMI, and includes progressive increases in: (a) gluconeogenic enzymes, refl ecting impaired FoxO1 phosphorylation

owing to elevated aPKC on WD40/ProF and impaired IRS-1-dependent Akt activity; (b) lipogenic enzymes refl ecting increased aPKC and mTOR; and (c) aPKC activity, refl ecting increased expression/levels of PKC-iota, and activa-tion of total aPKC by hyperinsulinemia and increased ceramide.

Supported By: U.S. Department of Veterans Affairs

1924-PInsulin-stimulated Tyrosine Phosphorylation of Phospholipase C Is Impaired in Type 2 Diabetic Skeletal MuscleDANJUN MA, XIANGMIN ZHANG, YUE QI, ABDULLAH MALLISHO, RODNEY BERRY, MICHAEL CARUSO, DIVYASRI DAMACHARLA, JEFFREY HOROWITZ, BERHANE SEYOUM, ZHENGPING YI, Detroit, MI, Ann Arbor, MI

The prevalence of type 2 diabetes (T2D) has reached 340 million people worldwide while its pathogenesis is incompletely understood. Phospho-lipase C, Gamma 1 (PLCG1) hydrolyzes phospholipids into diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), and is activated by multiple tyrosine kinases. Additionally, PLCG1 is a substrate of the protein-tyrosine phosphatase PTP1B, a known negative regulator of insulin signaling that can dephosphorylate the insulin receptor. Tyrosine phosphorylation of PLCG1 (e.g., pTyr771) activates PLCG1 and plays a key role in various downstream signaling pathways. Nonetheless, role of tyrosine phosphorylation of PLCG1 in skeletal muscle in T2D patients is unreported. In the present work, we have recruited 5 lean controls and 5 T2D participants. Each participants un-derwent a 2-hour hyperinsulinemic-euglycemic clamp to assess insulin sen-sitivity, and two skeletal muscle biopsies were obtained, one before (basal) and the other at the end of the clamp (insulin-stimulated). No signifi cant dif-ferences were observed for gender and age between lean controls and T2D subjects. In T2D subjects, body mass index, percent body fat, fasting plasma insulin and glucose were signifi cantly higher, whereas the M-values, a mea-sure of insulin sensitivity, were signifi cantly lower compared to lean con-trols. Western blot analysis of pTyr771 of PLCG1 in human skeletal muscle biopsies (with or without in vivo insulin infusion) indicated that the pTyr771 level increased signifi cantly in response to insulin in lean controls (6.5 fold increase insulin vs. basal, P<0.05), but not in T2D patients. In addition, the protein abundance of PLCG1 didn’t change between lean and diabetic par-ticipants. These results implied that the ability of insulin to stimulate pTyr771 of PLCG1 in human skeletal muscle is impaired in T2D patients compared to lean controls. This abnormality may contribute to the development of skel-etal muscle insulin resistance and T2D.

Supported By: American Diabetes Association (7-13-TS-35 to Z.Y.)

1925-PThe Insulin Receptor Mediates Insulin’s Clearance by Liver, Muscle, and KidneyRICK I. MEIJER, EUGENE J. BARRETT, Charlottesville, VA

Insulin is taken up by peripheral tissues from the plasma. Whether the in-sulin receptor mediates tissue-specifi c insulin clearance in vivo, or whether this is dependent on passive diffusion over the endothelial layer has not been reported. Using the synthetic peptide insulin receptor inhibitor S961, we measured the initial (fi rst 5 min) rate of clearance of intravenously inject-ed ([125I]TyrA14)-insulin by skeletal muscle, liver and kidney in healthy rats in the absence and presence of insulin receptor blockade. We further investi-gated whether 4 weeks of high fat diet (HFD) affects the initial plasma clear-ance of ([125I]TyrA14) insulin. Pre-treatment with S961 for 60 min prior to administration of labeled insulin raised plasma ([125I]TyrA14) insulin concen-tration by ~5-fold (p < 0.001) demonstrating receptor dependency for initial plasma insulin clearance. Uptake by skeletal muscle (p < 0.01), liver (p < 0.05) and kidney (p < 0.001) were each inhibited by receptor blockade, undoubtedly contributing to the decline in plasma insulin clearance. The initial plasma insulin clearance was not signifi cantly affected by HFD, nor was hepatic or muscle specifi c clearance. Surprisingly, HFD increased renal clearance by > 50% (p < 0.01) suggesting a signifi cant role for renal insulin clearance in limiting the hyperinsulinemia that accompanies HFD. We conclude that the insulin receptor is a major mediator of initial plasma insulin clearance and for its initial clearance by liver, kidney and muscle. HFD feeding increases renal insulin clearance suggesting a signifi cant role for the kidney to limit the systemic hyperinsulinemia that accompanies HFD feeding.

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1926-PHow to Transform a Metabolic Syndrome Score in an Insulin Sen-sitivity Value?MICHEL P. HERMANS, EVARISTE BOUENIZABILA, K. DANIEL AMOUSSOU-GUENOU, SYLVIE A. AHN, MICHEL F. ROUSSEAU, Brussels, Belgium, Brazzaville, Congo, Republic of the, Cotonou, Benin

The metabolic syndrome (MetS) predicts cardiovascular risk and incident type 2 diabetes mellitus (T2DM). The presence of a MetS is defi ned by the clustering of ≥3 out of 5 cardiometabolic criteria (hyperglycemia; hyperten-sion; enlarged waist; low HDL-cholesterol; and hypertriglyceridemia), each of which is connected with insulin resistance (IR). It is not known whether the severity of MetS, ranked from the sextet of scores’ range [0/5 to 5/5], is linearly related to reduced insulin sensitivity (IS) and/or lesser hyperbolic product across the glycemic spectrum.

We analyzed 839 adults (54 normoglycemic; 785 with abnormal glucose homeostasis, among whom 711 T2DM), whose IS was assessed together with their cardiometabolic phenotype.

There was a signifi cant gradient according to interval-scale MetS score in insulinemia; BMI; (visceral) fat; hepatic steatosis; and macroangiopathy. There was an inverse linear relationship between increasing MetS scores and decreased IS, allowing to defi ne an IR-predicting linear equation: IS (%) = [-15.1*MetS score] + 109.4 (R2 = 0.221). For each MetS category, mean IS values did not signifi cantly differ between groups of patients across the gly-cemic spectrum. The hyperbolic product (β-cell function x IS) and/or its loss rate were inversely related to MetS severity.

In conclusion, IS is linearly and inversely related to MetS severity across the 6 scores. This novel way to exploit information intrinsic to the MetS cri-teria provides an easy and costless means to quantify IS across the glycemic spectrum. Moreover, MetS severity is associated with a lesser hyperbolic product, and a higher loss of the latter.

1927-PPossible Involvement of Undercarboxylated Osteocalcin upon Reg-ulating Insulin Secretion in Patients with Type 2 DiabetesYUICHI TAKASHI, YOKO MATSUZAWA, JUN SAITO, MASAO OMURA, TETSUO NISHIKAWA, Yokohama, Japan

We examined the effect of undercarboxylated osteocalcin (ucOC) on the ability of insulin secretion, evaluated by glucagon loading test (GLT) and meal tolerance test (MTT). UcOC has been shown to regulate insulin secretion in rodents. However, data on the correlation between ucOC and glucose metabolism in humans is limited and controversial. We recruited 50 patients (41 men and 9 postmenopausal women) with type 2 diabetes under written informed consent, and performed both GLT and MTT, and analyzed the relationship between ucOC and C-peptide response (CPR). All patients were free of insulin therapy and drugs known to infl uence bone metabolism, such as vitamin D, bisphosphonate. Number of patients who were treated with DPP-4 inhibitor, sulfonylurea, glinide, metformin, thiazolidinedione and alpha-glucosidase inhibitor were 17, 19, 15, 19, 4 and 12 patients. The av-erage of age, duration of diabetes, BMI, and HbA1c of the subjects were 59.2 y.o., 7.8 years, 26.2 kg/m2 and 9.4%, respectively. UcOC was shown to correlate positively with CPR in GLT and CPR after meal taking (r = 0.32, p = 0.025; r = 0.29, p = 0.047). Furthermore, we focused on the patients with HbA1c less than 8.0% because we thought they had little infl uence of glucose toxicity. UcOC was shown to correlate positively more strongly with CPR after glucagon loading and CPR in GLT (r = 0.60, p = 0.0088; r = 0.67, p = 0.0025) than with CPR after meal taking (r = 0.51, p = 0.035). In both groups, ucOC was not shown to correlate with CPR before glucagon loading or meal taking. It is suggested that ucOC correlate with not basal but bolus insulin secretion and may directly induce insulin secretion from β-cells without in-volving gut-related insulin stimulation in patients with type 2 diabetes. UcOC is suggested to be a new marker to evaluate the reserve capacity of β-cell function, such as the bolus insulin secretion ability.

1928-P20/(Fasting C-Peptide × Fasting Plasma Glucose) Is a Novel Index of Insulin Resistance with Small Effect of Hepatic Insulin ClearanceTSUYOSHI OKURA, YOUHEI FUJIOKA, RISA NAKANISHI, HIDEKI SHIOCHI, KEISUKE SUMI, KYOKO SHOJI, AYUMI MURAWAKI, KAZUHIKO MATSUZAWA, SCHOICHIRO IZAWA, CHIEKO SAKAI, MASAHIKO KATO, SHIN-ICHI TANIGUCHI, KAZUHIRO YAMAMOTO, Yonago, Japan

We developed a simple and new insulin resistance index derived from a glucose clamp and a meal tolerance test (MTT) in Japanese patients with type 2 diabetes mellitus.

Twenty patients [mean fasting plasma glucose (FPG) 7.3 mmol/L, HbA1c 7.5%, BMI 28.4 kg/m2] underwent a MTT and a glucose clamp. Participants were given a test meal (460 kcal). Plasma glucose, insulin, and C-peptide im-munoreactivity (CPR) were measured at 0, 30, 60, and 120 min. HOMA-IR and Hepatic Insulin Clearance (HIC: AUC-Insulin/AUC-CPR ratio) were calculated from the MTT results. The glucose disposal rate (GDR) was measured during hyperinsulinemic-euglycemic glucose clamps.

The mean GDR in all patients was 5.08 mg·kg-1·min-1. The index 20/(F-CPR × FPG) was correlated strongly with GDR (R=0.72), better than HOMA-IR (R=−0.53). 20/(F-CPR × FPG) was able to estimate GDR, we would like to name this index “CPR-IR.” The median value of HIC in all patients was 6.0. In the patients with low hepatic insulin clearance (HIC<6.0), HOMA-IR and CPR-IR were correlated equally with GDR (R=-0.68 and 0.69). In the patients with high hepatic insulin clearance (HIC>6.0), whereas HOMA-IR did not cor-relate with GDR (R=-0.21), CPR-IR correlated with GDR (R=0.64).

CPR-IR is a simple and effective index of insulin resistance in the patients with T2DM, and performs better than HOMA-IR with small effect of hepatic insulin clearance.

Table. The Correlation between GDR and CPR-IR, HOMA-IR.CPR-IR HOMA-IR

All patients R=0.72 R=-0.53Low hepatic insulin clearance (HIC<6.0) R=0.69 R=-0.68High hepatic insulin clearance (HIC>6.0) R=0.64 R=-0.21

INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

Guided Audio Tour: Insulin Secretion In Vivo (Posters: 1929-P to 1934-P), see page 13.

& 1929-PLimitations of Standardized C-Peptide Kinetics for Estimation of β-Cell ResponsivityRON T. VARGHESE, FRANCESCA PICCININI, MEERA SHAH, CHIARA DALLA MAN, ROBERT A. RIZZA, CLAUDIO COBELLI, ADRIAN VELLA, Rochester, MN, Padova, Italy

Standard kinetics of C-Peptide clearance enable deconvolution of insu-lin secretion from changing plasma C-Peptide concentrations. However, it is uncertain if standard parameters apply to all conditions under which β-cell function is measured. As part of a series of experiments examining the mechanisms underlying prediabetes, we studied 60 nondiabetic individuals (38.7±2.2 years, BMI 28.3±0.8 Kg/M2) on 2 occasions in random order using an oral glucose challenge. On one occasion, free fatty acid (FFA) elevation to cause insulin resistance, was achieved by infusion of intralipid + heparin. On the other study day subjects received the same amount of glycerol pres-ent in the intralipid. On a 3rd study day, after a euglycemic clamp where somatostatin inhibited endogenous insulin secretion, subjects received a C-Peptide bolus to calculate individual kinetic for clearance. β-cell responsivity (ϕTotal) in the presence or absence of FFA elevation was estimated using the oral C-Peptide minimal model using either standard or individual C-Peptide kinetics. ϕTotal estimated using individual kinetics correlated well with those estimated using standard kinetics in the presence (46±4 vs. 46±4 10-9 min-1, R2 =0.59, p<0.01) or absence (56±4 vs. 53±4 10-9 min-1, R2 =0.66, p<0.01) of FFA elevation. Analysis of the components of ϕTotal reveals generally worse correlation for ϕDynamic in the presence (R2 =0.42, p<0.01) compared to the absence (R2 =0.68, p<0.01) of FFA elevation. Similarly, ϕStatic in the presence (R2 =0.50, p<0.01) and absence (R2 =0.76, p<0.01) of FFA elevation. The great-est discrepancies arise in basal β-cell responsivity during FFA elevation (ϕBasal - R2<0.01, p=0.71) compared to glycerol infusion (R2 =0.79, p<0.01). We conclude that results obtained with standard C-Peptide kinetics correlate well with those obtained using individual kinetics. However individual pa-rameters are required for optimal measurement of ϕBasal at least in response to acute decreases in insulin action.

Supported By: National Institutes of Health (R01DK078646)

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