1665-p insulin action—adipocyte biology · a rabbit antiserum against pparγ pser492 was prepared...

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INSULIN ACTION—ADIPOCYTE BIOLOGY

reduced Xbp1 mRNA to 0.78 ± 0.7(N=4, p=N.S.), while addition of DC 15 min prior to tBH treatment has no apparent effect at 2.45 ± 1.4 (N=4, p=N.S.). Similarly, treatment of islets with DCA at 1 µg/mL (DCA) for 24 h increased Xbp1 mRNA to 2.04 ± 1.06 folds over control (N=4). Pre-incubation with DCA for 20 h before addition of tBH effectively reduced Xbp1 mRNA to 0.22 ± 0.14 (N=4, p=N.S.) while addition of DCA15 min prior to tBH treatment had no apparent effect at 3.75 ± 2.06 (N=4, P=N.S). Our results show that by using spliced Xbp1 mRNA expression as a marker, DC and DCA show the potential application for improving islet quality before transplantation.

Supported By: Hecht Foundation; University of British Columbia Hospital Foundation


Guided Audio Tour: Novel Pathways and Mechanisms in Adipose Devel-opment and Function (Posters: 1663-P to 1669-P), see page 13.

& 1663-PRegulation of PPARγ by a Novel PhosphorylationHEE KWAN WON, TAE KEUN OH, YONG HEE LEE, Daejeon, Republic of Korea, Cheongju, Republic of Korea

Mammalian ste20-like kinase (MST) signaling pathway is conserved from Drosophila to mammals and is essential for the proper regulation of organ growth, apoptosis and tumorigenesis. MST binds a scaffolding protein, SAV1 through the coiled-coil domain in the C-terminus and phosphorylates and activates SAV1. Previously, it was shown that MST2 and SAV1 interact with and activate peroxisome proliferator-activated receptor γ (PPARγ), a master regulator of adipogenesis. They were also shown to induce adipocyte differentiation. To further characterize this phenomenon, we sought to identify and analyze the phosphorylation of PPARγ by MST2 kinase. Using mass spectrometric analysis of PPARγ protein which was co-expressed with MST2 and SAV1 and immunoprecipitated, we detected a novel phosphorylation at Ser492 in C-terminal region of PPARγ protein that was co-expressed with MST2 and SAV1. A rabbit antiserum against PPARγ pSer492 was prepared and it detected a strong phosphorylation in PPARγ-WT but not in SA. Mutation of Ser492 to Ala (SA) resulted in a signifi cant decrease in transcription activity of PPARγ compared to WT. Mutation of Ser492 to Glu (SE) increased the basal activity of PPARγ but had no effect on rosiglitazone-induced activation, indicating that PPARγ-SE is a partial phosphorylation-mimicking mutant. Interaction of PPARγ with PGC1β, one of PPARγ coactivators, was increased by rosiglitazone. However, SA mutant showed signifi cantly inhibited interaction with PGC1β, indicating the identifi ed Ser phosphorylation may be important for the binding of PPARγ to its coactivators. Finally, SA mutation signifi cantly decreased PPARγ-induced adipocyte differentiation of 3T3-L1 cells. In this study, a novel Ser492 phosphorylation in PPARγ was presented and this phosphorylation may be a novel regulatory mechanism of PPARγ activation by MST pathway.

& 1664-PCHOP Modulates Adipose Tissue Macrophage PolarizationTORU SUZUKI, JUNHONG GAO, KEIICHI KONDO, YASUSHI ISHIGAKI, JUNTA IMAI, TETSUYA YAMADA, HIDEKI KATAGIRI, Sendai, Japan, Morioka, Japan

Although the C/EBP homologous protein CHOP is well known to play important roles in ER stress-related cellular apoptosis and several other metabolic disorders, its roles in adipose tissue macrophage polarization and the resultant insulin resistance in high fat diet-induced obesity are unclear. We thus examined the effects of CHOP defi ciency on peripheral insulin resistance during high fat feeding of mice. Glucose infusion rates during hyperinsulinemic euglycemic clamp procedures were markedly higher in CHOP-/- mice than CHOP+/+ mice fed a high fat diet for 6 weeks, indicating CHOP defi ciency to prevent insulin resistance development. There were no signifi cant differences in body weight and plasma lipid parameters. Although white adipose tissue (WAT) weights and cell diameters were slightly greater in CHOP-/- mice, serum adiponectin levels were signifi cantly higher in CHOP-/-mice. Quantitative RT-PCR revealed for adipose tissue-resident macrophages (ATM)-related genes revealed that expression of the M1 markers, IL-1β,CCR7 and TNF-α,were signifi cantly decreased, while the M2 markers, such as CD163, IL-10 and CD206 were signifi cantly up-regulated in CHOP-/- mice, suggesting M2-type ATMs to predominate, resulting in suppression of obesity-induced infl ammation in WAT. Interestingly, in CHOP-/- mice, the expressions of key molecules for macrophage polarization from M1 to M2, such as PPARγ, IL-4 and IL-13, were signifi cantly increased in WAT, in both the fl oating adipocyte fraction and the stromal vascular fraction of WAT. In differentiated 3T3-L1 cells, knockdown of CHOP signifi cantly suppressed tunicamycin-induced down-regulation of PPARγ, IL-4 and IL-13. These fi ndings indicate the novel role of

CHOP which enhances obesity-induced macrophage polarization to M1 in WAT, leading to insulin resistance.

& 1665-PInhibition of PDGF Receptor Improves Insulin Resistance by Suppression of Chronic Infl ammation via Reduction of MCP1 Expression in the Adipose Tissue of Type 2 Diabetes MiceYASUHIRO ONOGI, TSUTOMU WADA, KAMIYA CHIE, KENTO INATA, YOKO ISHII, MASAKIYO SASAHARA, HIROSHI TSUNEKI, TOSHIYASU SASAOKA, Toyama, Japan

Adipose tissue shows hypoxic and infl ammatory phenotype characterized by the infi ltration of adipose tissue macrophages with progression of obesity. In diet-induced obese (DIO) mice, we found upregulation of platelet derived growth factor (PDGF) and its receptors in epididymal white adipose tissue (EWAT). Therefore, we investigated the pathophysiological role of PDGF in the development of adipose tissue infl ammation and insulin resistance.

The mRNA expressions of PDGF and its receptors were increased in EWAT of DIO and db/db mice. In RAW264.7 macrophages, CoCl2, chemical hypoxia mimetic, induced PDGF expression, which was accompanied by increased expression of HIF1α. These indicate that macrophage could produce PDGF in response to hypoxia in adipose tissue of obese mice. To investigate the role of PDGF signaling in adipose tissue, we administrated the PDGFR tyrosine kinase inhibitor (AG1296, 1 mg/kg/day) to m+/m+ and db/db mice for 4 weeks. Treatment with AG1296 did not affect body weight and fat mass on MRI imaging in m+/m+ and db/db mice. In contrast, glucose tolerance was apparently, and insulin sensitivity was slightly and signifi cantly improved by AG1296 administration in db/db mice. The histological analysis of EWAT revealed that AG1296 treatment decreased the adipocyte size in db/db mice. Moreover, AG1296 treatment decreased expressions of infl ammation-related genes, such as MCP1, TNFα and F4/80, in EWAT of db/db mice. To clarify the mechanism by which AG1296 improved insulin resistance in db/db mice, we differentiated 3T3-L1 adipocytes with or without AG1296 treatment. AG1296 treatment markedly decreased MCP1 gene expression without inhibiting adipocyte differentiation. These fi ndings suggest that higher expression of PDGF and its receptors in adipose tissue would be implicated in the development of adipose tissue infl ammation and insulin resistance in obesity.

& 1666-PEwing Sarcoma Gene, EWS, Plays Important Roles in Brown Adipocyte Differentiation and Mitochondria BiogenesisJUN HONG PARK, SEAN BONG LEE, New Orleans, LA

Obesity and its related diseases have become an enormous health issue not only to the rapidly growing adult population but also in young children. Thus, intense efforts are now underway to better understand the mechanisms of adipogenesis and metabolism. Ewing sarcoma gene (Ews) encodes a ubiquitously expressed RNA binding protein with proposed roles in transcription and splicing, but its function is largely unknown.

To study the physiological role of Ews, we have generated Ews-null mice by gene targeting. Interestingly, Ews-null mice exhibited signifi cant disruption of early brown adipocyte differentiation during embryogenesis. Interscapular and subscapular BATs from Ews-null newborn pups do not contain lipid droplets nor express Ucp1, a functional marker of BAT. Ews mutant brown preadipocytes fail to undergo adipocyte differentiation upon adipogenic stimuli due to lack of Bmp7 expression, an early brown adipogenic factor. We demonstrate that Ews interacts and functions as a transcriptional cofactor of Ybx1, Y-box binding protein 1, which activates Bmp7 transcription. Bmp7 expression is greatly diminished in the Ews-defi cient embryonic (E15.5) interscapular brown preadipocytes. Depletion of either Ews or Ybx1 leads to loss of Bmp7 expression and brown adipogenesis. Complementation of Ews in Ews-null preadipocytes restores Bmp7 transcription and brown adipogenesis. Ews haploinsuffi ciency leads to reduced brown fat gene expression following rosiglitazone or β3-agoinist stimulation. Ews-null BATs and brown adipocytes ectopically express myogenic genes. Remarkably, Ews-null BATs contained abnormal mitochondria which were undergoing degradation. In the absence of EWS, we observed reduced number and function of mitochondria in brown preadipocytes and myoblasts. These results demonstrate that Ews is essential for early brown cell-fate determination and mitochondria abundance and function.

Supported By: Tulane University (to S.B.L.)

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& 1668-PArp2/3 Complex Is a Critical Regulator of Adipocyte DevelopmentWULIN YANG, SHERMAINE THEIN, WEIPING HAN, Singapore, Singapore

Dramatic actin cytoskeleton remodeling, including disruption of stress fi bers across the cell body and assembly of cortical actin structure at the plasma membrane, occurs during adipocyte development. We previously showed that disruption of stress fi bers by the actin severing protein Cofi lin is a prerequisite step in adipogenesis. However, it remains unclear whether actin nucleation and assembly into the cortical structure is essential for adipocyte maturation. In this study, we investigated a potential role of cortical actin assembly, which is controlled by the actin nucleation complex Arp2/3, in adipogenesis by using 3T3-L1 adipocytes with stable gene expression or knockdown. We found that cortical actin structure formation started with accumulation of patchy F-actin near plasma membrane during adipogenesis. Depletion of the Arp2/3 complex by Arp3 or p21arc knockdown strongly impaired adipocyte differentiation, as evidenced by reduced lipid accumulation and downregulated adipogenic markers. Moreover, in the 3T3-L1 cells with stable depletion of the Arp2/3 complex, F-actin aggregation at the plasma membrane was suppressed and cortical actin structure poorly developed after adipogenic induction. These results show that Arp2/3 complex, which controls cortical actin structure formation, is a crucial regulator during adipocyte differentiation.

Supported By: Agency for Science Technology and Research (A*STAR)

& 1669-PEffects of All-Trans Retinoic Acid (ATRA) on White Adipose Tissue (WAT) in a Model of Obesity-related Type 2 Diabetes (T2D)SANA HASAN, RONDA SIMPSON, CYRUS V. DESOUZA, FREDERICK G. HAMEL, ROBERT G. BENNETT, Omaha, NE

Obesity-related T2D is characterized by WAT dysfunction including adipocyte hypertrophy, low grade infl ammation and few brown-like (beige) adipocytes in WAT.

We have shown that treatment (tx) of db/db mice with ATRA decreased random and fasting blood glucose, promoted weight loss, and improved glucose tolerance. In the present study, we investigated the effect of ATRA on infl ammation and remodeling of WAT in 3 month old db/db mice (10 per group) treated with vehicle (corn oil) or ATRA at 0.6 mg/day, 5 days/week for 12 weeks by voluntary oral feeding. We monitored a subset of mice for 8 weeks after tx cessation.

ATRA caused a signifi cant reduction in adipocyte size which was maintained in the post tx phase (8229±165 µm2 control vs. 5775±285 µm2

ATRA vs. 5405±571 µm2 post tx, p<.0001). ATRA increased uncoupling protein 1 (UCP1) gene expression in visceral WAT (1.4±0.4 control vs. 13.4±5.0 ATRA) and UCP1 protein levels which were not retained post tx.

Obesity is characterized by WAT macrophage infi ltration; the source of infl ammatory cytokines associated with insulin resistance. WAT was examined for macrophages using the protein marker F4/80. ATRA resulted in a signifi cant decrease in the frequency of F4/80-positive crown-like structures (CLS), which was sustained post tx (1.02±0.24 CLS/um2 control vs. 0.15±0.55 ATRA vs. 0.32±0.23 post tx, p<.05). The expression of infl ammation-related genes including MCP-1, PAI-1, and TNF-α were not reduced with ATRA tx, however, they were signifi cantly suppressed in the post tx phase.

We have shown that long-term ATRA tx promotes the remodeling of WAT and induces UCP1-positive beige adipose in WAT. Further, ATRA attenuates infl ammation by reducing macrophage infi ltration and suppressing the expression of the infl ammation-related genes. Therefore, ATRA may be useful for obesity-related T2D by inducing the beige phenotype to promote weight loss, while simultaneously reducing infl ammation to improve WAT function and insulin resistance.

1670-PDexamethasone Promotes FKBP5 and CNR1 Gene Expression in Human Adipose Tissue—Two Factors That May Be Implicated in Insulin ResistanceMARIA J. PEREIRA, JENNY PALMING, MARIA K. SVENSSON, MAGNUS RIZELL, JAN DALENBÄCK, MÅRTEN HAMMAR, TOVE FALL, CHERNO O. SIDIBEH, PER-ARNE SVENSSON, JAN W. ERIKSSON, Uppsala, Sweden, Gothenburg, Sweden, Mölndal, Sweden

Glucocorticoid excess promotes central obesity and insulin resistance (IR). To identify potential novel mechanisms for glucocorticoid-induced IR we investigated the effects of dexamethasone on gene expression in human adipose tissue.

Subcutaneous and omental adipose tissue, obtained from 25 non-diabetic subjects (28-60 yrs; 20.7-30.6 kg/m2), was incubated without or with

dexamethasone (0.003-3 µM) for 24 h. Gene expression was assessed by microarray and real time-PCR. Protein levels were assessed by immunoblotting.

FKBP5 (FK506 binding protein 5) and CNR1 (cannabinoid receptor 1) were the most responsive genes to dexamethasone, with increased gene expression up to 7-fold in a dose-dependent manner in both subcutaneous and omental fat depots. The FKBP5 gene product, FKBP51 protein, also increased in a similar manner following dexamethasone exposure in both fat depots. However, FKBP51 protein was 10-fold higher in the omental than in the subcutaneous fat depot, whereas the mRNA levels were similar. FKBP5 gene expression in subcutaneous fat depot was positively correlated with, HOMA-IR and subcutaneous adipocyte diameter, while change in gene expression produced by dexamethasone was negatively correlated with markers related to IR, i.e. HOMA-IR, BMI and HbA1c (p<0.05). Interestingly, FKBP5 SNPs were found to be signifi cantly associated with type 2 diabetes and with diabetes-related phenotypes (2-h OGTT glucose, HDL-C and triglycerides) in large population-based samples. The CNB1 gene expression in the subcutaneous fat depot, similar to FKBP5, was positively correlated with clinical markers of IR and adiposity, i.e. HOMA-IR and BMI (p<0.05).

In conclusion, dexamethasone promotes gene expression of FKBP5 and CNR1 in adipose tissue, two genes that may be implicated in glucocorticoid-induced as well as other forms of IR. This study gives support to the concept of peripheral cannabinoid receptors as novel targets for antidiabetic drugs.

Supported By: AstraZeneca; University of Gothenburg (LUA/ALF ALFGBG-11379)

1671-PObesity-related Adipose Tissue Stiffening Sensitizes Adipocytes to a TNFα-induced Proinfl ammatory ResponseHIROYA KOBAYASHI, LIKANG CHIN, YUKIKO BANDO, YOSUKE SHIKAMA, PAUL A. JANMEY, MAKOTO FUNAKI, Tokushima, Japan, Philadelphia, PA

Crosstalk between adipocytes and infi ltrated macrophages enhances proinfl ammation in obese adipose tissues, which contributes to develop insulin resistance. We previously reported that adipose tissues stiffen in obesity, which is presumably attributable to fi brosis reported by others. Little is known yet about the effect of tissue stiffening on obesity-induced proinfl ammation in adipose tissues. In this study adipokine secretion by 3T3-L1 adipocytes treated with TNFα, one of major cytokines produced by infi ltrated macrophages, was measured in cells that were seeded on polyacrylamide gels with stiffness designed to mimic that of either lean (250 Pa) or obese (750 Pa) adipose tissues. When 3T3-L1 adipocytes were seeded on 250 Pa gels, TNFα treatment had little if any effect on MCP-1 (Monocyte Chemotactic Protein-1) secretion On the other hand, TNFα-induced MCP-1 secretion was signifi cant in cells on 750 Pa gels. Adipocytes have been known to infl ate in obese adipose tissues. To investigate the effect of adipocyte infl ation on TNFα-induced MCP-1 secretion by adipocytes, 3T3-L1 cells on polyacrylamide gels were loaded with palmitate, which increased the adipocyte size. Palmitate-loading further increased TNFα-induced MCP-1 secretion by adipocytes on 750 Pa gels, but not by cells on 250 Pa gels. When adipocytes were seeded on glass, MCP-1 secretion level was signifi cantly higher than that by cells on 250 Pa gels even without TNFα treatment, suggesting that adipocytes are infl amed on rigid materials such as glass. These results suggest that tissue stiffening observed in obese adipose tissue may make adipocytes competent to respond to TNFα provided by neighboring macrophages, and that this effect is further enhanced by adipocyte infl ation.

1672-PProteomics Analysis of Subcutaneous Adipocytes Reveals Novel Abnormalities in Human Insulin ResistanceXITAO XIE, ZHENGPING YI, SANDEEP SINHA, BEN BOWEN, PAUL LANGLAIS, LAWRENCE J. MANDARINO, CHRISTIAN MEYER, Tempe, AZ, Detroit, MI, Phoenix, AZ, Orlando, FL

Adipocytes play a major role in insulin resistance. Identifi cation of the underlying factors has largely been focus oriented. Using an unbiased proteomics approach, the present study was designed to provide a more global view of adipocyte changes in insulin resistance that can be useful in pointing out new research directions.

Baseline biopsies of abdominal adipose tissue were obtained from 10 insulin-resistant (IR) and 13 insulin-sensitive (IS) non-diabetic subjects, based on predefi ned criteria of insulin sensitivity by euglycemic clamp. Global protein abundance of isolated adipocytes was determined by 1-D SDS-PAGE for protein separation followed by HPLC-ESI-MS/MS for protein assignment with quantifi cation by a normalized spectral abundance factor method.

IR and IS were of similar age and gender distribution, but IR had ~2.5-fold lower insulin sensitivity and a greater BMI (p < 0.01). Proteomics analysis

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assigned a total of 1,261 proteins, of which 383 were detected in >50% of all subjects. Of these, 94 were altered in abundance by a factor >1.5 in IR, of which 30 were signifi cant (P < 0.05). Of the 19 proteins with lower abundance, 9 were mitochondrial, of which nearly all were involved in energy homeostasis; others of interest include fatty acid binding protein (FABP) 4 and FABP5, profi lin, which prevents actin polymerization, and abhydrolase domain containing 5, which functions in phosphatidic acid biosynthesis and regulates triacylglycerol storage through activation of the phospholipase PNPLA2. Of the 11 proteins with higher abundance, most were involved in microtubules and the cytoskeleton; cytochrome b5 reductase 1 was the only mitochondrial protein, which is involved in desaturation and elongation of fatty acids and cholesterol biosynthesis. We conclude that changes in adipocyte mitochondria, lipid metabolism, microtubules and the cytoskeleton are associated with insulin resistance in humans.

Supported By: ADA (1-09-CR-39)

1673-PRelation of Adipocyte Mitochondria to Insulin Sensitivity and Lipid Metabolism in HumansXITAO XIE, SANDEEP SINHA, CASSANDRA WOLF, CHRISTIAN MEYER, Tempe, AZ, Phoenix, AZ, Orlando, FL

Abnormalities in adipocyte mitochondria have been implicated in playing an important role in dysregulated lipid metabolism and insulin resistance. However, the relation of adipocyte mitochondria to lipid metabolism and insulin sensitivity has not been evaluated in humans. In the present study, we therefore examined the mitochondrial copy number (mtDNA/nDNA) in abdominal subcutaneous adipocytes across a wide range of plasma lipids, insulin sensitivity (measured by euglycemic clamps) as well as adiposity in 18 non-diabetic individuals (6 M/12 F; age 22-68 years; BMI 20.4-37.6 kg/m2).

Compared to the group of individuals with mtDNA/nDNA in the upper 50th percentile, that in the lower 50th percentile had a signifi cantly greater male/female ratio (p < 0.05), greater BMI, lower HDL and lower insulin sensitivity (all p < 0.04); moreover, there was a trend for reduced suppression of plasma free fatty acids (FFA) during the clamp (p < 0.08). Age, percent body fat, LDL, VLDL and triglycerides did not differ. Simple linear regression analysis of all subjects’ data showed signifi cant correlations between mtDNA/nDNA and BMI (r= -0.473, p < 0.05) and HDL (r = 0.567, p < 0.015), and a trend for correlations of mtDNA/nDNA with LDL (r = -0.433, p < 0.073), VLDL (r =-0.438, p < 0.07), insulin sensitivity (r = 0.409, p < 0.092) and percent suppression of plasma FFA during the clamp (r = 0.465, p < 0.051). When data were adjusted for differences in BMI using ANCOVA, the correlations of mtDNA/nDNA with lipids and insulin sensitivity no longer showed statistical signifi cance or trend; moreover, mtDNA/nDNA did not differ between men and women.

We conclude that the abdominal adipocyte mitochondrial copy number correlates inversely with BMI and favorably, albeit not independently of BMI, with aspects of lipid metabolism and insulin sensitivity in humans.

Supported By: ADA (1-09-CR-39)

1674-PIncreased Browning of White Adipose Tissue Contributes to the Benefi cial Metabolic Effects of Inhibition of the Cannabinoid Regu-lating System In VivoMORVARID KABIR, MALINI IYER, JOYCE M. RICHEY, QIANG WU, DARKO STEFANOVSKI, ORISON O. WOOLCOTT, STELLA P. KIM, CATHRYN M. KOLKA, VIORICA IONUT, RICHARD N. BERGMAN, Los Angeles, CA

The CB1 receptor antagonist, rimonabant (RIM) improves deleterious effects of a high fat diet (HFD) by reducing body fat and increasing insulin sensitivity. We recently showed that RIM increased expression of UCP1 and the novel hormone irisin, secreted by muscle. The current study examines the longitudinal effects of RIM on genes involved in the process of fat browning (PGC1-α, Prdm16, FGF21, Dio2, Cidea, Cox2, and Elov3) in the subcutaneous (SC) and visceral (VIS) fat depots. Animals were fed a HFD (52% fat) for 6 weeks followed by an additional 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) and after 16 weeks of HFD +/- RIM. Consistent with irisin results, RIM increased PGC1-α, expression in the VIS depot by 3 fold (P<0.001) and in the SC by 2 fold (P<0.01). The same trend was observed for Prdm16, DIO2, Cox2 (VIS: by 4, 1.77, 3.17 fold respectively (P<0.05), SC: 3, 2.5, 5.7 fold respectively (P<0.05)). Elov3, Cidea FGF21 gene expression increased only in the VIS depots (by 2.5, 4 and 2 fold respectively (P<0.05). Thus, CB1-R antagonist increased expression of genes involved in browning of fat to a higher extent in the VIS fat depot than the SC depot of fat-fed dog. Increased browning of fat, particularly in the visceral fat depot, appears to be an important mechanism by which the cannabinoid system regulates body fat and metabolism.

1675-PIntra-abdominal Transplantation of Subcutaneous Adipose Tissue in Mice Prevents High-Fat Diet-induced Infl ammation and Glucose IntoleranceSAMANTHA L. HOCKING, REBECCA L. STEWART, AMANDA E. BRANDON, ELLA STUART, EURWIN SURYANA, SINEAD BLABER, MICHAEL MEDYNSKYJ, BEN HERBERT, GREGORY J. COONEY, MICHAEL M. SWARBRICK, Sydney, Australia

In mice, intra-abdominal transplantation of subcutaneous (inguinal), but not visceral (epididymal) adipose tissue protects against insulin resistance, hepatic lipid accumulation and obesity. The underlying mechanisms have not been determined.

We hypothesised that transplantation of subcutaneous fat prevents chronic infl ammation associated with a high-fat diet (HFD). Inguinal and epididymal depots were implanted into 7 wk old male C57BL6/J mice (subQ→vis and vis→vis mice, respectively). Glucose tolerance and skeletal muscle glucose uptake were assessed after 13 wks; and cytokines were measured with a Bio-Plex Mouse Cytokine Assay (Bio-Rad).

SubQ→vis mice displayed improved glucose tolerance and lower fasting insulinaemia relative to sham and vis→vis mice (both P<0.05). Skeletal muscle glucose uptake did not differ between groups (P=0.25).

Transplantation of either type of fat suppressed plasma IL-2 and IL-5 levels; while subQ→vis transplantation alone prevented HFD-induced increases in plasma IL-10, IL-17 and TNF-α. Vis→vis mice displayed elevated plasma IL-6, as well as increased liver TG content and reduced hepaticSocs3 expression (all P<0.05). Therefore, subQ→vis and vis→vis transplantation exert opposing effects on infl ammation and hepatic TG accumulation.

All data mean±SEM. *P<0.05 vs. sham, †P<0.01 vs. subQ→vis, ‡P<0.05 vs. subQ→vis.

Plasma Cytokine Concentrations at 4 and 10 Weeks Post-transplantation.Timepoint 4 weeks — — — 10 weeks — —Plasma Concentration (pg/ml)

Chow (n=5)

HFD Sham (n=12)

HFD SubQ→vis

(n=9)

HFD Vis→vis (n=10)

HFD Sham (n=12)

HFD SubQ→vis

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HFD Vis→vis (n=10)

IL-2 3.0±1.1* 12.6±1.3 8.2±2.9 10.0±2.7 19.1±1.7† 11.0±1.8 12.3±1.0IL-5 3.9±1.1* 9.1±1.2 5.2±0.6* 7.6±0.9 12.0±0.7† 8.8±0.9 9.9±0.8IL-6 2.0±0.7 4.2±0.6 3.5±0.4 5.9±1.5 6.5±0.7 4.9±0.8 10.1±2.0‡IL-10 9.8±3.9* 22.7±3.1 11.2±3.2* 9.8±1.7* 32.8±3.2‡ 21.1±1.9 30.6±2.6‡IL-17 4.7±2.5 10.1±1.1 5.9±0.7 9.1±1.9 14.9±1.6† 8.4±0.9 14.3±1.7‡TNF-α 61±24 104±9 71±12 108±23 149±12‡ 102±13 148±13‡

Supported By: NHMRC

1676-PDPP-4 Inhibitor Anagliptin Exerts Anti-infl ammatory Effects on Macrophages, Adipocytes, and Mouse Liver by Suppressing LPS-Induced Nf-κB ActivationTAKANORI SHINJO, MISAKI IWASHITA, HIDEYUKI SAKODA, MIDORI FUJISHIRO, TAKESHI YAMAMOTOYA, AKIFUMI KUSHIYAMA, FUSANORI NISHIMURA, TOMOICHIRO ASANO, Hiroshima, Japan, Tokyo, Japan

DPP4 expression reportedly increases in chronic infl ammatory conditions including obesity, suggesting an association of DPP4 with infl ammation. In this study, we investigated the effects of the DPP4 inhibitor anagliptin on the LPS-induced infl ammatory response in RAW264.7 macrophages, 3T3-L1 cells co-cultured with macrophages and mouse livers.

First, we demonstrated that 24h incubation with LPS markedly increased infl ammatory cytokine mRNA expressions (TNF-α, IL-1β, IL-6 and IL-12) in RAW264.7 macrophages, and that these increases were signifi cantly suppressed by co-incubation with anagliptin (10ng/ml). Interestingly, another DPP4 inhibitor, sitagliptin (10ng/ml), had no such effect. This may suggest that the anti-infl ammatory effect of anagliptin occurs via a mechanism independent of DPP4. As a molecular mechanism underlying the anti-infl ammatory effect of anagliptin, LPS-induced degradation of IκBα and phosphorylation of p65, JNK and p38, as well as NF-κB and AP-1 promoter activities, were suppressed in RAW264.7 macrophages by incubation with anagliptin, indicating the suppressive effect of anagliptin on both NF-κB and AP-1 signaling pathways. Subsequently, the effect of anagliptin on 3T3-L1 adipocytes co-cultured with macrophages was examined. When RAW267.4 macrophages were stimulated with LPS or TNFα, infl ammatory adipokine expressions in the co-cultured 3T3-L1 adipocytes were upregulated while anagaliptin suppressed these responses. Finally, LPS was administered to mice alone or with anagliptin and the effects of anagliptin on LPS-induced hepatic NF-κB activity were thereby examined. While LPS injection markedly elevated hepatic NF-κB activity, anagliptin attenuated the degree of this

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INSULIN ACTION—CELLULAR AND MOLECULAR METABOLISM

elevation. These profi les are potentially benefi cial in terms of preventing exacerbation of diabetes and cardiovascular events.

1677-PAdipose ChREBP-β Expression Increases after a Hypercaloric Diet and Is Associated with Induction of GLUT4 ExpressionMURAT KILICARSLAN, BARBARA B. KAHN, KARIN E. KOOPMAN, JOHANNES A. ROMIJN, MARIËTTE T. ACKERMANS, AART J. NEDERVEEN, MATTHAN W. CAAN, SUSANNE E. LA FLEUR, MARK A. HERMAN, MIREILLE J. SERLIE, Amsterdam, Netherlands, Boston, MA

Adipose tissue expression of the Carbohydrate Responsive Element Binding Protein (ChREBP)-β, a recently discovered isoform, is regulated by GLUT4 and predicts insulin-stimulated glucose disposal. While adipose ChREBP-β expression is downregulated in obesity, the direct effects of excess nutrient ingestion are unknown. To study the effect of caloric excess on subcutaneous adipose tissue (SAT) expression of CHREBP and GLUT4, 35 healthy men (BMI 22.2 [19.6-24.9] kg/m2) consumed a hypercaloric, high-sugar or high-fat-high-sugar diet (40% extra calories) or a control (eucaloric) diet for 6 weeks. Before and after the diet, we performed a 2-step hyperinsulinemic-euglycemic clamp, and measured SAT and liver fat by MRS. SAT biopsies were obtained to measure gene expression by qPCR. Prior to the diet, both SAT ChREBP-α and ChREBP-β correlated with GLUT4 expression (rs = 0.427, P = 0.010 and rs = 0.576, P < 0.001, resp.). ChREBP-β, but not -α expression inversely correlated with BMI (rs = -0.510, P = 0.002) and amount of SAT (rs = -0.437, P = 0.011) and liver fat (rs = -0.376, P = 0.028). ChREBP-β expression correlated positively with basal endogenous glucose production (EGP) (rs = 0.411, P = 0.014), but not with peripheral insulin sensitivity. After the hypercaloric diet, insulin sensitivity did not change, ChREBP-α trended towards an increase (P = 0.069) and ChREBP-β increased signifi cantly (P = 0.045). The change in ChREBP-β correlated strongly with the change in GLUT4 expression (rs = 0.572, P = 0.001). In the control diet group, insulin sensitivity and ChREBP and GLUT4 expression did not change.

To conclude, SAT ChREBP-β is linked to hepatic glucose metabolism. We show that overeating induces SAT ChREBP-β expression in association with increased GLUT4 expression, suggesting a role for ChREBP-β as an early adaptive response in adipose-tissue handling of nutrient excess.

1678-PA New Role for Adipose Steroid Receptor Coactivator 1 (SRC1): Decreasing Expression of SRC1 in Adult Sd Rats Increases Insulin Stimulated Skeletal Muscle Glucose UptakeJENNIFER L. CANTLEY, DANIEL F. VATNER, THOMAS GALBO, ANILA MADIRAJU, MAX C. PETERSEN, RACHEL J. PERRY, NAOKI KUMASHIRO, FITSUM GUEBRE-EGZIABHER, MITCHELL STACY, DONALD P. DIONE, ALBERT J. SINUSAS, VARA PRASAD MANCHEM, SANJAY BHANOT, JONATHAN S. BOGAN, GERALD I. SHULMAN, VARMAN T. SAMUEL, New Haven, CT, Carlsbad, CA

SRC1 infl uences numerous physiological processes, including those regulating glucose homeostasis. SRC1-/- mice were reported to have decreased expression of hepatic gluconeogenic enzymes and rate of endogenous glucose production (EGP). To determine whether SRC1 is a key regulator of hepatic glucose metabolism decreased SRC1 expression in normal, adult Sprague-Dawley rats. Regular chow-fed (RC) and high-fat fed (HFF) male SD rats were treated with an ASO against SRC1 or a control ASO for four weeks followed by metabolic assessments. SRC1 ASO specifi cally decreased SRC1 expression in the liver and WAT by ~80% without affecting expression in BAT or skeletal muscle. In contrast to SRC1-/- mice, in RC rats SRC1 ASO did not alter body weight, expression of gluconeogenic enzymes or the basal rate of EGP. However, fasting insulin concentration was ~ 50% lower, suggesting improvements in insulin sensitivity. In RC rats, SRC1 ASO increased insulin-stimulated whole body glucose disposal by ~30%, largely attributable to improved insulin-stimulated muscle glucose uptake. In HFF rats, SRC1 ASO also increased insulin-stimulated glucose disposal by ~50%. Muscle insulin receptor signaling, AMPK activity and hexokinase II expression were not changed by SRC1 ASO. However, we observed a ~7-fold increase in adipose expression of lipocalin-type prostaglandin D2 synthase (PGDS), a previously reported regulator of insulin sensitivity, and ~70% increase in plasma PGD2 concentration. Though PGD2 is a potent vasodilator, skeletal muscle tissue perfusion was not altered with SRC1 knockdown. Furthermore, though total GLUT4 content was unchanged, SRC1 ASO potentiated insulin mediated cleavage of Tether containing UBX domain for GLUT4 (TUG), a regulator of GLUT4 translocation. Conclusion: These studies suggest that adipose SRC1 may regulate the expression of PGDS which may then impact insulin stimulated muscle glucose uptake.

Supported By: NIH; U.S. Dept. of Veterans Affairs; HHMI

1679-PRole of Fyn Kinase in Adipocyte DifferentiationELENA TARABRA, CLAIRE C. BASTIE, Bronx, NY

We previously showed that Fyn kinase (Fyn) knockout mice are leaner than their control littermates and display reduced adiposity. Although others have reported that the Src kinase family might participate to adipose tissue differentiation, the role of Fyn during this process has not been fully clarifi ed.

We used 3T3-L1 adipocytes in which Fyn has been silenced (3T3-FynKD) and evaluate the expression of differentiation markers, as well as lipid accumulation. As reported by others, PPARgamma and Fabp4 gene expression levels as well as triglycerides content were reduced after 10 days of differentiation in 3T3-FynKD adipocytes compared to control cells. Importantly, we found that perilipin and AGTL protein levels were also decreased in the 3T3-FynKD cells, suggesting that reduced lipid accumulation was not a consequence of increased lipolytic events.

In order to pinpoint the differentiation step that is affected in adipocytes lacking Fyn, we treated 3T3L1-FynKD cells and their controls with Rosiglitazone, a specifi c activator of PPARgamma known to increased adipocyte differentiation. Unexpectedly, PPARgamma expression and lipid accumulation were partially rescued upon rosiglitazone treatment, suggesting that endogenous activation of PPARgamma might be impaired in 3T3L1-FynKD adipocytes. Together, these data suggest that Fyn might primary be involved in the later events leading to fully differentiated adipocytes but is not exclusively fundamental for the adipogenesis process.

Supported By: NIH/NIDDK

1680-PLong-Range Transactivation of C/EBPa Gene Expression by PPARγ through Distal Enhancers during Adipocyte DifferentiationTOMOHISA AOYAMA, HIRONORI WAKI, TOSHIMASA YAMAUCHI, KEN-ICHI WAKABAYASHI, TSUYOSHI INOUE, MASAHIRO NAKAMURA, JING YU, KAZUMI TAKE, WEI SUN, MASATO IWABU, MIKI OKADA-IWABU, KOHJIRO UEKI, YOUICHIRO WADA, SHUICHI TSUTSUMI, TATSUHIKO KODAMA, JURO SAKAI, HIROYUKI ABURATANI, TAKASHI KADOWAKI, Tokyo, Japan

Adipogenesis is controlled by a cascade of transcription factors. Although the regulation of C/EBPα gene expression by PPARγ is crucial, the mechanistic basis of this regulation is poorly understood. Here, we employed ChIP-seq and identifi ed distal PPARγ binding sites (+3, +19, +22, +24, +50, +53kb) in downstream region of the C/EBPα gene in differentiated 3T3-L1 adipocytes. These regions contain DR-1 motifs that bind to the PPARγ/RXRα heterodimer in a gel-shift assay and are functional in a luciferase assay, and exhibit increased histone H3 acetylation and chromatin accessibility (as judged by FAIRE-qPCR) during differentiation. Moreover, we noted that a multifunctional insulator protein CTCF, bind to the distal enhancers and the promoter. Chromosome conformation capture (3C) assays showed that these distal enhancers and the C/EBPα promoter organize specifi c interaction and create a long-range loop structure, which is enhanced upon differentiation. RNA-mediated depletion of either PPARγ or CTCF resulted in decreased loop formation, transcriptional regulation of C/EBPα and differentiation. Our fi ndings indicate that PPARγ and CTCF-dependent DNA loop formation between the distal enhancers and the promoter is required for optimal transactivation of C/EBPα gene expression and differentiation.


Guided Audio Tour: Integration of Insulin Signaling and Metabolism (Posters: 1681-P to 1687-P), see page 13.

& 1681-PAdipocyte-Specifi c Deletion of Akt2 Leads to Systemic Insulin ResistanceABIGAIL L. SHEARIN, BOBBY R. MONKS, WILLIAM J. QUINN III, MORRIS J. BIRN-BAUM, Philadelphia, PA

The relationship between obesity and the development of hepatic insulin resistance is not well understood. One model proposes that insulin resistance fi rst develops in fat depots and is propagated to other insulin target tissues. To test this, we have generated a mouse model with genetic disruption of insulin signaling in fat cells by deleting Akt2 in an adipocyte-specifi c manner (F2KO). F2KO mice maintain normal adipose depot size and glucose tolerance. However, compared to wild type (WT) mice, they exhibit hyperinsulinemia when fasted and after a glucose challenge. Glucose infusion rates (GIR) during a hyperinsulinemic-euglycemic clamp study were signifi cantly lower in F2KO mice. At the 100 minute time point, average GIR was 51.45 mg/kg/min in WT mice (n=3) and 5.77 mg/kg/min in F2KO mice

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(n=5). Glucose disposal rates were 60.0 mg/kg/min in WT and 23.7 mg/kg/min in F2KO mice. Moreover, insulin’s ability to suppress hepatic glucose production (HGP) was severely blunted compared to WT mice. Basal HGP in WT and F2KO mice were 19.0 mg/kg/min and 18.6 mg/kg/min, respectively. During the clamp, WT mice HGP was 8.6 mg/kg/min while F2KO mice HGP was 16.8 mg/kg/min. Suppression of HGP was 54% in WT mice and only 10% in F2KO mice. These results suggest that insulin resistance in the fat can reduce insulin sensitivity in liver and muscle.

Supported By: NIDDK

& 1682-PIdentifi cation of Insulin’s Novel Action on Endothelin β Receptor Expression and Ca2+- Calmodulin-eNOS Activation to Reduce AtherosclerosisKYOUNGMIN PARK, AKIRA MIMA, QIAN LI, CHRISTIAN RASK-MADSEN, PAUL L. HUANG, PING HE, GEORGE L. KING, Boston, MA, Morgantown, WV

Enhancing insulin’s signaling in the endothelial cells (EC) via the specifi c overexpression of IRS1 using VE-cadherin promoter (ECIRS1) greatly decreased atherosclerosis in ApoE-/- mice. By genomic array and immunoblot analysis, endothelin receptor β (ETBR) was identifi ed as being signifi cantly increased by the enhanced insulin action in the EC of ECIRS1 mice. Insulin increased ETBR expression by the activation of IRS1/PI3K/Akt pathway using gain and deletion techniques. Further, ETBR expressions (mRNA and protein) were signifi cantly decreased by 34±13% and 53±23% when comparing endothelium from ApoE-/- mice on western diet (WD) and high fat diet (HFD) mice to normal chow, which was prevented in ECIRS1 mice on HFD. Expression of ETBR mRNA was reduced by 63±23% in EC of the mammary artery from obese diabetic subjects vs. non-diabetic control. Insulin increased ETBR expression and NO production by more than 3.6 fold in cultured EC isolated from ECIRS1 mice vs. ApoE-/- mice as measured by time lapse confocal immunofl ourescent microscopy. Potential novel mechanism for insulin to activate eNOS independent of AKT phosphorylation of eNOS was confi rmed when insulin was still able to increase NO production by 2 fold in EC cultured from mice with specifi c knock in of eNOS with Ala replacing Ser at 1176 specifi cally in the EC. The enhanced insulin action on ETBR and NO in EC with Ala 1176 (eNOS) can be inhibited by ETBR antagonist, BQ-788 and the knock-down of ETBR using siRNA. Detailed studies on Ca++ uptake and NO production showed that the enhanced insulin action in EC from ECIRS1 mice was due to activation of ETBR- Ca++-calmodulin pathway to activate eNOS independent of its p-Ser 1176 of eNOS. This study provided the fi rst demonstration that insulin can activate eNOS through the ETBR/Ca++/calmodulin pathway independent of p-eNOS (1176) as a novel mechanism of insulin’s action to decrease atherosclerosis even in severe insulin resistant and diabetic states.

Supported By: ADA (7-12-MN-40); NIDDK (5R01DK05310513)

& 1683-PThe PRC1 Component, Bmi1, Regulates Insulin SensitivityCOREY E. CANNON, DAVID N. GROFF, MORRIS J. BIRNBAUM, DORIS A. STOF-FERS, Philadelphia, PA

Insulin resistance in peripheral tissues is a defi ning feature of type 2 diabetes. Bmi1, as part of the Polycomb Repressive Complex 1 (PRC1), is responsible for repression of the Ink4a/Arf locus, whose products are notable regulators of cell cycle progression. De-repression of this locus in Bmi1-/-

mice results in early and severe diabetes due to impaired beta cell cycling; however, early postnatal lethality prevents assessment of metabolic effects later in life. Bmi1+/- mice survive the postnatal period and, surprisingly, are protected from age-induced insulin resistance as measured by insulin tolerance test (P< 0.01) and hyperinsulinemic-euglycemic clamp, with no impact on weight gain or body composition. During the clamp, glucose infusion rate increased from 31.8±2.5 mg/kg/min to 66.5±6 mg/kg/min (p<0.01) and glucose disposal rate from 42.04±1.7 mg/kg/min to 70.23±5.6 mg/kg/min (p<0.01) in Bmi1+/+ compared to Bmi1+/- littermates, in association with a trend towards increased glucose uptake to muscle (180±27.1 nmol/g/min in Bmi1+/+ vs. 281.9±39.8 nmol/g/min in Bmi1+/-; p=0.06) but not adipose tissue. Bmi1+/- mice suppressed hepatic glucose production 77.5±2% in response to insulin as compared to 39.6±3.6% in Bmi1+/+ (p<0.001). Basal AKT phosphorylation was lower in Bmi1+/- liver and muscle tissue, as measured by western blot analysis. Consistent with increased insulin sensitivity, we observed that AKT is 4.1 fold more readily phosphorylated in response to insulin in Bmi1+/- liver and 2.1 fold more in muscle tissue. Our data defi ne a new role of Bmi1 in regulating pathways governing insulin sensitivity.

Supported By: NIDDK (R015R01DK068157 to D.A.S.)

& 1684-PRole of Aryl Hydrocarbon Receptor in Insulin Resistance and Metabolic DisordersCANXIN XU, CHUN WANG, KATHY BOTTUM, CASSIE JAEGER, STACEY KRAGER, SHELLEY TISCHKAU, Springfi eld, IL

Aryl hydrocarbon receptor (AhR), a basic helix-loop-helix-PAS domain containing nuclear receptor, plays an important role in large aspects of physiological and toxicological activities. Recent studies have shown that AhR is involved in xenobiotic-induced insulin resistance and diet-induced adiposity. However, the underlying mechanisms are largely unknown. This study is to demonstrate the effects of AhR status on insulin signaling, lipid metabolism and glucose homeostasis. AhR activation by its agonists induced IRS-1 protein degradation through ubiquitination pathway and attenuated insulin signaling pathway, and AhR knockdown alleviated AhR agonist-induced IRS-1 degradation and preserved insulin signaling. Interestingly, AhR heterozygous (AhR+/-) and AhR knockout (AhR-/-) male mice showed an elevated IRS-1 level with enhanced insulin signaling, which protected against high-fat diet (HF, 60% fat diet)-induced insulin resistance, adiposity, hepatic steatosis and infl ammation. Taken together, these data suggest that AhR activation induces IRS-1 degradation which leads to impair insulin signaling, on the other hand, AhR defi ciency and attenuation preserve insulin signaling which protects diet-induced insulin resistance and metabolic disorders. Therefore, AhR signaling pathway may provide a novel treatment option for metabolic disorders.

& 1685-PMitochondrial Fission Mediates Ceramide-induced Metabolic Disrup tion in Skeletal Muscle and Accumulates with Hyper insulin-emiaBENJAMIN T. BIKMAN, Provo, UT

Ceramide is a sphingolipid that serves as an important second messenger in an increasing number of stress-induced pathways. Ceramide has long been known to affect the mitochondria, altering both morphology and physiology. The purpose of this work is to identify novel regulators of muscle ceramide accrual and determine the impact of ceramide on skeletal muscle mitochondrial structure and function. Regarding ceramide metabolism, we found that induced hyperinsulinemia increases ceramide accrual in cultured myotubes and whole skeletal muscle. We also found that, functionally, ceramide induces a rapid and dramatic division of mitochondria in ceramide-treated cells. This effect is likely a result of increased Drp1 action, as ceramide increased Drp1 expression and Drp1 inhibition prevented ceramide-induced mitochondrial fi ssion. Further, we found that ceramide treatment reduced mitochondrial O2 consumption (i.e., respiration) in cultured myotubes and permeabilized red gastrocnemius muscle fi ber bundles. Ceramide treatment also increased H2O2 levels and reduced Akt/PKB phosphorylation in myotubes. However, inhibition of mitochondrial fi ssion via Drp1 knockdown completely protected the myotubes and fi ber bundles from ceramide-induced metabolic disruption, including maintained mitochondrial respiration, reduced H2O2 levels, and unaffected insulin signaling. Collectively, these data suggest that hyperinsulinemia from exogenous insulin treatments increases muscle ceramide, relevant for type 2 diabetes. Moreover, the forced and sustained mitochondrial fi ssion that results from ceramide accrual may alter metabolic function in skeletal muscle, which is a prominent site not only of energy demand (via the mitochondria), but also of ceramide accrual with weight gain.

& 1686-PThe PDE-4 Inhibitor Rofl umilast Reduces Weight Gain, Enhances Insulin Sensitivity, and Prevents Hepatic Steatosis in Mice by Increasing MitochondrogenesisFLORIAN KAHLES, JULIA MÖLLMANN, CHRISTER BÄCK, ANA LIBERMAN, KATHA-RINA HESS, HANNES M. FINDEISEN, STEFAN KRÜGER, CORINNA LEBHERZ, FRANK TACKE, NIKOLAUS MARX, MICHAEL LEHRKE, Aachen, Germany

Purpose: Rofl umilast is a new PDE-4 inhibitor with anti-infl ammatory properties which is clinically used to reduce the exacerbation of chronic bronchitis. Interestingly, rofl umilast was also found to reduce body weight and improve glucose metabolism of humans with yet elusive mechanism.

Methods and Results: We therefore treated C57B6 mice on western diet (n=15/group) for a period of 12 weeks with rofl umilast (added to the diet, 21mg/kg).

This treatment led to a signifi cant reduction in body weight (28 g (rofl umilast) vs. 32g (control), p<0.001 ) despite a trend to increase food consumption in the rofl umilast group. This was paralleled by a decrease

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in hepatic steatosis (50% cholesterol reduction; p<0.01; 30% triglyceride reduction; p<0.05) and hepatic macrophage infi ltration (10% F4/80 positive cells of CD45 positive cells in the rofl umilast group versus 20% in control; p<0.05). Rofl umilast treatment caused decreased plasma AST (38% reduction; p<0.05) and ALT (49% reduction ;p<0,05) levels compared to controls. In addition, rofl umilast ameliorated glucose tolerance (mean difference in glucose during glucose tolerance test of 100 mg/dl between groups at 30, 60 and 90 min; each p<0.01) and improved insulin sensitivity in vivo (mean glucose difference during insulin tolerance test of 80 mg/dl at 30, 60, 90 and 120 min; each p<0.001). This was confi rmed by an increased insulin signalling and AKT-Phosphorylation in liver and adipose tissue (p<0.05 in between groups). Furthermore rofl umilast increased the hepatic expression of the insulin independent glucose transporter GLUT1 (p<0.05) and of the transcriptional coactivator PGC1a (p<0.05) which is a master regulator of mitochondrogenesis leading to increased oxidative capacity.

Conclusions: PDE-4 inhibition holds promise as a new concept for body weight reduction and anti-diabetic therapy which can be attributed to increased mitochondrogenesis and oxidative capacity.

& 1687-PPeroxiredoxin6, a Novel Player in the Pathogenesis of Diabetes MellitusFRANCESCA PACIFICI, ROBERTO ARRIGA, GIAN PIO SORICE, BARBARA CAPUANI, DONATELLA PASTORE, ANDREA COPPOLA, SARA CARATELLI, ALFONSO BELLIA, GIULIA DONADEL, MASSIMO FEDERICI, PAOLO SBRACCIA, MANFREDI TESAURO, GIUSEPPE SCONOCCHIA, DAVID DELLA MORTE, ANDREA GIACCARI, AUGUSTO ORLANDI, DAVIDE LAURO, Rome, Italy

Oxidative stress is considered one of the main risk factor of type 2 diabetes (T2D). T2D develops when insulin resistance (IR) is associated to a defect of glucose mediated insulin secretion. The relationships between antioxidants enzymes and glucose homeostatis have been established, highlighting the important role of antioxidants systems. Furthermore, there are no data linking glucose homeostatis and peroxiredoxin 6 (PRDX6), a new antioxidant enzyme capable to neutralize peroxides and phospholipid hydroperoxides. The aim of this study is to determine the role of PRDX6 in the physiopathology of T2D using PRDX6 knockout (-/-) and wild type (wt) mice.

Glucose and insulin tolerance were evaluated respectively with intraperitoneal glucose and insulin tolerance test. Peripheral insulin sensitivity was analyzed by euglycemic hyperinsulinemic clamp. Insulin signaling at skeletal muscle level was studied by using Western blot analysis. Infl ammatory and lipids parameters were evaluated. Our data revealed that PRDX6-/- mice developed a phenotype similar to early phase of T2D with reduced glucose dependent insulin secretion and increased levels of IR. After stimulation with an infusion of insulin 1 U/kg into portal vein, we observed signifi cant alteration in insulin signaling which led to reduction of glucose uptake. The key molecule involved in this modifi cation is IRS1, which resulted less phosphorylated and activated in PRDX6-/- mice compared to wt. Morphological changes with a reduction in islets of Langerhans volume were observed of PRDX6-/- but not in wt. Serum concentrations of triglycerides, VLDL, HDL resulted altered with the presence of NASH defi ned with NASH score suggesting the presence of diabetic dyslpidemia in PRDX6-/- mice. We, also, observed a pro-infl ammatory status in PRDX6-/- mice which can contribute to the onset of DM.

For the fi rst time we demonstrated the involvement of PRDX6 in the physiopathology of T2D, opening interesting perspectives in fi nding new therapeutics options for DM care.

Supported By: Rome Foundation (PRIN2012)

1688-PDoes Carnosine Play a Role in Obesity Related Insulin Resistance in Humans?BARBORA DE COURTEN, MAXIMILIAN DE COURTEN, JOZEF UKROPEC, INGE EVERAERT, WIM DERAVE, BARBARA UKROPCOVA, Clayton, VIC, Australia, Copenhagen, Denmark, Bratislava, Slovakia, Ghent, Belgium

Carnosine is a naturally present dipeptide (mostly in the muscle) in humans and over-the counter available food additive. There is a growing body of evidence from animal studies for a role of carnosine supplementation in the prevention and treatment of diabetes. Mechanisms include reduction of chronic low-grade infl ammation, glycation and oxidative stress. Therefore, we studied a relationship between muscle carnosine and obesity/diabetes measures as there are currently no data available in humans.

We measured muscle carnosine levels from vastus lateralis muscle biopsies (HPLC), adiposity (DEXA), subcutaneous and visceral adipose tissue (MRI), insulin resistance (euglycaemic glucose clamp), blood pressure and

lipid profi le in 9 age matched lean men, 9 obese men, and 9 men with impaired glucose tolerance, as well as in 9 male patients with diet-only treated type 2 diabetes (age 45±7y; mean±SD, range of BMI 23-35 kg/m2).

Muscle carnosine levels increased with glucose intolerance (3.5±1.0 and 4.7±1.1 mmol/kg wet weight of skeletal muscle for lean and type 2 diabetics, respectively) and were positively related to % body fat (r=0.34, p=0.046), greater subcutaneous (r=0.38, p=0.02) and negatively related to plasma HDL (r=-0.34, p=0.048). There were no correlations with systolic and diastolic blood pressure (both p>0.3). Interestingly, there was a negative relationship with insulin sensitivity in non-diabetics (r=-0.53, p=0.005) but tendency of a positive relationship in diabetics (r=0.37, p=0.3, p=0.004 for interaction).

These fi rst human cross-sectional data suggest that muscle carnosine increases with increasing insulin resistance. This might be due to a compensation for increasing obesity and decreases then when the compensation fails in type 2 diabetics. Although our data is suggestive of the role of carnosine in the development of type 2 diabetes, only intervention studies targeting insulin resistance in people without diabetes can establish the role of carnosine in etiology of type 2 diabetes.

Supported By: Slovak Academy of Sciences

1689-PEffects of Metformin on Metabolic and Infl ammatory Insulin Signal-ing Pathways in Vascular Smooth Muscle CellEUGENIO CERSOSIMO, XIAOJING XU, CURTIS TRIPLITT, NICOLAS MUSI, San Antonio, TX

To examine the effects of metformin on metabolic & infl ammatory insulin signaling in vascular smooth muscle cells (VSMCs) we assessd the activities of PI-3 kinase (Akt) & AS160, MAP-kinase (Erk) & p38 and mTOR in human coronary artery VSMCs cultured in media with high glucose or palmitate. VSMCs were exposed to glucose (5 & 25mM) or palmitate (200 µM), ± metformin (1 mM) for 24 hrs followed by 20 min insulin treatment (100nM). After homogenization, the active (phosphorylated)/total protein ratio was calculated with Western blotting, expressed in arbitrary units (AU); viable cells were confi rmed with α-actin immunohistochemistry.

In VSMCs pre-treated with metformin, the insulin-induced 4-fold increase in p-Akt/Akt (0.6±0.1 vs. 2.5±0.3 AU) in 5mM of glucose (5G) was attenuated to a 2.5-fold increase (1.0±0.1 vs. 2.4±0.3 AU) in 25mM of glucose (25G) [p<0.05]; the rise in p-Akt/Akt in media containing palmitate (PAL) was also blunted (0.25±0.05 vs. 0.40±0.1) [p<0.05 vs. 5G]. Insulin-induced increase in p-AS160/AS160 was 1.5±0.2 vs. 2.5±0.3AU in 5G, 0.9±0.3 vs. 2.8±0.3AU in 25G and 1.5±0.2 vs. 3.5±0.3AU in PAL. Insulin induced a rise in p-Erk/Erk (1.1±0.2 vs. 1.6±0.2AU) at 5G, which was blunted by metformin in 25G (0.9±0.1 vs. 1.0±0.2AU) and PAL (2.2±0.3 vs. 2.5±0.2AU). While p-p38/p38 ratio increased with insulin from 2.2±0.3 vs. 2.8±0.3AU at 5G, it decreased at 25G from 2.0±0.3 to 0.9±0.1AU (p<0.001 vs. 5G) and remained the same at PAL (1.0±0.3 to 1.4±0.3AU, p<0.01 vs. 5G) with metformin pre-treatment. Insulin increased p-mTOR/mTOR at 5G (0.2±0.1 to 0.5±0.1AU ) and at 25G (0.5±0.2 to 0.7±0.1AU) in VSMCs pre-treated with metformin.

Conclusions: In VSMCs exposed to high glucose & palmitate metformin had little effect on the metabolic insulin signaling, whereas proliferative & infl ammatory signals were attenuated. These data suggest that metformin could reduce the atherogenic response of coronary artery VSMCs in insulin resistance conditions.

Supported By: Krokonsky Foundation

1690-PEndothelial Cell Insulin Resistance Promotes Intestinal Tumor Forma -tionXUANCHUN WANG, CHRISTIAN RASK-MADSEN, Boston, MA

Obesity and type 2 diabetes are associated with cancer of the colon and other organs. Although hyperinsulinemia is associated with cancer it is unkown whether insulin resistance in cancer cells or tumor stroma can itself affect tumor formation. Insulin resistance in endothelial cells is an important aspect of endothelial dysfunction in humans with obesity and type 2 diabetes and we have previously published that mice with endothelial cell knockout of the insulin receptor gene (Insr) have 4-fold increased leukocyte adhesion to endothelial cells because of endothelial cell upregulation of vascular cell adhesion molecule-1 (VCAM-1). Since infl ammation promotes tumor formation we examined whether these changes affects tumor susceptibility. We deleted the Insr gene in endothelial cells of mice with the multiple intestinal neoplasia mutation [Apc(Min/+)] using the cre/loxP system under control of the VE-cadherin/Cdh5 promoter. In these mice (Cdh5/M), insulin receptor protein (INSR) was undetectable in primary culture of endothelial cells isolated from intestinal mucosa from Cdh5/M

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mice but INSR was not different in bone marrow or circulating leukocytes between genotypes. There was no difference between glucose and insulin tolerance or plasma insulin in the two groups. Flow cytometry of single-cell suspensions derived from enzymatic digestion of the small intestine showed a 6.2-fold increase in CD45+ cells (leukocytes) in Cdh5/M mice compared to controls (p=0.007). This was explained by enlarged populations of CD11c+ F4/80- (dendritic) cells and T-cell receptor-β+ cells (T-cells). The number of tumors in the small intestine was increased by 52% (76±6 and 50±6 tumors in Cdh5/M and controls, respectively, p=0.01). We conclude that insulin resistance in endothelial cells promotes intestinal tumor formation, likely by recruiting immune cells to intestinal tissue. Improving endothelial cell insulin resistance may reduce cancer risk in obesity and type 2 diabetes.

1691-PCaveolin-1 Phosphorylation Is Required for Vascular Endothelial Insulin Uptake and Impaired by Insulin ResistanceHONG WANG, AILEEN X. WANG, KEVIN AYLOR, EUGENE J. BARRETT, Charlottes-ville, VA

Caveolae mediate trans-endothelial transport (TET) of proteins including insulin. As insulin entry to muscle interstitium is rate-limiting for its metabolic action, defi ning the route and regulation of its entry is critical. We previously reported that inhibiting insulin signaling by wortmannin or PD98059 or inhibition of Src by PP1 or treatment with TNFα or IL6 signifi cantly impaired insulin uptake by bovine aortic endothelial cells (bAECs). Here we examined whether insulin-stimulated tyrosine (Tyr) 14 phosphorylation of caveolin-1(CAV1), a structural protein of caveolae, was required for the fi rst step of insulin TET, its uptake by vascular endothelial cells (ECs). We also tested whether in vivo high-fat diet (HFD)-induced insulin resistance affected CAV1 Tyr14 phosphorylation and insulin uptake by rat ECs. Insulin stimulated CAV1 Tyr14 phosphorylation in both bAECs and freshly isolated rat ECs. Insulin also activated Src by phosphorylation at Tyr416. In bAECs inhibiting either Src or Tyr phosphorylation inhibited both basal and insulin-stimulated EC CAV1 Tyr14 phosphorylation. Inhibiting MAPK but not PI3-K, or exposure of bAECs to TNFα or IL6 decreased while inhibiting protein tyrosine phosphatase 1B increased insulin-stimulated CAV1 Tyr14 phosphorylation. Overexpressing Flag-tagged CAV1Y14F inhibited while overexpressing wild type Flag-tagged CAV1 increased both insulin-stimulated CAV1 Tyr14 phosphorylation and bAEC fl uoroisothiocyanate (FITC)-labeled insulin uptake. Feeding rats HFD for 1 week eliminated insulin-stimulated rat EC CAV1 Tyr14 phosphorylation and inhibited EC FITC-insulin uptake by ~50%. Finally, inhibition of Src inhibited CAV1 Tyr14 phosphorylation and FITC-insulin uptake by freshly isolated rat ECs. We conclude that insulin facilitates its own uptake by ECs through stimulating Src-dependent CAV1 Tyr14 phosphorylation and both HFD in vivo and pro-infl ammatory cytokines in vitro inhibit this process.

Supported By: ADA (1-11-BS-06); NIH (DK057878, DK073059)

1692-PMethyl Donor Defi ciency Due to Low Vitamin B12 Impairs Insulin Signalling in Human Adipocytes by Down Regulating Akt Pathway via PTEN and TRB3ANTONYSUNIL ADAIKALAKOTESWARI, PHILIP VOYAIS, SUDHESH KUMAR, PHILIP G. MCTERNAN, PONNUSAMY SARAVANAN, GYANENDRA TRIPATHI, Coventry, United Kingdom, Warwick, United Kingdom

Vitamin B12 and folate are needed for the synthesis of methionine, which is the precursor of S-adenosylmethionine, a key methyl donor for DNA methylation. Data from human longitudinal studies and animal models suggest a causative role for B12 and folate in the development of insulin resistance, potentially mediated by adiposity. We therefore investigated the role of B12 in insulin signalling pathway and its underlying molecular mechanisms in human adipocytes. Primary human pre-adipocytes were differentiated in various B12 concentrations (1) Control: (B12-500nM); (2) LowB12 (0.15nM) (3) NoB12: (B12-0nM). mRNA and protein expression were characterized by qRT-PCR and western blotting, respectively. Glucose uptake using 2deoxy-D-[3H] glucose was measured by scintillation counting. Adipocytes cultured in B12 defi cient conditions showed reduced basal and insulin stimulated glucose uptake (p<0.05). mRNA expression of insulin receptor substrate (IRS1,2) and protein expression of phospho-Aktser473 (p<0.001) were down regulated. Downstream targets of Akt such as phospho-GSK3-β (Glycogen synthase kinase) and phospho-FOXO1 (Forkhead box protein O1) were down regulated and phospho-S6K (p70-S6 kinase) was upregulated (p<0.001). The negative regulators of Akt, PTEN (phosphatase and tensin homologue deleted on chromosome 10) and TRB3 (mammalian homolog of drosophila tribbles) were upregulated (p<0.05). Adipocytes treated with PTEN inhibitor (bpV(phen)) or transfected with Trb3 siRNA alleviated Akt inhibition and

restored insulin stimulated glucose uptake (p<0.01). Our study provides evidence that adipocytes cultured in B12 defi cient conditions develop insulin resistance due to impaired Akt signalling cascade, which appears partly through activation of PTEN/Trb3 and subsequent inhibition of Akt. This link between methyl donor defi ciency and insulin resistance opens new insights into the pathogenesis of metabolic disorders.

Supported By: George Eliot NHS Trust; UHCW NHS Trust

1693-PLoss of Insulin Signaling in Intestinal Epithelial Cells Impairs Glucose Transport but Does Not Protect Against Tumor FormationMAX-FELIX HAERING, SIEGFRIED USSAR, C. RONALD KAHN, CHRISTIAN RASK-MADSEN, Boston, MA

Both obesity and type 2 diabetes are associated with increased risk of cancer. It has been proposed that this link may be partly explained by hyperinsulinemia, which is a risk factor for colorectal cancer and other malignancies. Our aim was to determine whether insulin action promotes intestinal tumor formation. Mice with deletion of the insulin receptor gene in intestinal epithelial cells were generated using the cre/loxP system under control of the villin-1 promoter (VILIRKO mice). In isolated intestinal epithelium from VILIRKO mice, 2-deoxyglucose uptake was reduced by 53% compared to controls. As a result, glucose tolerance in VILIRKO mice was better than in controls. To study tumor formation we introduced the multiple intestinal neoplasia mutation [Apc(Min/+)] in VILIRKO mice by breeding. In these animals [VILIRKO/Apc(Min/+)] we expected a decrease in tumor formation because insulin can promote proliferation and because increased glucose uptake in tumor cells has been linked to increased glycolytic fl ux, augmented anabolic metabolism, and increased tumor growth. Surprisingly, we found no change in tumor number or size in the small intestine at 16 weeks of age (31±3 and 27±3 tumors in VILIRKO/Apc(Min/+) mice versus controls, respectively). When fed a high-fat diet for 12 weeks, animals in the two groups became equally hyperinsulinemic. However, tumor number did not change (31±3 and 25±3 tumors, respectively). In the colon, tumor number was no different between genotype or diet groups. Indices of tumor proliferation (Ki67 immunostaining) and markers of infl ammation (TNF-α, CD68, or IL-6 mRNA) were also similar between groups. We conclude that insulin signaling in intestinal epithelial cells has an important role for glucose transport and whole-body glucose homeostasis. However, loss of insulin signaling in intestinal epithelium has no effect on tumor formation, indicating that the link between hyperinsulinemia and cancer risk may be indirect.

1694-PEffects of Varying Degrees of Intermittent Hypoxia on Pro infl am-matory Cytokines and Adipokines in Rats and 3T3-L1 AdipocytesQING HE, QINGCHAN YANG, WENYAN NIU, JING FENG, YAN WANG, BAOYUAN CHEN, Tianjin, China

Obstructive sleep apnea (OSA) is a common condition characterized by repeated episodes of upper airway obstruction. Intermittent hypoxia (IH), resulted from recurring episodes of upper airway obstruction, the hallmark feature and the most important pathophysiologic pathway of OSA, is believed to be the most important factor causing systemic infl ammation which may play the key role in the development and progression of insulin resistance (IR). We hypothesized that the recurrence of IH might result in cellular and systemic infl ammation, which was manifested through the levels of proinfl ammatory cytokines and adipokines after IH exposure, and because IR and glucose intolerance are linked with infl ammation tightly, this infl ammatory situation may implicate an IR status.

We developed an IH 3T3-L1 adipocyte and rat model respectively, recapitulating the nocturnal oxygen profi le in OSA. In animal model, arterial blood gas, IL-6, TNF-α, adiponectin, leptin, blood glucose and insulin levels were analyzed. In IH cells, NF-κB DNA binding reactions, HIF-1α, glucose transporter-1, IL -6, TNF-α, adiponectin, leptin mRNA transcriptional activities and protein expressions were measured.

When rats in different group were exposed to different levels of hypoxia situation, the minimum PO2 was dramatically reduced. At the end of 8-week treatment, both insulin and blood glucose levels in RIH-1 group and both Glut-1 and NF-κB DNA binding activities in CIH-1 group were the highest. In cellular and animal models, the mRNA and protein levels of TNFα, IL-6 and leptin were the highest in CIH-1/RIH-1 group while the lowest for adiponectin.

The results from this study shows that oxidative stress and the release of pro-infl ammatory cytokines/adipokines are associated with IH closely and are proportional to the severity of IH. Our results may implicate the clinical

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relationships between OSA and IR, as IR and glucose intolerance are linked with infl ammation.

Supported By: NSFC

1695-PMuscle-specifi c TRIB3 Overexpression Produces Weight Gain and Insulin Resistance in Mice, and Exacerbates Glucose-induced Insulin Resistance in DiabetesWEI ZHANG, RAVI H. JARIWALA, TEAYOUN KIM, MENGRUI WU, W. JOHN GARVEY, LING TIAN, DENNIS STEVERSON, QINGLIN YANG, YUCHANG FU, W. TIMOTHY GARVEY, Birmingham, AL

A component of insulin resistance in diabetes is induced by hyperglycemia itself (glucose toxicity). We previously showed in vitro that glucose-induced insulin resistance (GIIR) was dependent upon TRIB3 and involved glucose metabolism via the hexosamine biosynthetic pathway. We also reported in mice that TRIB3 expression was altered during short-term nutrient excess and fasting, supporting its role as a nutrient sensor and metabolic regulator. In the current study, we investigated the In Vivo impact of TRIB3 on GIIR and systemic metabolism in Muscle-Specifi c TRIB3 Overexpressing (MOE) mice. When compared with wild type (WT), MOE exhibited abnormal expression of genes regulating metabolism in response to short-term fasting and re-feeding, indicative of abnormal fuel metabolism. With 16 weeks’ high fat feeding, MOE developed higher body weight (46.5±3.1 vs. 40.9±3.5g: p<.05) with signifi cantly impaired glucose tolerance (2-hr GTT glucose: 248±27 vs. 191±19 mg/dl: p<.01) when compared with WT. Insulin resistance in MOE was accompanied by decreased AKT phosphorylation in muscle. To determine the role of TRIB3 in GIIR, hyperglycemia was induced by streptozotocin. Serial insulin tolerance tests demonstrated that MOE mice developed a greater degree of insulin resistance than WT after 3 weeks’ exposure to hyperglycemia (1-hr ITT glucose: 137±26 vs. 90±6 mg/dl: p<.01), which was accompanied by a signifi cant decrease in insulin-stimulated glucose oxidation in muscle (P<0.01). In conclusion, our results indicate that: 1) Muscle-specifi c overexpression of TRIB3 impairs the molecular regulation of fuel metabolism and results in greater weight gain and insulin resistance; 2) Muscle TRIB3 overexpression exacerbates insulin resistance during hyperglycemia, supporting TRIB3 as a critical facilitator of glucose-induced insulin resistance in vivo and pointing to TRIB3 as a novel target for treatment of glucose toxicity.

Supported By: ADA (1-13-IN-19); U.S. Dept. of Veterans Affairs; University of Alabama, Birmingham (P30-DK079626); NIH (DK-038765, DK-083562)

1696-PInsulin Signaling Attenuates Autophagic Flux via an Akt-dependent but mTOR Complex 1-independent Mechanism in CardiomyocytesQUAN-JIANG ZHANG, YI ZHU, DEBORAH JONES, E. DALE ABEL, Iowa City, IA, Salt Lake City, UT

Altered autophagy may contribute to diabetic cardiomyopathy. To investigate mechanisms by which insulin signaling regulates autophagic fl ux (AF) in cardiomyocytes (CMs), H9C2 CMs were starved for 2 hrs in nutrient defi cient medium (ND, glucose-free Hank’s Balanced Salt Solution + 0.5 mM pyruvate), and insulin (Ins-100 nM) was added for 1 hr after 1hr starvation. Akt1/2 kinase (Akti-2uM) and ERK1/2 inhibitors (PD0325901-2 uM), and Rapamycin (Rap-15 ng/ml) were added to the ND medium, respectively. mTOR was also silenced by siRNA, and CMs starved for 2 hr ± Ins. Mice with inducible cardiac-specifi c mTOR defi ciency were fasted for 24 hrs and hyperinsulinemic euglycemic clamps performed. Under ND conditions, AF was induced in H9C2 CMs as evidenced by increased LC3-II (1.00+/- 0.06 in nutrient replete (NR) vs. 2.40±0.19 in ND, P <0.01), and was reduced by Ins (1.37±0.11 vs. ND, P<0.05). ERK inhibition had no effect on basal AF in NR or ND conditions, nor did it infl uence the ability of insulin to suppress AF. By contrast, inactivation of Akt with Akti, increased basal AF in NR conditions (1.71±0.26 vs. NR, P<0.05) and ND-initiated AF was not further augmented. Akti, blocked the attenuation of ND-initiated AF by Ins . Inhibition of mTORC1 with Rap, modestly activated AF in NR conditions, but did not further augment ND-initiated AF. Importantly, Ins repressed ND-initiated AF in the presence of Rap (1.79±0.16 vs. 2.81±0.34 in ND+Rap, P<0.05), which was recapitulated in CMs with siRNA silencing of mTOR (1.35±0.12 vs. 2.63±0.08 ND+mTOR siRNA, P<0.05). Fasting markedly induced AF in mouse hearts evidenced by increased LC3-II (1.00±0.27 in fed hearts vs. 5.77±0.06 in fasted hearts, P<0.01). Hyperinsulinemic clamps signifi cantly attenuated AF in WT (1.92±0.18 vs. fasted hearts, P<0.05), and mTOR defi cient hearts (1.81±0.13 vs. fasted hearts, P<0.05). These data suggest that insulin suppresses autophagy in CMs via an Akt-dependent but mTORC1-independent mechanism.

Supported By: NIH; AHA

1697-PDietary Restriction of Branched Chain Amino Acids Improves Insulin Sensitivity in Zucker-Obese Rats via Reversal of Mitochondrial OverloadPHILLIP J. WHITE, AMANDA L. LAPWORTH, JIE AN, LIPING WANG, OLGA ILKAYEVA, ROBERT D. STEVENS, CHRISTOPHER B. NEWGARD, Durham, NC

Comprehensive metabolic profi ling of clinical samples has uncovered a branched-chain amino acid (BCAA)-related metabolic signature that is associated with insulin resistance and predictive of incident diabetes and intervention outcomes. However, the role of this metabolite cluster in the pathogenesis of diabetes remains poorly understood. We evaluated the impact of dietary BCAA restriction on insulin sensitivity and metabolic profi les in Zucker-obese rats, which like humans exhibit a spontaneous elevation of BCAA and related metabolites relative to Zucker-lean controls. Zucker-obese and Zucker-lean rats were fed standard chow (SC), or an isonitrogenous SC with 45% restriction in BCAA for 15 weeks. BCAA restriction lowered circulating BCAA in Zucker-obese rats to the levels of Zucker-lean rats. Zucker-obese rats have striking depletion of glycine and arginine levels in skeletal muscle relative to Zucker-lean animals, and feeding of the BCAA restricted diet restored Gly and Arg to normal. Zucker-obese animals also exhibited a dramatic accumulation of medium and long chain fatty acyl CoA species in skeletal muscle relative to lean animals, and these levels were normalized by feeding of the BCAA-restricted diet. BCAA restriction also caused a clear improvement in insulin sensitivity in Zucker-obese rats, as measured by hyperinsulinemic-isoglycemic clamp, and by increased rates of glucose uptake in muscle and liver. These data support a model wherein elevated circulating BCAA contribute to development of obesity-associated insulin resistance via interference with effi cient lipid oxidation.

Supported By: NIH (P01DK58398); Pfi zer, Inc.

1698-PReceptor for Advanced Glycation End Products Is a Negative Regulator of Glucagon ProductionBROOKE E. HARCOURT, CHRISTOPHER LEUNG, DANIELLE J. BORG, DOMENICA A. MCCARTHY, LISA TOM, SOFIANOS ANDRIKOPOULOS, TIMOTHY J. KIEFFER, JOSEPHINE M. FORBES, Woolloongabba, Australia, Melbourne, Australia, Heidelberg VIC, Australia, Vancouver, BC, Canada

Type 2 diabetes is associated with enhanced hepatic gluconeogenesis exacerbating hyperglycaemia as a result of increased production and release of glucagon by pancreatic alpha cells. We recently identifi ed a nuclear isoform of the receptor for advanced glycation end products (RAGE), with DNA regulatory capacity to bind consensus sequences in the promoter region of the preproglucagon gene. This project investigated the role of RAGE in the transcriptional control of preproglucagon.

Male wild-type (WT) and RAGE defi cient (RAGE-/-) mice were fed chow or a high fat diet for 16 weeks. RAGE-/- gained more body weight and % adipose tissue than corresponding WT mice (WT Control Δ 2.9 (±2.1) vs. RAGE-/- Chow (Δ 8.3 (±2.1) p<0.05, WT HFF Δ 11.0 (±21.6) vs. RAGE-/- HFF (Δ 15.4 (±2.2) p<0.05,). Fasting plasma glucose and insulin concentrations were increased in RAGE -/- mice and were exacerbated by obesity as a result of high fat feeding. Insulin tolerance testing (i.pITT) revealed RAGE-/- had increased plasma glucose concentrations. Oral and intraperitoneal glucose tolerance testing revealed glucose intolerance and hyperinsulinaemia as well hyperglucagonemia in RAGE-/- mice (AUC Glucagon, WT Chow, 742.6 (± 0.8) v AUC RAGE-/- Control, 828.7 (±0.7), p<0.01). Plasma lactate concentrations during i.pGTT were elevated in RAGE-/- mice (AUC Lactate, WT Chow, 41.9 (±0.5) v AUC WT HFF 53.3 (±0.7) p<0.05). RAGE expression in pancreatic islet alpha cells was decreased following high fat feeding. Nuclear RAGE binding activity in the promoter region of the preproglucagon gene was decreased with high fat feeding and obesity in the context of hyperglucagonaemia, suggesting negative regulation of the preproglucagon gene by RAGE binding.

Loss of RAGE expression in pancreatic islet alpha cells perturbed hyperglucagonaemia in obesity by impairing homeostatic mechanisms to suppress preproglucagon expression. RAGE may be an important regulatory protein for alpha cell glucagon production and hepatic gluconeogenesis.

1699-PErythropoietin and Glucose Metabolism in Differentiating Erythroid Progenitor CellsHEATHER M. ROGERS, MAWADDA ALNAEELI, RUIFENG TENG, CONSTANCE TOM NOGUCHI, Bethesda, MD, Zanesville, OH

Erythropoietin (Epo) is known for its erythropoietic activity. We previously showed that Epo treatment in wild type C57Bl/6 mice increased hematocrit

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accompanied by decreased fasting blood glucose levels, glucose tolerance improvement and fat mass reduction. This raises the possibility that Epo stimulated erythropoiesis increases glucose uptake and contributes to decreased blood glucose. Using cultures of primary human erythroid progenitor cells, we found that Epo stimulation increased glucose uptake in a dose dependent manner (1 - 10 U/ml) with erythroid differentiation, peaking at day 8-10 and decreasing with terminal differentiation. Glut1 followed EpoR expression and the pattern of glucose uptake, peaking when EpoR expression and Epo response were maximal, and decreasing with terminal differentiation. This indicates that Epo stimulation regulates glucose uptake and Glut1 expression in erythroid progenitor cells, dependent on the stage of differentiation. Interestingly, Epo treatment in mice with EpoR restricted to hematopoietic tissue showed the expected increase in hematocrit, but little or no change in blood glucose level or improvement in glucose tolerance, indicating that the marked decrease in fasting blood glucose with Epo treatment in WT mice is due largely to specifi c Epo response in non-hematopoietic tissue. However, further examination of glucose level on Epo response revealed that high glucose concentration inhibited erythroid progenitor cell proliferation during differentiation, but increased hemoglobinization, indicating premature hemoglobin accumulation and differentiation. The maximal peak of EpoR and Glut1 expression decreased with high glucose concentration. This response of erythroid progenitor cells to elevated glucose suggests that elevated blood glucose concentration contributes to a blunted Epo response leading to Epo resistance. These fi ndings provide us with new insight on the interplay between erythropoiesis and glucose metabolism.

1700-PThe Regulation of Wnt Signaling Pathways by Insulin and Free Fatty Acids in Skeletal Muscle and Adipose Tissue of Healthy HumansMONIKA KARCZEWSKA-KUPCZEWSKA, AGNIESZKA NIKOLAJUK, MAGDALENA STEFANOWICZ, NATALIA MATULEWICZ, MAREK STRACZKOWSKI, Bialystok, Poland, Olsztyn, Poland, Białystok, Poland

The Wnt signaling plays an important role in embryonic development, cell proliferation, cell cycle, differentiation, apoptosis and tissue homeostasis. The Wnt ligands bind to frizzled (Fzd) receptors and activate canonical (β-catenin dependent) and non-canonical (β-catenin independent) pathways. Dysregulation of Wnt signal transduction is associated with many pathophysiological states including metabolic disorders. The aim of the present study was to assess skeletal muscle and adipose tissue expression of genes associated with Wnt signaling pathways in young healthy population, their regulation by hyperinsulinemia and serum free fatty acids elevation and their relationship with insulin sensitivity. We studied 20 healthy male subjects (mean age 25.20±3.15 years, mean BMI 26.47±4.64 kg/m2). The biopsies of vastus lateralis muscle and subcutaneous adipose tissue were performed at baseline and after 6 hours of euglycemic hyperinsulinemic clamp with or without Intralipid/heparin infusion. The participants were divided into subgroups of high (high-IS) and low insulin sensitivity (low-IS). The muscle mRNA expression of β-catenin, Fzd coreceptor- LDL receptor-related protein 6 (Lrp6), Dishevelled 2 (Dsh2) and adipose tissue mRNA expression of Lrp6 were higher in low-IS, whereas adipose tissue mRNA of Dsh2 and glycogen synthase kinase 3 β (GSK3β) were lower in low-IS. Hyperinsulinemia resulted in a decrease in muscle and adipose tissue Lrp6, adipose tissue c-Myc and transcription factor 7 - like 2 (TCF7L2) expression and an increase in muscle and adipose tissue Dvl1/2/3 expression. Most of these changes disappeared after free fatty acids elevation and adipose tissue c-Myc expression was even up-regulated by free fatty acids. Our data indicate that Wnt canonical signaling is increased in insulin resistant subjects and insulin could inhibit β-catenin dependent pathway and mediate switching between canonical and non-canonical pathways.

Supported By: Program Innovative Economy (UDA-POIG.01.03.01-00-128/08)

1701-PVascular Mitochondrial Adaptations to Nutrient Stress in DiabetesKATE GEARY, AMY KELLER, LESLIE A. KNAUB, PENN MCCLATCHEY, JANE REUSCH, Denver, CO, Aurora, CO

Defi ning the mechanisms leading to accelerated cardiovascular disease in DM is crucial. Mitochondrial dysfunction in the vasculature leads to smooth muscle cell (VSMC) proliferation, enhanced migration, and impaired vasoreactivity. Optimally regulated mitochondrial dynamics are needed to respond to metabolic stress. We hypothesized that DM causes disequilibrium of mitochondrial fi ssion and fusion leading to cellular dysfunction. Primary VSMC from DM (Goto Kakizaki-GK) and control (Wistar-W) rats were tested. VSMC were incubated in glucose 5mM or 25 mM (high glucose-HG) for 1, 4

or 24 hrs. We evaluated fi ssion (Fis1, Drp1) and fusion (Opa1, Mfn1, Mfn2) proteins, mitochondrial content and structure, ROS, respiration, membrane potential (Ψm), and transition pore (mPTP) opening. Relative to W, GK exhibited a signifi cant ↓ in VDAC1 (p<0.05), altered respiration (OROBOROS O2K), and ↑ basal H2O2 production. Baseline PGC-1a was reduced (p<0.05) and preliminary data shows ↓ Mfn2. At 4 hrs, W signifi cantly increased Mfn1 (p<0.05), DRP1 (p<0.05) and superoxide (p<0.05), suggesting a coupled fi ssion and fusion response. GK demonstrated discordant changes in fi ssion and fusion with ↓ Opa1 (p<0.05) and MFN1 (p<0.05) but ↑ fi ssion with time (p<0.05) until 24 hrs when Fis1 signifi cantly decreased (p<0.05). GK superoxide did not change. HG led to a hyperpolarized Ψm and a closed mPTP state in GK but depolarized Ψm and ↑ mPTP opening in W. Quantitative confocal imaging revealed an acute fusion response after 1 hr HG exposure in the GK and W with loss of mitochondrial mass in GK at 4 hrs. In summary, GK VSMC in-vitro exhibit a defi nitive change in mitochondrial phenotype at baseline that is exacerbated by HG stress. HG provokes different alterations in the balance of fi ssion and fusion in GK vs. W. Membrane hyperpolarization and impaired mPTP opening pose a plausible mechanism driving mitochondrial responses in DM vasculature.

Supported By: U.S. Dept. of Veterans Affairs

1702-PNew Pathway of the Renin-Angiotensin System as a New Diabetes TargetJIN-KUI YANG, CHANG LIU, Beijing, China

It is now apparent that blocking the renin-angiotensin system (RAS) has important and direct effects on the development of islet cell dysfunction associated with type 2 diabetes mellitus (T2DM). This system has been extended recently by the addition of a novel axis consisting of the angiotensin-converting enzyme 2 (ACE2), the heptapeptide angiotensin-(1-7) (Ang-(1-7)), and the G-protein-coupled receptor, Mas. The new pathway, ACE2/Ang-(1-7)/Mas, acts as a negative regulator of the RAS. Interestingly, a functional receptor for the severe acute respiratory syndrome (SARS) coronavirus is ACE2. We found that ACE2 expression in the endocrine tissues of the pancreas suggests that SARS virus may damage islet cells and cause acute insulin-dependent diabetes. ACE2-knockout mice displayed a selective decrease in fi rst-phase insulin secretion, as well as a progressive impairment of glucose tolerance with age. Furthermore, we reported that the interactions among RAS-related genes were associated with T2DM. Recently, we found that ACE2/Ang-(1-7)/Mas axis of the RAS can protect against oxidative stress, increase insulin secretion of islet β-cells and improve insulin resistance (Fig.1). In conclusion, The ACE2/Ang-(1-7)/Mas axis may play an important role in preventing T2DM and may become a new target for T2DM therapeutics.

Supported By: NSFC (81200641, 81270918, 81070644)

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INSULIN ACTION—GLUCOSE TRANSPORT AND INSULIN RESISTANCE IN VITRO


1703-PTrace Fasting Glucose Fluxes with Unstressed Catheter Approach as Platform in Type 1 Diabetic RatsYINGLI LU, SHICHUN DU, HUI WU, XIAO XU, Shanghai, China

Blood glucose concentrations of type 1 diabetic rats are vulnerable, especially to stress and trauma. The present study aimed to investigate the fasting endogenous glucose production and skeletal muscle uptake of STZ-induced type 1 diabetic rats by infusion of [6, 6-2H2] glucose and [1-14C]-2-deoxy-glucose as isotope tracers using an unstressed vein and artery implantation of catheters at the tails of the rats as a platform. Streptozotocin (STZ, 65 mg/kg) were administered to the rats at about 10 weeks old. Physiological parameters such as body weight, food and water intake along with plasma glucose and insulin were monitored. The approach of catheters of vein and artery at the tails of the rats was established before the isotope tracer injection. Dynamic measurement of fasting endogenous glucose production was assessed by continuously injecting stable isotope [6, 6-2H2] glucose, while skeletal muscle glucose uptake by bolus injecting radioactively labeled [1-14C]-2-deoxy-glucose. STZ induced type 1 diabetic rats displayed polydipsia, polyphagia, polyuria along with overt hyperglycemia and hypoinsulinemia. In parallel, the STZ induced type 1 diabetic rats also had enhanced fasting endogenous glucose production (121 ± 15 vs. 42 ± 5 µmol/kg/min, P < 0.01) and reduced glucose uptake in skeletal muscle (0.11 ± 0.01 vs. 0.25 ± 0.05 µmol/g/min, P < 0.01) compared to non-diabetic rats. These data demonstrate that STZ-induced type 1 diabetic rats display the classic hyperglycemia traits. The dual catheters implantation at the tails of the rats together with isotope tracers injection is a save time, unstressed and feasible approach to explore the glucose metabolism in animal models in vivo.

1704-PRestructuring Intestinal Flora with Sleeve Gastrectomy in Diabetic RatsXIAOFEI HUANG, PAN WENG, HUIXIN ZHANG, YINGLI LU, Shanghai, China

As a complicated symbiotic system, intestinal fl ora is closely related to human health, as well as the balance of metabolism and energy, even it leads to the development of type 2 diabetes if it disorders. Sleeve gastrectomy is one of approaches of bariatric surgery, and could improve blood glucose control in type 2 diabetes patients. This study was to explore whether restructuring intestinal fl ora after sleeve gastrectomy in diabetic rats. 10 male GK diabetic rats were operated, 5 of them underwent sleeve gastrectomy (SG group, N=5), and 5 of them underwent sham operation (SO group, N=5). Meanwhile 10 male SD rats also were done sleeve gastrectomy as control. The animals’ weight and FBG had measured regularly. Fecal samples were collected then amplifi cated intestinal fl ora 16S rDNA, the composition changes of intestinal fl ora were detected by DNA sequence analysis. In diabetes GK rats, weight, fasting blood glucose decreased signifi cantly one month after sleeve gastrectomy compared with the sham operation group(P <0.05). However, there was no signifi cant change for weight and blood glucose in normal SD rats post-operation. The intestinal fl ora of GK rats reduced in the proportion of Firmicutes and increased in the proportion of Bacteroidetes after sleeve gastrectomy. Sleeve gastrectomy decreased blood glucose level in diabetes rats obviously as well as altered the composition of the intestinal fl ora. The change of type of dominant microorganisms in intestinal fl ora might play an important role in the metabolism homeostasis of glucose.

1705-PTrace Glucose Homeostasis with Sleeve Gastrectomy in Non-Diabetic and Diabetic RatsPAN WENG, ZHEN CANG, CHAOXIA ZHU, YINGLI LU, Shanghai, China

To observe the effect of sleeve gastrectomy on food intake, body weight, glucose homeostasis by isotope tracer and alteration of GLP-1 in non-diabetic and diabetic Rats. 12 fi ve months-aged male GK rats with glucose (15.16 ± 0.23 mmol/L) and 12 fi ve months-aged male SD rats with glucose (5.6 ± 0.14 mmol/L) ,each were divided into 2 groups: surgical group (GK-SG, N=6; SD-SG, N=6) with sleeve gastrectomy and sham surgery group (GK-sham, N=6; SD-sham, N=6). Monitor the food intake, body weight and blood glucose in each group. At one week before and one month after the operation,trace the rate of glucose appearance, the rate of glycerol appearance and gluconeogenesis (GNG) with infusion of 3-3H-glucose and U-13C-glycerol. Serum GLP-1 levels were measured by radioimmunoassay. The weight of GK-SG group was lower than that of the GK-sham group and food take showed obvious decrease (17.23 ± 2.5 vs. 24.17 ± 3.6g); Fasting and random glucose

in GK-SG group was taken reduce signifi cantly after operation; The rate of glucose appearance, glycerol appearance and gluconeogenesis in GK-SG group had been showed a decrease signifi cantly p<0.05 compared GK-sham group; GLP-1 levels in GK-SG group increased; There were no signifi cant differences between SD-SG and SD-sham group in body weight, blood glucose, food intake, the rate of glucose appearance, glycerol appearance and gluconeogenesis after sleeve gastrectomy. Sleeve gastrectomy could control food intake and weight gain, it also inhibit the gluconeogenesis and increase GLP-1 levels to improve hyperglycemia for diabetic GK rats.

1706-PGLP-1 Improves Palmitate-induced Insulin Resistance of Human Skeletal Muscle Myotubes through SIRT1JA YOUNG JEON, EUN SUK HA, SUNG-E CHOI, TAE HO KIM, SO-YEON AHN, SO YOUNG OCK, SEUNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, YUP KANG, KWAN WOO LEE, Suwon, Republic of Korea, Incheon, Republic of Korea

The target molecules for diabetes treatment have been identifi ed. Glucagon like peptide-1 (GLP-1) is known to have a potent glucose-dependent insulinotropic action on the pancreas as well as extrapancreatic actions. SIRT1, as an NAD-dependent deacetylase, has been demonstrated in the role of protecting ageing-related diseases and benefi cial effects of metabolic homeostasis. We investigated whether the actions of GLP-1 were mediated by SIRT1 activation in skeletal muscle cells under palmitate induced-insulin resistance. After confi rming the action of GLP-1 to palmitate-induced insulin resistance, we determined 2-NDBG uptake, GLUT4 mRNA, and the phosphorylation levels of protein related to insulin signal pathway in human skeletal muscle myotubes (HSMM) exposed to palmitate and compared to those in HSMM exposed to palmitate and GLP-1 simultaneously. To elucidate whether SIRT1 contributed to GLP-1 action in the palmitate-induced insulin resistance, we compared the levels of proliferator-activated receptor-γ-co-activator 1α (PGC1α) deacetylation in HSMM exposed to palmitate and GLP-1 for 24 hours. Moreover, when exposed to SIRT1 inhibitor (EX527) in the same conditions, we demonstrated the changes of GLUT4 mRNA expression and insulin signaling. GLP-1 restored the reduction of glucose uptake, GLUT4 mRNA levels, and GLUT4 promoter activity by palmitate in human skeletal muscle myotubes. GLP-1 reduced acetylation of PGC1α in HSMMs over expressed PGC1α. This result suggested that GLP-1 activated a deacetylase, such as SIRT1. SIRT1 inhibitor suppressed GLUT4 mRNA expression by GLP-1 in HSMMs. SIRT1 inhibitor prevented phosphorylation of protein related to insulin signal pathways by GLP-1 in HSMMs. This suggested the expression of GLUT4 mRNA and the activation of insulin signaling by GLP-1 were associated with SIRT1.Our data suggests GLP-1 improved palmitate-induced insulin resistance in HSMM by the activation of SIRT1.

1707-PA Modulator of TAZ (TM-25659) Attenuates Insulin Resistance in C2 Myotubes and C57BL/6J Mice Skeletal MuscleKWAN WOO LEE, JA YOUONG JEON, JONG-GAB JEONG, EUN SUK HA, SUNG-E CHOI, SANG-A LEE, YUP KANG, MYUNG AE BAE, JIN HEE AHN, HANA JEONG, EUN SOOK HWANG, SEUNG JIN HAN, DAE JUNG KIM, Suwon, Republic of Korea, Seoul, Republic of Korea

The transcriptional co-activator with PDZ-binding motif (TAZ) inhibits adipocyte development by intervention with peroxisome proliferator-activated receptor (PPAR)-gamma. We investigated the effects of TAZ modulator on skeletal muscle function in C2 myotubes and in C57BL/6J mice. In a previous study, Jang et al identifi ed 2-butyl-5-methyl-6-(pyridine-3-yl)-3-[2’-(1H-tetrazole-5-yl)-biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine] (TM-25659) as a TAZ modulator and was shown to decrease weight gain in a high fat diet-induced obese model. In our present study, we confi rmed TM-25659 considerably inhibits palmitate-induced insulin resistance on C2 myotubes. We measured insulin signaling and glucose uptake using immunoblotting and 2-NBDG uptake. To elucidate the preventing mechanism of TM-25659 on palmitate-induced C2 myotubes insulin signaling, we performed real-time PCR with pro-infl ammatory cytokines (TNF-alpha, IL-1-beta, IL-6 and MCP-1) mRNA. Moreover,we showed that TM-25659 signifi cantly suppresses high fat diet (HFD) induced insulin resistance in C57BL/6J mice skeletal muscle. Male C57BL/6J mice that were fed a HFD for 8 weeks were randomly assigned for an additional 6 weeks to 3 groups: normal diet (ND), HFD, HFD+TM-25659. After treatment, oral glucose tolerance test (OGTT) and intraperitoneal insulin tolerance test (IPITT) were performed to evaluate anti-diabetic effects. After sacrifi ce, tissue extracts of the soleus muscle were obtained for quantifi cation of insulin signaling through Akt phosphorylation proteins by immunoblotting. Consequently, the TM-25659-treated group showed improved HFD-reduced glucose tolerance, increased insulin sensitivity, and

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increased AKT phosphorylation protein. Also, TM-25659 down-regulated expression of genes involved in pro-infl ammation. These results suggest TAZ modulator, TM-25659, play a benefi cial role in controlling insulin resistance in obese and diabetic conditions.

1708-PBlocking Central Galanin Receptors Attenuated Skeletal GLUT-4 Translocation in Diabetic RatsLE BU, XUSHENG CHANG, XIAOHUI SHI, SHEN QU, Shanghai, China

Recent research has shown that activation of peripheral galanin receptors promotes glucose transporter 4 (GLUT4) translocation and improves insulin sensitivity. However, its central effects on insulin sensitivity have not been elucidated. In this study, male Wistar rats pretreated with 30 mg/kg streptozotocin i.p. were used as models of type 2 diabetes mellitus (T2DM) when blood glucose concentrations exceeded 11.1 mmol/l. To further understand the central effect of galanin on insulin sensitivity, its antagonist M35 was injected into the cerebral ventricle in type 2 diabetic rats. Running on a treadmill was used to stimulate circulating Gal secretion and central Gal mRNA expression. Glucose infusion rates and muscle GLUT-4 expression were measured after training. The results showed that M35 signifi cantly decreased glucose infusion rates in euglycemic-hyperinsulinemic clamp tests as well as 2-deoxy-[3H]D-glucose uptake in skeletal muscle. M35 also lowered glucose GLUT4 concentration in plasma membranes and total cell membranes of myocytes. and the ratios of the GLUT4 contents in M35 groups were lower than those of each diabetic control. These results suggest that endogenous Gal, acting through its central receptor, may facilitate GLUT4 translocation from cytoplasmic vesicles to the cell surface of myocytes to accelerate glucose uptake in muscle and improve insulin sensitivity in control and diabetic rats. Gal and its relative agents are potential targets for treatment of type 2 diabetes mellitus and its complications.

Supported By: NSFC (81370093)

1709-PInsulin Increases Skeletal Muscle Ceramide BiosynthesisMELISSA SMITH, TREVOR TIPPETTS, MADELINE ANDERSON, ZACHARY HOLUB, REX SAITO, ADAM SWENSEN, JOHN PRINCE, BENJAMIN T. BIKMAN, Provo, UT

Insulin resistance and hyperinsulinemia, at least initially, are the foundation of type 2 diabetes. Due to greater clinical attention focused on regulating blood glucose levels, and many obvious symptoms being the result of hyperglycemia, the effects of chronic hyperinsulinemia are often overlooked. As a result of this perspective, type 2 diabetics are commonly treated with insulin, with common side effects being increased weight gain and exacerbated insulin resistance. Given the well-established role of ceramide as a mediator of insulin resistance, we sought to determine the effect of prolonged hyperinsulinemia on muscle ceramide biosynthesis. C2C12 myotubes treated with insulin for prolonged periods of time experienced a signifi cant increase in ceramide accrual and expression of enzymes involved in ceramide biosynthesis. Knock down of the rate-limiting biosynthetic step resulted in the expected reduction in ceramide biosynthesis with insulin treatments and a concomitant increase in triacylglycerol accrual, indicating altered nutrient. In animals, mice received vehicle (saline) or insulin injections daily for 14 days. Insulin-injected animals had signifi cantly higher skeletal muscle ceramide levels at the conclusion of the study period. These studies provide evidence that insulin is a potent regulator of ceramide metabolism, which reveals a mechanism whereby hyperinsulinemia and exogenous insulin treatment in type 2 diabetics exacerbates existing insulin resistance.

1710-PPharmacological PKC Activation Inhibits AMPK by Phosphorylation at Ser485/491 in Muscle and Liver CellsKIMBERLY COUGHLAN, RUDY J. VALENTINE, NEIL B. RUDERMAN, ASISH K. SAHA, Boston, MA

Our lab has previously shown that exposure to excess nutrients inhibits AMP-activated protein kinase (AMPK) activity and causes insulin resistance in insulin-sensitive tissues, such as liver and muscle. Recent studies suggest that an inhibitory phosphorylation site on AMPK’s α subunit (Ser485/491) may play an important role in its diminished activity, though the upstream signaling regulating this site is poorly understood in insulin-responsive tissues. Since various protein kinase C (PKC) isoforms are activated by excess nutrition and contribute to insulin resistance, we examined whether PKC activation may inhibit AMPK through Ser485/491 phosphorylation. C2C12 myotubes and HepG2 hepatocytes were treated with the PKC activator PMA (10-200nM) for 5min-6hrs. Cells were treated with the PKC inhibitor Gö6983 (1-20µM) for 1hr prior to PMA treatment (50nM, 30min). PMA treatment signifi cantly

increased p-AMPK Ser485/491 at all concentrations and timepoints. Maximal phosphorylation was seen by 10min and maintained up to 6hrs. Pre-treatment with Gö6983 dose-dependently decreased PMA induced p-AMPK Ser485/491. Phosphorylation of AMPK at Ser485/491 through a PKC mediated mechanism represents a novel way by which AMPK may be inhibited, and thus contribute to insulin resistance in muscle and liver.

1711-PAMPK Activators Improve Glucose Uptake and Lipid-induced Infl ammation in Primary Myotubes from Older SubjectsSANGEETA GHOSH, RAWEEWAN LERTWATTANARAK, NICOLAS MUSI, San Antonio, TX

Approximately 60% of older subjects suffer from glucose metabolism abnormalities (IGT, T2DM), associated with low grade infl ammation and decreased insulin-mediated muscle glucose uptake. Thus, strategies to enhance muscle glucose uptake and insulin sensitivity could to improve their glucose metabolism. Activation of the energy-sensing enzyme AMPK promotes glucose uptake, improves insulin sensitivity, and reduces infl ammation. We tested the hypotheses that pharmacologic AMPK activation would (i) directly increase glucose uptake; (ii) improve insulin responsiveness; and (iii) ameliorate lipid-induced infl ammatory responses in myotubes from older (age=74±6 y; n=6) and younger (age=24±4 y; n=6) lean, normal-glucose tolerant subjects. Results: Insulin-stimulated (100 nM for 20 min) glucose uptake was reduced in myotubes from older subjects (18% of young; P<0.05). Metformin alone (1 and 2 mM for 24 h) signifi cantly increased glucose uptake (19-50% from baseline depending on dose) in both groups (P=NS between groups). Pre-treatment (24 h) with metformin (1 and 2 mM) and AICAR (1 and 2 mM) signifi cantly enhanced insulin-stimulated glucose uptake (10-50% over insulin alone, depending on dose) in both groups (P=NS between groups). Stearate (200 µM for 12 h) induced an infl ammatory response evidenced by decreased IκBα protein and increased JNK and p38 phosphorylation in both groups. Addition of 1 and 2 mM AICAR (but not metformin) partially prevented the infl ammatory response caused by stearate in both groups. Summary: (i) Aging is associated with reduced insulin-mediated glucose uptake; (ii) Myotubes from older subjects respond normally to AMPK activators (e.g. metformin); (iii) AICAR enhances insulin responsiveness and (iv) Activation of AMPK with AICAR (but not metformin) prevents the infl ammatory response caused by saturated fatty acids. Conclusion: Activation of AMPK may be a useful strategy to improve glucose metabolism and infl ammation in older subjects.

Supported By: NIH

1712-PWITHDRAWN

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INSULIN ACTION—SIGNAL TRANSDUCTION, INSULIN, AND OTHER HORMONES

1713-PFRET-based Measurement of Intracellular Glucose in Real Time Reveals Differential Targeting of Class I GLUT Transporters by HIV Protease InhibitorsTHOMAS E. KRAFT, MONIQUE HEITMEIER, MARINA PUTANKO, PAUL W. HRUZ, St. Louis, MO

Several fi rst generation HIV protease inhibitors (PIs) are known to act as potent reversible antagonists of GLUT4. Since this initial discovery, the number of PIs has markedly increased, several additional GLUT isoforms have been identifi ed, and differential inhibitory effects of PIs on GLUT activity have been recognized. To directly compare the ability of individual PIs to target each of the known GLUTs, a sensitive high throughput FRET-based assay system was developed to measure acute changes in intracellular glucose levels in real time. HEK293 cells stably overexpressing individual human GLUTs were transfected with the glucose nanosensor FLII12Pglu-700µδ6 (FLIP). After starving hGLUT FLIP cells in glucose-free media, 50 µM ritonavir, indinavir or darunavir was added. FRET was then measured immediately following addition of 5 mM glucose. Cytocholasin B (20 µM) was used as a positive control for the global inhibition of glucose transport. As expected, preincubation of hGLUT FLIP cells with indinavir resulted in isoform-selective inhibition of hGLUT4 (~75%) over hGLUT1. Indinavir also potently inhibited glucose uptake by hGLUT2, but failed to inhibit glucose uptake by cells overexpressing hGLUT3. Darunavir inhibited glucose uptake in hGLUT2 and hGLUT4 FLIP-expressing cells, sparing hGLUT1 and hGLUT3. Ritonavir resulted in the near complete inhibition of glucose uptake by hGLUT4 as well as hGLUTs-2, and -3 FLIP cells, but had no effect on hGLUT1. These data support differential isoform-selectivity of PI mediated effects on glucose uptake. The development of a high throughput cell-based assay that assesses glucose uptake by individual GLUTs in real time will greatly assist ongoing efforts to identify compounds that modulate GLUT transporter activity for use in elucidating the pathogenesis of diabetes, diabetic complications and other diseases in which dysregulation of glucose homeogenesis plays an essential role.

1714-PEffect of Liraglutide on GLUT4 Translocation in Mouse Skeletal Muscle CellsZHU LI, LIMING CHEN, WENYAN NIU, Tianjin, China

Liraglutide is an analog with 97% homology to human glucagon-like peptide (GLP-1) and acts as a GLP-1 receptor agonist. Right now, most studies about the early benefi ts of GLP-1 analogues are focus on weight losing and cell protecting. As another characteristic of T2DM and glucose intolerant, insulin resist is expressed as the alleviation of the prompting effect of insulin on transportation of glucose by GLUT4 in skeletal muscle cells. Stimuli increasing glucose uptake are all through enhancing the redistribution of GLUT4 to the plasma membrane. So the amount of translocation of GLUT4 can be considered as matching the glucose uptake. Thus, we wanted to investigate the effects of liraglutide on GLUT4 translocation in C2C12 cells overexpressing GLUT4myc. C2C12GLUT4myc cells were divided into the control group, insulin group (100nMm/L), liraglutide group (100nM/L), liraglutide group (1000nM/L) and AICAR (AMPK agonist) group (2mmol/L).The amount of GLUT4myc on the cell surface was measured by enzyme-linked immunosorbent assay (ELISA) after stimulation or AMPK inhibitor compound C incubated cells for 30min prior to stimulation. The phosphorylation of AMPK, AKT, PAS and GLUT4 expression were tested by Western blotting. Statistical analyses were carried out using SPSS software (San Diego, CA). Two groups were compared using Student’s paired t-test and more than two groups were compared using analysis of variance (ANOVA) with Tukey’s post hoc analysis.

The amount of GLUT4myc on the cell surface in liraglutide groups were more than the control group (P<0.05). The expression of GLUT4 had no changed in liraglutide groups compared with the control group. The phosphorylation of AMPK and PAS was raised in liraglutide groups compared to the control group (P0.05). The phosphorylation of ACC in liraglutide groups was declined after AMPK inhibitor compound C incubated cells for 30min (P<0.05). This study suggests that liraglutide may induce GLUT4 translocation of mouse skeletal cells by activation of AMPK.


Guided Audio Tour: Hormonal and Cellular Signaling in Diabetes (Posters: 1715-P to 1722-P), see page 17.

& 1715-P20/(Fasting C-Peptide × Fasting Plasma Glucose) Is a Novel Index of Insulin Resistance in Patients with Type 2 Diabetes MellitusTSUYOSHI OHKURA, HIDEKI SHIOCHI, YOUHEI FUJIOKA, RISA NAKANISHI, KAZUHIKO MATSUZAWA, SHOICHIRO IZAWA, HIROKO OHKURA, 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-fi ve patients [mean age: 54 years, fasting plasma glucose (FPG) 7.3 mmol/L, HbA1c 7.1%, body mass index 27.6 kg/m2] underwent a MTT and a glucose clamp. Participants were given a test meal (450 kcal). Plasma glucose and insulin were measured at 0 (fasting), 30, 60, 120, and 180 min. Serum C-peptide immunoreactivity (CPR) was measured at 0 (fasting; F-CPR) and 120 min. Homeostasis model assessment of insulin resistance (HOMA-IR) and insulin sensitivity indices (ISI) were calculated from the MTT results. The glucose infusion rate (GIR) was measured during hyperinsulinemic-euglycemic glucose clamps. The mean GIR in all patients was 5.5 mg·kg-1·min-1. The index 20/(F-CPR × FPG) was correlated strongly with GIR (r = 0.82, P < 0.0001). HOMA-IR (r = −0.70, P < 0.0001) and ISI (r = 0.74, P < 0.0001) were also correlated with GIR. 20/(F-CPR × FPG) was able to estimate GIR (Figure 1). 20/(F-CPR × FPG) is a simple and effective index of insulin resistance, and performs better than HOMA-IR and ISI in Japanese patients with type 2 diabetes mellitus. Our results suggest that 20/(F-CPR × FPG) is able to estimate GIR. We would like to name this index ‘CPR-IR’.

Supported By: Japan Diabetes Society

& 1716-PDifferential Roles of Insulin and IGF-1 Receptor Intracellular Domains in Cellular SignalingWEIKANG CAI, ANDRE KLEINRIDDERS, C. RONALD KAHN, Boston, MA

The insulin receptor (IR) and IGF-1 receptor (IGF1R) are widely expressed throughout the body. Despite the high homology between these receptors and their downstream signaling pathways, IR and IGF1R have strikingly distinct physiological functions in physiology and pathophysiology, which are mirrored by loss-of-function studies in mice with IR signaling being more involved in metabolic control and IGF1R signaling more critical for growth regulation. The molecular mechanisms underlying these differential functions remain unknown. In the present study, we have created preadipocytes in which both IR and IGF1R have been genetically inactivated (DKO) and reconstituted them with normal IR, normal IGF1R, two chimeric receptors: one with the IR-extracellular domain (ECD) fused to the IGF1R-intracellular domain (ICD) (hereafter called IR-IGF1R) and the other with IGF1R-ECD fused to IR-ICD (called IGF1R-IR). Upon hormonal stimulation, cells expressing either the normal IR or IGF1R showed higher activation of ERK1/2 than cells with either chimeric receptor. Cells with receptors containing the IR-ICD (i.e., IR or IGF1R-IR) had higher receptor autophosphorylation and a trend toward increased phosphorylation of IRS2 than cells with IGFR1-ICD. In contrast, cells with IGF1R ICD showed higher phosphorylation of Shc, a trend toward increased Akt phosphorylation, and increased activation of the p70 S6K1-S6 pathway. The latter cells also had signifi cantly shorter population doubling time, smaller cell size and were more potent in differentiating into mature

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adipocytes than cells expressing receptors with IR-ICD. Together, these data indicate that the intracellular domains of IR and IGF1R have divergent roles in regulating downstream signaling pathways. These differences lead to differential substrate utilization, different effects on downstream enzymes and ultimately differences in protein synthesis, cell proliferation and differentiation.

& 1717-PPituitary Function Directs the Catabolic Response to TNF-α—Studies of Protein, Glucose, and Fatty Acid Metabolism in Hypo-pituitary and Healthy SubjectsERMINA BACH, ANDREAS B. MØLLER, JENS O.L. JØRGENSEN, MIKKEL H. VENDELBO, NIELS JESSEN, STEEN B. PEDERSEN, THOMAS S. NIELSEN, NIELS MØLLER, Aarhus, Denmark

In humans TNF-α generates infl ammatory responses and insulin resistance, lipolysis and protein breakdown. It is unclear whether these TNF-α induced metabolic changes depend on intact hypothalamo-pituitary stress hormone responses triggering release of cortisol and growth hormone.

We sought to defi ne the effects of TNF-α on glucose, protein and lipid metabolism in hypopituitary patients (HP) (without intact hypothalamo-pituitary axis) and healthy controls (CTR) in a randomized, placebo controlled, single-blinded design.

We studied eight HP and eight age and gender matched CTR twice during 4-h basal and 2-h hyperinsulinemic euglycemic clamp conditions with isotope dilution to measure phenylalnine, urea, palimtate and glucose fl uxes together with muscle biopsies and fat biopsies in each period during infusion of saline or TNF-α (12 ng/kg/h) for 6 h.

Overall TNF-α infusion signifi cantly increased cortisol and GH levels in CTR but not in HP. TNF-α increased phenylalanine fl uxes in both groups, but signifi cantly more so in CTR, and raised urea fl ux by 40 % in CTR without any alteration in HP. Endogenous glucose production (EGP) was elevated in CTR compared to HP after TNF-α administration, whereas insulin sensitivity assessed by glucose infusion rates remained similarly unaffected in both groups. TNF-α increased whole body palmitate fl uxes and decreased palmitate specifi c activity in CTR, but not in HP without any statistical difference between groups. We did not detect signifi cant effects TNF-α on insulin signaling in muscle or lipase expression in fat although there was a trend towards decreased G(0)/G(1) Switch Gene 2 (G0S2) adipose tissue mRNA in CTR.

In summary TNF-α increased both urea and amino acid fl uxes and EGP signifi cantly more in CTR compared to HP, suggesting that in humans intact pituitary function with boosted cortisol and GH release is an integrated component of the catabolic response to TNF-α.

& 1718-PThe Role of miRNAs in Methylglyoxal-induced Endothelial Insulin ResistanceCECILIA NIGRO, PAOLA MIRRA, GREGORY A. RACITI, IMMACOLATA PREVEN-ZANO, ALESSIA LEONE, FEDERICA ZATTERALE, FRANCESCA FIORY, THOMAS H. FLEMING, PIETRO FORMISANO, FRANCESCO BEGUINOT, CLAUDIA MIELE, Naples, Italy, Heidelberg, Germany

Insulin plays a hemodynamic action on endothelium. An imbalance between insulin-dependent ET1 and NO production predisposes to the onset of diabetes-associated vascular complications. Hyperglycaemia promotes insulin-resistance in part through the generation of advanced glycation end products (AGEs). Increasing evidences show that hyperglycaemia modifi es endothelial expression of several miRNAs, suggesting their potential role in the impairment of endothelial insulin sensitivity. We have recently shown that the AGEs precursor methylglyoxal (MGO) induces insulin resistance in mouse aortic endothelial cells (MAEC). Here we evaluated MGO effects on endothelial insulin sensitivity in vivo and miRNAs expression.

C57/BL6 mice were injected ip with MGO. Insulin signaling was analyzed in isolated aortae. NO and ET1 serum levels were measured by colorimetric and ELISA assays, respectively. To increase MGO levels MAEC were incubated with the Glyoxalase 1 inhibitor SpBrBzGSHCp2 and miRNAs were evaluated by miScript miRNA PCR Array and RTqPCR.

In murine aortae MGO impairs both insulin-stimulated IRS1, Akt and eNOS activation, thereby blunting NO release, whilst both ERK1/2 activation and ET1 release are constitutively increased. In MAEC, treatment with SpBrBzGSHCp2 reduces the expression of miR34a, miR126-5p, miR190a-5p, miR214-3p, miR450a-5p, miR30d and miR181a. Interestingly, miR190a-5p and miR214-3p are known to modulate the expression of the Akt regulators PTEN and PHLPP, respectively. In SpBrBzGSHCp2-treated MAEC, miR214-3p levels are decreased by 77% thereby leading to a 2-fold increase of its target PHLPP2.

In conclusion, MGO impairs endothelial insulin action in vivo leading to a shift of NO/ET1 balance. In vitro our data indicate that MGO may play its harmful effect on vascular insulin sensitivity by modifying specifi c miRNA expression, suggesting these RNA molecules to be considered a new tool for the development of strategies to preserve endothelial function in diabetic subjects.

Supported By: MIUR (PRIN 2009FATXW3_003)

& 1719-PAkt-dependent Phosphorylation of Hepatic FoxO1 Is Compart-mentalized on a WD40/Propeller/FYVE Scaffold and Selectively Inhibited by Atypical PKC in High-Fat Fed and ob/ob MiceMINI P. SAJAN, MILDRED ACEVADO-DUNCAN, MARY STANDAERT, ROBERT IVEY, MACKENZIE LEE, ROBERT FARESE, Tampa, FL

Initiating and sustaining mechanisms that impair gluconeogenic enzymes and spare lipogenic enzymes in obesity are obscure. Here, we examined insulin signaling to Akt and atypical PKC (aPKC) in liver and muscle, and hepatic enzyme expression in two diet-dependent obesity models: (a) mice fed a moderate (40% of calories) high-fat (HF) diet over 10 weeks, and (b) 5 month-old obese non-diabetic ob/ob mice. In HF and ob/ob mice, resting and insulin-stimulated Akt and atypical PKC (aPKC) activities were diminished in muscle; however, in liver, Akt and aPKC activities were increased. Despite elevated hepatic Akt activity, FoxO1 phosphorylation, which diminishes gluconeogenic enzyme expression, was impaired, but, in contrast, Akt-dependent phosphorylation of glycogenic GSK3β and lipogenic mTOR was elevated. Diminished Akt-dependent FoxO1 phosphorylation was traceable to reduced Akt activity associated with scaffold protein, WD40/Propeller/FYVE (ProF), which, in adipocytes, has been found to bind Akt, aPKC and FoxO1, and facilitate Akt-dependent FoxO1 phosphorylation. In contrast to Akt, levels and activity of aPKC associated with ProF was markedly increased. Moreover, selective but partial inhibition of hepatic aPKC with small molecule agents during the 10 week period reduced aPKC association with ProF, restored ProF-associated Akt activity, restored FoxO1 phosphorylation, and diminished expression of gluconeogenic enzymes. With inhibition of hepatic aPKC, hepatic lipogenic enzyme expression also diminished, and Akt and aPKC activities in muscle improved, as did glucose intolerance, weight gain, hepatosteatosis and hyperlipidemia. Our fi ndings show that, in liver, Akt-dependent FoxO1 phosphorylation occurs on the ProF scaffold and is selectively inhibited in both initial and later phases of diet-dependent obesity by increases in activity of co-compartmentalized aPKC.

Supported By: U.S. Dept. of Veterans Affairs

& 1720-PThe Close Relationship between Insulin Resistance and FGF21 Resistance in IGT SubjectsTAE HO KIM, JA YOUNG JEON, EUN SUK HA, SUNG-E CHOI, SO-YEON AHN, SO YOUNG OCK, SEUNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, YUP KANG KANG, KWAN WOO LEE, Incheon, Republic of Korea, Suwon, Republic of Korea

Decreased insulin sensitivity is a major characteristic of impaired glucose tolerance (IGT). Despite numerous studies, the pathogenesis of insulin resistance has not been well established. FGF-21 is a key mediator of the fasted state, which contributes to the regulation of lipolysis in white adipose tissue and substrate utilization in the liver. In addition, FGF-21 increases insulin-independent glucose uptake in mice 3T3-L1 adipocytes and primary human adipocytes. Recently, FGF-21 was also shown to directly enhance skeletal muscle glucose uptake, providing additional points of regulation that may contribute to the benefi cial effects of FGF-21 on glucose homeostasis. We investigated the mechanisms involved in the induction of FGF21 resistance in human skeletal muscle of IGT patients. We recruited 10 IGT subjects and 6 subjects with normal glucose tolerance (NGT) for this study. Whole-body insulin-mediated glucose uptake was determined using a euglycemic hyperinsulinemic clamp test. A percutaneous biopsy sample of the vastus lateralis muscle was obtained from 15 to 20 cm above the knee using a Bergstrom needle. We used western blotting to determine levels of FGF21, beta-klotho, active FGF receptor, and FRS2α in skeletal muscles from IGT subjects and normal glucose tolerant subjects. Levels of FGF21 were signifi cantly increased in skeletal muscles from IGT subjects, but levels of active FGF receptors and FRS2α were signifi cantly decreased in skeletal muscles from IGT subjects as compared to NGT subjects. Levels of beta-klotho were signifi cantly decreased in skeletal muscles from IGT subjects as compared to NGT subjects. In conclusion, FGF-21 resistance apparently was involved in insulin resistance in skeletal muscle in IGT subjects.

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& 1721-PA Novel Oxide Transport Chain via Aquaporins Controls Pathway-Selective Insulin ResistanceXIANGDONG WU, KEVIN JON WILLIAMS, Philadelphia, PA

Patients with type 2 diabetes (T2DM) and the atherometabolic syndrome develop fatty liver and poor glucose handling. These effects share a common origin—namely, pathway-selective insulin resistance and responsiveness (SEIRR). We recently reported that NADPH oxidase-4 (NOX4) forms a tight complex with superoxide dismutase-3 (SOD3). Thus, insulin stimulates NOX4 to generate O2· -, the same product as other NOX enzymes. NOX4 hands O2· - to SOD3 for effi cient dismutation into H2O2. This H2O2 inactivates PTEN, a molecule that otherwise blocks signaling from class I PI3Ks to AKT. Inactivation of PTEN allows normal, balanced insulin signaling.

Surprisingly, SOD3 is a cell-surface or extracellular enzyme. How does the H2O2 it generates after insulin stimulation enter the cell to inactivate PTEN? We hypothesized a role for metabolic channeling via aquaporins, which facilitate transmembrane diffusion of small, uncharged molecules. Here, they would act as peroxoporins.

In McArdle hepatocytes treated with a new, non-toxic, global inhibitor of aquaporins, AgNO3, we found that 10nM insulin failed to stimulate an intracellular H2O2 burst. As in T2DM liver, insulin stimulated these cells to produce robust amounts of an unusual monophosphorylated form of AKT at Thr308 (pT308-AKT) with only weak phosphorylation at Ser473. Downstream targets showed continued insulin-stimulated phosphorylation of TSC2 (lipogenesis), but poor insulin-stimulated phosphorylation of FOXO1 (gluconeogenesis). Activation of ERK remained insulin-responsive.

We next surveyed four aquaporins, AQP1, 3, 8, and 9. AQP3 and 8 had been reported to transport H2O2. Knockdowns of only AQP1 and AQP3 produced SEIRR. AQP8 and 9 knockdowns had no effect.

Our work identifi es a novel oxide transport chain, NOX4-SOD3-AQP-PTEN, as a master regulator of insulin signaling. Defects in this oxide transport chain may be the cause of SEIRR: how insulin still drives hepatic lipogenesis, fatty liver, and ERK, yet fails to control glucose.

Supported By: ADA (1-13-BS-209); Temple University; Ruth & Yonatan Ben-Avraham Fund, Swedish Heart-Lung Foundation

& 1722-PDysfunctional Protein Phosphatase 2A in Human Skeletal Muscle in Type 2 DiabetesXIANGMIN ZHANG, DIVYASRI DAMACHARLA, MICHAEL CARUSO, MONIQUE LEWIS, DANJUN MA, ZHAO YANG, YUE QI, RODNEY BERRY, BERHANE SEYOUM, ZAHER MSALLATY, WISSAM AL-JANABI, REBECCA TAGETT, SORIN DRAGHICI, JEFFREY F. HOROWITZ, ZHENGPING YI, Detroit, MI, Dearborn, MI, Ann Arbor, MI

Protein phosphatase 2A (PP2A), a major serine/threonine phosphatase, regulate multiple cellular events such as insulin signaling. PP2A can inactivate/activate multiple kinases (e.g. Akt, GSK3), and emerging evidence suggests hyperactivation of PP2A in liver, muscle, retina and islets under the duress of glucolipotoxicity. Nonetheless, most experiments regarding PP2A activity and regulation were carried out in cell/animal models. Whether these fi ndings can be translated into humans is currently unknown. Recently, we identifi ed the catalytic subunit of PP2A (PP2Ac) and the 65 kDa regulatory subunit A alpha (PP2Aa) as novel IRS1 interaction partners in human skeletal muscle, and demonstrated increased interactions of these PP2A subunits with IRS1 in muscle of type 2 diabetic subjects (T2D) compared to lean controls (LC). Here, we further explored the activity and regulation of PP2A in T2D. 1st, we assessed overall PP2A activity in muscle biopsies from 4 LC and 4 T2D subjects. The PP2Ac activity was signifi cantly lower after 2h insulin infusion in LC, while unchanged in T2D between basal and 2h biopsies. 2nd, we determined the level of carboxylmethylation of leucine309 (mLeu309) of PP2Ac (known to activate PP2A) from 4 LC and 3 T2D. There is a trend of decrease for mLeu309 in LC after 2h insulin infusion. However, insulin did not reduce mLeu309 in T2D at all. 3rd, proteomics analysis on muscle biopsies from 5 participants revealed 89 PP2Ac interaction partners, including PP2Aa, and protein phosphatase methylesterase 1 that activates PP2Ac through mLeu309 of PP2Ac. In summary, these results provided the 1st experimental evidence that insulin’s ability to suppress PP2A activity (possibly through suppressing mLeu309 of PP2Ac) is impaired in T2D muscle, and provided the largest PP2Ac interaction network in skeletal muscle in humans. These novel fi ndings may facilitate the design of new drugs to restore abnormal PP2Ac activity and improve insulin signaling, resulting in the treatment/prevention of T2D.

Supported By: NIH/NIDDK (R01DK081750)

1723-PSex Differences Modulate Relationships among Obesity, Insulin Resistance, and Obstructive Sleep ApneaALICE LIU, JAMES CARDELL, DANIT ARIEL, CINDY LAMENDOLA, FAHIM ABBASI, SUN H. KIM, VANESSA TOMASSO, HAFASA MOJADDIDI, KAYLENE GROVE, CLETE KUSHIDA, GERALD M. REAVEN, Stanford, CA

Although obstructive sleep apnea (OSA) is associated with obesity and insulin resistance, the relationships among these variables have not been fully explored, nor is it known if they are modifi ed by sex differences. We evaluated adiposity and measured insulin sensitivity in overweight/obese, non-diabetic adults with untreated OSA.

Volunteers were recruited consecutively from the Stanford Sleep Medicine Center and local communities. Apnea-hypopnea index (AHI) > 5 events/hour during polysomnography met criteria for OSA; higher AHI indicated more severe OSA. Minimum (minO2) and mean oxygen saturation (meanO2), and oxygen desaturation index (ODI) were also reported. Insulin-mediated glucose uptake was quantifi ed by measuring steady-state plasma glucose (SSPG) levels during the insulin suppression test. Body mass index (BMI) was calculated and waist circumference (WC) measured.

Men (n=70) did not differ from women (n=39) with respect to mean age (50 vs. 52 years), BMI (30.7 vs. 30.5 kg/m2), SSPG (177 vs. 159 mg/dL), or AHI (34.9 vs. 31.7 events/hour), but had higher WC (107 vs. 98 cm, p<0.001). Among men, BMI was highly correlated with WC (r=0.79, p<0.001) and SSPG (r=0.41, p<0.001), but not with any OSA-related measures. WC correlated with SSPG (r=0.32, p<0.01), ODI (r=0.30, p<0.05), and inversely with minO2 (r=-0.28, p<0.05). SSPG was signifi cantly associated with all four OSA measures: AHI (r=0.33, p=0.006), ODI (r=0.40, p=0.001), minO2 (r=-0.24, p<0.05), and meanO2 (r=-0.26, p<0.05). In multivariate analysis, only SSPG (but neither adiposity index) was independently associated with AHI or ODI. Results were quite disparate among women. Whereas SSPG correlated with no OSA measures, BMI was independently associated with minO2 or meanO2.

In conclusion, insulin resistance rather than adiposity was a predictor of OSA severity or hypoxia in men, whereas the opposite was observed in women. Differences in sex modulate the relationships among obesity, insulin resistance, and OSA.

Supported By: NIH/NIDDK (5K23DK088877); NHLBI (5U01HL108647)

1724-PCommon Food Additive Carrageenan Impairs Insulin Signaling by Increasing Expression of GRB10 in Addition to Activation of Infl am-ma tionSUMIT BHATTACHARYYA, LEO FEFERMAN, JOANNE K. TOBACMAN, Chicago, IL

In human hepatic epithelial cells and in C57BL/6J mice, exposure to low concentration for short duration of the common food additive carrageenan leads to glucose intolerance, insulin resistance, and impaired insulin signaling due to stimulation of infl ammation through TLR4 and ROS, leading to increased phosphorylation of IRS-1, an inhibitor of downstream insulin signaling (Diabetologia 2012;55:194-203). However, inhibition of carrageenan-induced infl ammation did not completely reverse the effect of carrageenan exposure on phosphorylation of AKT Ser473. To isolate the signaling mechanism responsible for this additional inhibitory effect on p-AKT, studies in human HepG2 cells and in C57BL/6J mice were performed. Cells were exposed to λ-carrageenan (Sigma) 1 µg/ml x 24h and mice were exposed to κ-λ carrageenan 10 µg/ml in their water supply for 18 days. Following carrageenan, expression of Growth receptor binding (Grb)-10 protein was determined by QPCR, Western blot, and ELISA, which showed signifi cant increases in expression in HepG2 cells and mouse liver. Grb10 inhibits downstream insulin signaling by binding to IRS-1, and silencing of GRB10 inhibited the carrageenan-induced decline in the p-Tyr IRS-1 increase in response to insulin. When GRB10 was silenced by siRNA, the carrageenan-induced decline in p-(Ser473)-AKT response to insulin was less, and the combination of GRB10 silencing, BCL10 silencing, and Tempol, a ROS inhibitor, completely reversed the carrageenan-induced decline in the response of p-AKT to insulin. Following carrageenan exposure, GRB10 promoter activity, detected by Renilla luciferase assay, increased, due to carrageenan-induced activation of GATA-2 binding to the GRB10 promoter, as demonstrated by chromatin immunoprecipitation assay. These fi ndings further support the potent role of carrageenan as an inhibitor of insulin signaling and potential etiologic role in clinical diabetes.

Supported By: ADA (1-12-BS-216)

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1725-PHypoadiponectinemia and Resultant APPL1 Downregulation Con-tributes to Vascular Insulin Resistance in Normotensive Young SHRsHAIFENG ZHANG, WENJUAN XING, XIANGYAN LIANG, LELE JI, RONG LI, FENG GAO, Xi’an, China

Aims: Vascular insulin resistance contributes to elevated peripheral vascular resistance and subsequent hypertension. Clinical observation has revealed that a low plasma adiponectin concentration is associated with hypertension. This study aimed to determine whether and how hypo-adiponectinemia induces vascular insulin resistance in prehyper tension.

Methods: Four-week-age prehypertensive spontaneously hypertensive rats (ySHRs) and adiponectin knockout mice (APNKO) were used to evaluate the role of hypoadiponectinemia in insulin-induced vasodilation.

Results: Mesenteric arteriole segments of ySHRs showed a markedly reduced vasorelaxation response to insulin compared with those of age-matched Wistar-Kyoto controls (WKY) (21.6 % ± 3.2 % vs. 41.6 % ± 4.4 % to 1 µmol/L insulin, n=8, P < 0.05). Adiponectin levels in serum (7.52 ± 0.86 vs. 4.89 ± 0.49 µg/ml, P < 0.05) and APPL1 expression in mesenteric arterioles of ySHRs were signifi cantly reduced. Moreover, eNOS phosphorylation and NO production in vascular tissue were reduced, while ERK1/2 phosphorylation and ET-1 secretion were augmented in ySHRs. APNKO mice also showed markedly decreased APPL1 expression and vasodilation evoked by insulin. Treatment of ySHRs in vivo with the globular domain of adiponectin (gAd) for 1 week increased insulin-induced vasodilation and APPL1 expression in arterioles, and restored the balance between insulin-stimulated Akt/eNOS/NO and ERK1/2/ET-1 pathway. In cultured human umbilical vein endothelial cells, gAd upregulated APPL1 expression in a dose-dependent fashion. Suppression of APPL1 expression with siRNA blunted the modulation of insulin signaling by gAd (all P < 0.05).

Conclusion: Hypoadiponectinemia induces APPL1 downregulation in resistance vessels, contributing to the development of vascular insulin resistance by reciprocally modulating the Akt/eNOS/NO and ERK1/2/ET-1 pathways in vascular endothelium in prehypertensive SHRs.

Supported By: NSFC (81270330, 81270401)

1726-PInsulin Regulates Proangiogenic Signaling by Downregulating CITED2 in Endothelial CellsXUANCHUN WANG, SAM LOCKHART, LARS M. RASMUSSEN, 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 diabetes. FoxO transcription factors, which are inhibited by insulin, are known to be potently antiangiogenic. Our aim was to identify genes involved in angiogenesis and regulated by insulin through FoxO in endothelial cells. FoxO1/3/4 knockout mice develop hemangiomas in liver but not in lung. A previous study identifi ed FoxO targets among genes that were changed by FoxO1/3/4 knockout in liver endothelial cells but unchanged in lung endothelial cells, as assayed by gene arrays. We now measured expression of these FoxO target genes by real-time PCR in endothelial cells after adenoviral-mediated expression of wild-type FoxO1 or a constitutively active FoxO1 mutant combined with insulin treatment (10 nM for 4, 8, or 16 hours). Cited2, Ctgf, and Adm mRNA were upregulated by wildtype or mutant FoxO1 and downregulated by insulin in conditions without FoxO1 overexpression or with expression of wildtype, but not mutant, FoxO1. Spry2, Klf6, Bmper and Ccnd1 were regulated by insulin but not by FoxO1, whereas Fbn1 and Sdpr were regulated by FoxO1 but not by insulin. Nine genes were neither regulated by insulin nor by FoxO1. Cited2, one of the genes most robustly regulated by insulin, encodes CBP/p300-interacting transactivator 2 (CITED2) which inhibits transcription of hypoxia-inducible factor-1α and other genes. Insulin downregulated Cited2 mRNA by 70% and this effect was abrogated by pretreatment with LY294002 and MK2206 (inhibitors of phosphoinositide 3-kinase and Akt respectively). siRNA-mediated knockdown of CITED2 increased tube formation on Matrigel and sprouting from Cytodex 3 microbeads. We conclude that insulin may promote angiogenesis through downregulation of CITED2 via Akt-dependent inhibition of FoxO1. Enhancing insulin sensitivity of this pathway or directly inhibiting CITED2 may improve neovascularization in patients with type 2 diabetes.

1727-PUmbelliferone Increases Insulin Sensitivity and Adiponectin Secre-tion in High-Fat Diet/Streptozotocin-induced Type 2 Diabetic RatsJARINYAPORN NAOWABOOT, PATCHAREEWAN PANNANGPETCH, NUNTIYA SOMPARN, SUPAKATE SAENTAWEESUK, Pathumthani, Thailand, Khon Kaen, Thailand

Umbelliferone (UMB) is a natural product of the coumarin family. Its therapeutic effects are anti-infl ammation, anti-oxidation, antitumor and antihyperglycemia. However, the mechanism of its antihyperglycemic effect is still unclear. Therefore, this study was aimed to investigate the antidiabetic mechanism of UMB in high-fat diet/streptozotocin-induced type 2 diabetic rats. Diabetes was induced by feeding high-fat diet (45% fat) for four weeks followed by a single injection of 35 mg/kg STZ intraperitoneally. Normal control rats were treated with 5% Gum Arabic and diabetic rats were treated with UMB (10 and 30 mg/kg) or 5% Gum Arabic or pioglitazone (10 mg/kg) for eight weeks. After eight weeks of treatments, the levels of fasting blood glucose (FBG) in 10 and 30 mg/kg UMB treated groups were signifi cantly reduced. The UMB treatment also signifi cantly improved the intraperitoneal glucose tolerance test of diabetic rats as compared with diabetic control rats. Interestingly, in the 30 mg/kg UMB treated diabetic group, serum insulin level was signifi cantly increased and the HOMA-IR value, an indicator of insulin resistance, was signifi cantly reduced (26.2 ± 2.9 and 61.4 ± 9.5 for UMB treated and diabetic control groups, respectively). In addition, the level of adiponectin was signifi cantly increased in diabetic group treated with 30 mg/kg UMB. These fi ndings suggest that umbelliferone is effective in reducing hyperglycemia in type 2 diabetes and the antihyperglycemic activity of umbelliferone may be mediated via its insulin sensitivity improving and adiponectin secretion stimulating activities.

Supported By: Thailand Research Fund; Thammasat University

1728-PInvestigating Endocrine Effects of Insulin on β-CellsROHIT N. KULKARNI, JIANG HU, TERESA MEZZA, Boston, MA

Insulin receptors and proteins in its signal transduction pathway have been reported to play a critical role in the regulation of β cell survival, proliferation and secretory function. Designing experiments aimed at directly evaluating the effects of insulin (exogenous or endogenous) on β cells have been challenging due to potential effects of insulin in the local milieu. Further, the constant washout of insulin by normal circulation likely precludes accumulation of local insulin to impact β cell receptors. To investigate how insulin regulates downstream signaling proteins ,we designed two experimental approaches in healthy C57/B6 mice.. To examine physiological stimulus we performed an oral glucose (1 gm/kg) tolerance or saline (n=6). In an independent experiment, to explore the potential endocrine effects of circulating insulin we performed either a 15-min saline infusion (sham clamp-SC) or a hyperinsulinemic clamp (HC) (150 mUI/kg priming followed by 2.5 mU·kg-1·min-1 insulin infusion rate) (n=6). Then, pancreases were rapidly dissected fi xed and sections were analyzed by IHC for alterations in pAKT, p70S6K, MAPK and FOXO1 co-stained also for insulin.

We observed a signifi cant increase in β cells that co-stained for pAKT, p70S6K and pERK in mice that underwent OGTT compared to those receiving saline (pAKT + 35.02±9.6% vs. 24.6±1.65%, p=0.05; p70S6K + 46.5±19.6% vs. 29.8±1.65%, p=0.04; MAPK+ 17.2±2.56 % vs. 8.5±0.95, p=0.04). Further, in the group receiving the 15-min HC, signaling proteins downstream of the insulin receptors were also increased compared to Sham clamp (pAKT+ 54.1±7.91 vs. 36.8±2.84%, p<0.01; p70S6K+ 55.2±6.2% vs. 36.6±2.04%, p<0.01; MAPK+ 14.2±0.97 % vs. 8.2±0.09, p=0.001 ). In contrast the number of nuclear FOXO1+ β cells was decreased in HC group compared to SC group (nuclear FOXO1+ 11.2±4.6 vs. 51.4±9.6%, p<0.01). These data suggest that signaling proteins downstream of the insulin receptors in β cells are more responsive to alterations in circulating levels of insulin via the endocrine pathway.

1729-PNew Insights into Atomic-Level Interactions between Akt1 and Pyruvate Dehydrogenase Complex in the Cardiac MitochondriaJAMES B. FIELDS, YUMAY CHEN, YU-HAN CHEN, CHARITY JUANG, DOUGLAS J. TOBIAS, PING H. WANG, Irvine, CA

Mitochondria dysfunction contributes to diabetic cardiomyopathy and its metabolic dysregulation. The aim of this study was to study how Akt1 forms signaling complexes with mitochondria proteins upon insulin stimulation and modulates bioenergetics. Inhibition of mitochondrial Akt decreased glucose uptake, and attenuated insulin-stimulated glucose uptake in cardiomyocytes. Activating Akt1 in the cardiac mitochondria increased the effi ciency of cell

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respiration by enhancing glucose uptake and glycolytic ATP production. We have identifi ed a panel of mitochondrial proteins as putative targets of Akt1 signaling, including the pyruvate dehydrogenase, the gate keeper of bioenergetic pathways. Phospho-Akt1 (pAKT1) co-immunoprecipitated with pyruvate dehydrogenase complex (PDC), consisting of E1α, E1β, E2, E3, and PDHX subunits. pAKT1 preferentially bound to the E3 subunit in cardiac mitochondria and activated PDC. We used Brownian dynamics (BD) and atomistic molecular dynamics (MD) computer simulations to model atomically detailed pAKT1-E3 complexes. BD simulation trajectories indicated the interactions were through several pairs of amino acids: the negatively charged side chains of Asp190, Glu355, Asp323, Glu322, and Asp325 on Akt1 and the positively charged side chains of Lys232, Lys385, Arg228, Lys224, and Lys265 on E3. The interaction required the NADH co-factor, and phosphorylation of Akt1 greatly enhanced the strength of the interaction. During MD trajectory, three additional salt bridges formed between Arg465, Asp353, and Glu184 of Akt1 and Glu77, Lys470, and Lys87, respectively, of the E3 domain, and some of the salt bridges from the initial (BD) model were disrupted during the MD. Mutations at specifi c amino acid residues signifi cantly reduced their interactions. These data provided new information on the mechanism of Akt1-E3 signaling complex formation in mitochondria, and new opportunities to develop novel therapeutic approach.

Supported By: NIH (R01HL096987)

1730-PMediobasal Hypothalamic PTEN Modulates Hepatic Insulin Resist-ance Independently of Food Intake in RatsTAKASHI SUMITA, Saitama, Japan

PTEN (phosphatase and tensin homolog) dephosphorylates phosphati-dylino sitol 3,4,5-triphosphate and antagonizes PI 3-kinase. Insulin acts in the mediobasal hypothalamus (MBH) not only to suppress food intake and weight gain, but also to improve glucose metabolism, via PI 3-kinase activation. Thus, blocking hypothalamic PTEN is a potential target for treating obesity as well as diabetes. However, genetic modifi cation of PTEN in specifi c neuronal populations in the MBH yielded complex results, and no postnatal intervention for hypothalamic PTEN has yet been reported. In order to elucidate how postnatal modifi cation of hypothalamic PTEN infl uences food intake as well as glucose metabolism, we bidirectionally altered PTEN activity in the MBH of rats by adenoviral gene delivery. Inhibition of MBH PTEN activity reduced food intake and weight gain, while constitutive activation of PTEN tended to induce the opposite effects. Interestingly, the effects of MBH PTEN intervention on food intake and body weight were blunted by high-fat feeding. However, MBH PTEN blockade improved hepatic insulin sensitivity even under high-fat fed conditions. On the other hand, constitutive activation of MBH PTEN induced hepatic insulin resistance. Hepatic Akt phosphorylation and the G6Pase expression level were bidirectionally modulated by MBH PTEN intervention. These results demonstrate that PTEN in the MBH regulates hepatic insulin sensitivity, independently of the effects on food intake and weight gain. Therefore, hypothalamic PTEN is a promising target for treating insulin resistance even in states of over nutrition.

1731-PEstimation of Insulin Resistance Assessed by Euglycemic Hyper-insulinemic Clamp in Japanese Type 2 Diabetic PatientsYUKO YAMAZAKI, MASANORI EMOTO, TOMOAKI MORIOKA, SATOSHI IMA-MURA, RYUTARO NUMAGUCHI, HIROMI URATA, KOKA MOTOYAMA, KATSUHITO MORI, SHINYA FUKUMOTO, TETSUO SHOJI, MASAAKI INABA, Osaka, Japan

The estimation of insulin resistance (IR) is an important issue for indi-vidualized approach for the treatment of diabetes, since IR differs widely among type of diabetes, ethnicity, and obesity. Only a few reports have proposed estimated glucose disposal rate (eGDR) as a surrogate index of insulin resistance measured by euglycemic hyperinsulinemic clamp (Clamp), a gold standard method in humans. However, it is not fully ascertained whether such estimation of IR is applicable to Asian people with type 2 diabetes, who are now extremely increasing. The aim of the study was to explore the estimation of GDR by Clamp from simple clinical parameters in 287 Japanese subjects with type 2 diabetes (T2D) as compared with 12 obese subjects with impaired glucose tolerance (IGT) and 38 healthy subjects (age, 53.3±12.8 (SD), 46.3±15.0, 38.1±20.2 years; BMI 25.2±3.9, 29.0±3.2, 23.7±4.2 kg/m2, respectively). GDR in all subjects was measured by Clamp, using an artifi cial pancreas (STG-22 or 55, Nikkiso, Tokyo). GDRs in both T2D and obese IGT were signifi cantly lower than that in healthy subjects (4.56±2.89, 4.09±2.07 vs. 9.33±5.74 mg/kg/min/ (mU/L), p<0.0001).

There was no signifi cant difference in GDR between T2D and obese IGT (p=0.899). GDR in T2D was signifi cantly inversely correlated with BMI, HOMA-R, triglyceride, or free fatty acid (FFA) and positively with HDL-cholesterol (HDL), respectively. Multiple regression analysis revealed that HOMA-R, BMI, FFA, HDL, and renal function signifi cantly and independently contributed to GDR in T2D (R2=0.503, p <0.0001). In conclusion, our data suggests that the equation of simple clinical parameters is able to estimate GDR measured by euglycemic hyperinsulinemic clamp in Asian people with type 2 diabetes, which may be a useful surrogate marker of IR.

1732-P“Metabolic” vs. “Mitogenic” Insulin Receptor Signaling by an Allo-steric Monoclonal Antibody: Specifi c Metabolic Pathway Bias or Partial Agonism?DANIEL H. BEDINGER, JOHN A. CORBIN, MARINA K. ROELL, IRA D. GOLDFINE, SEAN H. ADAMS, Berkeley, CA, Davis, CA

XMetA, an insulin receptor (IR) allosteric monoclonal antibody, is a partial agonist of the INSR, and has been previously reported to activate the “metabolic-biased” AKT kinase signaling pathway much more strongly than the “mitogenic” MAPK signaling pathway of ERK 1/2. To investigate the nature of this signaling bias, a detailed XMetA dose response study was conducted to evaluate specifi c phosphorylation patterns of IR, AKT, and ERK(1/2) in CHO cell lines over-expressing either the short or long form of the human insulin receptor (IR-A or IR-B respectively). These studies confi rmed that XMetA stimulates phosphorylation of AKT much more strongly than ERK(1/2). Studies with insulin revealed that while the hormone activated both pathways, the phosphorylation of AKT was more sensitive to the hormone than the phosphorylation of ERK (EC50 47 pM vs. 757 pM respectively in CHO-hIR-B cells), and the phosphorylation of the kinase regulatory loop of IR had an intermediate dose response (EC50 of 121pM). Maximal effective concentrations of XMetA elicit phosphorylation patterns similar to 30-100 pM insulin, which is suffi cient for robust AKT phosphorylation but shows little activity on ERK phosphorylation. These data indicate that the signaling bias of XMetA is in large part due to a convergence of pathway sensitivity and partial agonism, rather than a separate pathway-biased mechanism. The “metabolic bias” of partial IR agonists like XMetA, if recapitulated in vivo, may be a desirable feature in terms of therapeutics designed to regulate blood sugar while minimizing potential mitogenic outcomes of IR activation.

1733-PAssessment of Insulin Response in STZ Rats Using Continuous Glucose TelemetrySCOTT TIESMA, ROBERT BROCKWAY, TAMER COSKUN, LIBBEY O’FARRELL, MER VYN D. MICHAEL, AMY L. COX, KIMBERLY V. WHITE, HEATHER V. BOGIE, St. Paul, MN, Indianapolis, IN

Continuous glucose monitoring options have been very limited for research applications in pre-clinical models. Investigators have used periodic sampling of blood glucose to provide a coarse assessment of the effi cacy of novel anti-diabetic therapies. The present study employs a newly developed implantable glucose monitor (HD-XG, Data Sciences International) to evaluate the pharmacodynamic (PD) profi le of several commercially available insulins including the basal insulins Humulin® N and Lantus® and the mealtime insulin analogs Humalog®, Novolog®, and Apidra®. The implantable device is 1.4 cc and provides continuous temperature, activity, and arterial blood glucose readings for 4 wks or longer. Continuous assessment of blood glucose was performed in 12 Sprague Dawley rats in which diabetes was induced with streptozotocin (STZ). Data were recorded continuously for 26 d with averages reported every 10 sec. Daily reference glucose values were measured from tail vein blood samples using the Nova StatStrip Xpress glucometer. Following STZ administration, the rats experienced a transient period of hyperglycemia followed by a hypoglycemic period. Over the next 24 hrs, blood glucose steadily increased, and hyperglycemia stabilized with a distinct diurnal fl uctuation. Three days after STZ induction, the rats were divided into 2 groups that received either Humulin® N or Lantus® BID to determine an optimal basal insulin regimen. Rats in the Humulin® N group had more sustained glucose lowering so all rats were switched to Humulin® N for individualized dose adjustments. Following the basal insulin assessment, the rats were dosed twice (10 nmol/kg) with each of the three mealtime insulin analogs to assess the PD response. The implanted glucose sensors provided unprecedented time resolution of the glucose response for each insulin dose. These profi les will provide a reference against which to compare novel drugs under development.

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1734-PChromium-Insulin Reduced Insulin Clearance by Inhibition of Hepatic Insulin-degrading Enzyme in KKAy MiceZHONG Q. WANG, YONGMEI YU, XIAN H. ZHANG, JIMES KOMOROWSKI, Baton Rouge, LA, Purchase, NY

Insulin-degrading enzyme, also known as IDE is a human enzyme fi rst identifi ed by its ability to degrade the B chain of the hormone insulin. JDS-CRI-003 (CRI) is a novel form of insulin that has been conjugated with chromium (Cr) instead of zinc. In prior studies, CRI has shown enhanced blood insulin levels and glucose lowering effects compared to non-Cr conjugated insulin. Our hypothesis was that CRI enhances insulin’s effects by altering IDE. To test this hypothesis, we measured hepatic IDE content and other parameters in mice. Male KKAy mice at age of week 5 were randomly divided into three groups (n=8/group); Sham (saline), human insulin (Reg-In) and chromium conjugated human insulin (CRI) respectively. All animals were fed a normal chow diet. Interventions were initiated at doses of 2 U insulin/kg body weight daily for eight weeks. Plasma glucose and insulin were measured by glucose strips and human insulin ELISA kits. Hepatic IDE and insulin signaling proteins were determined by western blotting. Insulin tolerance tests at week 7 showed that both insulin treatments signifi cantly reduced glucose concentrations and increased insulin levels compared with the sham group, and there was no difference between two insulin groups. Moreover, CRI showed signifi cant reduction in glucose at 4 and 6 hrs whereas Reg-In did not, suggesting the effects of CRI on reducing glucose last longer than Reg-In (P<0.05). The insulin concentrations at 30 min and 60 min were signifi cantly higher in the CRI group than in the Reg-In group (P<0.05). Compared with the sham group, CRI treatment signifi cantly increased hepatic IRS-1 and Akt1 and reduced IDE protein abundance (P<0.01, P<0.05, and P<0.001, respectively), but Reg-In only signifi cantly increased Akt1 and slightly reduced IDE content (P<0.05 and P=NS). Our study indicates that CRI reduced plasma insulin clearance by inhibition of hepatic IDE content in KKAy mice.

Supported By: JDS Therapeutics, LLC

1735-PIncreased Betatrophin Concentrations in Type 2 and Type 1 Diabetes While Insulin Suppresses Betatrophin: Evidence of a Negative Feedback Mechanism?PARESH DANDONA, HUSAM GHANIM, KELLY GREEN, AJAY CHAUDHURI, MANAV BATRA, NITESH D. KUHADIYA, ANTOINE MAKDISSI, Buffalo, NY

Betatrophin is a peptide secreted by the liver which induces the proliferation of β-cells of the pancreatic islet and whose expression and secretion are increased in experimental insulin resistant states. Therefore, we have now investigated the concentrations of betatrophin in patients with T2DM and obesity. Fasting plasma betatrophin concentrations were measured in 19 normal weight healthy subjects, 18 patients with T1DM, 32 patients with T2DM, and 12 patients with T2DM and morbid obesity using an EIA kit. In normal subjects, mean betatrophin concentration was 0.61±0.18ng/ml; it was signifi cantly elevated in patients with T2DM (1.23±0.24ng/ml; p<0.01) and T1DM (0.93±0.19ng/ml; p<0.05). The highest concentrations were observed in patients with morbid obesity and T2DM (1.57±0.42ng/ml; p<0.01). Thus, the absence of insulin, as in T1DM, and the reduction of insulin action, as in T2DM, results in an increase of betatrophin. To elucidate this further, we investigated the effect of a low dose infusion of insulin on plasma concentrations of betatrophin. Ten patients with type 2 diabetes were infused with insulin (3.5U/h) for 24h and glucose (5%, 100 ml/h) to maintain euglycemia. Plasma concentrations of betatrophin fell signifi cantly within 2h by 16±11% and by 29±10% below the baseline (p<0.05) at 14h and continued to be suppressed until 24h. It is, thus, possible that insulin may exert a direct inhibitory effect on betatrophin secretion and concentrations through a negative feedback mechanism. Since also FFAs fell following insulin, it is possible that FFAs which induce insulin resistance, may also modulate the secretion and concentrations of betatrophin in plasma. These data support the concept that insulin and betatrophin have a negative feedback mechanism with betatrophin stimulating the population and function of the β-cell while insulin, a product of the latter, inhibits betatrophin secretion.

1736-PDiscovery of Novel GLP-1R Interacting ProteinsFEIHAN DAI, ALPANA BHATTACHARJEE, MICHAEL WHEELER, Toronto, ON, Canada

Glucagon-like-peptide-1 receptor (GLP-1R) agonists are used drugs to treat Type-2 diabetes. To facilitate future drug development based on the

premise of GLP-1R activation, we employed a novel membrane yeast two hybrid (MYTH) system. Using the unliganded human GLP-1R as bait and screening of a human fetal brain cDNA prey library, we discovered over 30 novel proteins that interacted with the receptor. When co-expressed with the hGLP-1R, approximately half of these interactors signifi cantly affected GLP-1-induced receptor activation, however, they all suppressed signalling. This could be explained by the fact that a brain cDNA library was used as prey or an unliganded/inactivated GLP-1R was used as bait. To address these issues two strategies were followed, fi rst, the use of human and mouse islet libraries in MYTH and second, an affi nity purifi cation mass spectrometry (AP-MS) method, using an activated GLP-1R expressed in mammalian cells. Using MYTH, we identifi ed 68 islet interactors. Second, we established a stable GLP-1R expressing HEK cell line by knock-in of hGLP-1R-strep-HA cDNA into the genome. Putative hGLP-1R/interactors protein complexes were purifi ed and analyzed by MS. In 3 independent screens we identifi ed 50 interactors of unliganded, 4 of liganded and 15 of liganded and unliganded hGLP-1R. In initial studies 12 interactors were selected based on membrane localization and screen specifi city. After overexpression in INS-1 beta cells, 2 interactors increased GLP-1 induced insulin secretion (GIIS) without effect on glucose-stimulated insulin secretion (GSIS), 2 increased both GIIS and GSIS while 1 decreased both GSIS and GIIS. Thus using both MYTH and AP-MS we have identifi ed novel factors in islets that enhance GIIS and may serve as targets to augment GLP-1R activity.

Supported By: Novo Nordisk A/S

1737-PSTARS Regulates the Actin Cytoskeleton and Mitochondrial Locali-zationAKIHISA MINO, MARY-ELIZABETH PATTI, Boston, MA

We previously reported that STARS (striated muscle activator of Rho signaling) is the top-ranking gene upregulated in muscle from patients with type 2 diabetes & correlated with insulin resistance. Moreover, experimental modulation of STARS regulates glucose uptake & mitochondrial oxygen consumption. Since STARS regulates Rho signaling, we hypothesized that STARS effects on metabolism might be linked to interactions with the actin cytoskeleton & mitochondrial localization. We observe that subcellular localization of mitochondrial Hsp60 in C2C12 myoblasts is regulated by insulin, predominantly cytosolic at baseline but increased perinuclear localization & intensity by insulin. We observed similar insulin-dependent Hsp60 localization in primary mouse myoblasts; Hsp60 also colocalized with mDia2, the formin-related Rho GTPase effector. These events were inhibited by nocodazole & latrunculin B. These insulin-stimulated effects were increased in STARS-null myoblasts. STARS-null myoblasts also were 2.8-fold smaller (180 vs. 500µm2, n=30 cells, p<0.001), with decreased actin-stress fi bers (13vs. 80% of wild type cells, n=50) and increased membrane ruffl ing (60 vs. 7% of wild type, n=50). Together, these data suggest STARS impact on metabolism is likely mediated by its regulation of RhoA, actin polymerization, and actin cytoskeleton-and microtubule-dependent mitochondrial subcellular localization and function.

Supported By: ADA (7-12-BS-145); Sanofi

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INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

1738-PSustained Action of Ceramide on Insulin Signaling in Muscle Cells: Implication of the Double-Stranded RNA Activated Protein KinaseRIMA HAGE HASSAN, ISABELLE HAINAULT, AGNIESZKA BLACHNIO-ZABIELSKA, RANA MAHFOUZ, OLIVIER BOURRON, PASCAL FERRÉ, FABIENNE FOUFELLE, ERIC HAJDUCH, Paris, France, Białystok, Poland

Intramyocellular accumulation of fatty acid derivatives like ceramide plays a crucial role in altering the insulin message. If short-term action of ceramide inhibits the protein kinase B (PKB/Akt), long-term action of ceramide on insulin signaling is less documented. Short-term treatment of either the C2C12 cell line or human myotubes with palmitate (ceramide precursor, 16h) or directly with ceramide (2h) induces a loss of the insulin signal through the inhibition of PKB/Akt. Extended periods of treatment with palmitate (48h) or ceramide (16h), however, shows an inhibition of insulin signaling through increased-IRS1 serine 307 phosphorylation. The double-stranded RNA-dependent protein kinase (PKR) could play a central role to mediate long-term ceramide effects on IRS1, as recent studies showed that PKR acts as a key modulator of metabolic infl ammation, insulin sensitivity and glucose homeostasis in obesity. Here, we show that both PKR mRNA and PKR phosphorylation are increased in muscle of high fat diet fed mice compared to control mice as well as in myotubes from diabetic patients compared to human control myotubes. These results are confi rmed in vitro in both human and C2C12 myotubes in response to either palmitate or ceramide. Pre-treatment of C2C12 or human myotubes with PKR inhibitors prevents the inhibitory effect of either palmitate or ceramide on IRS1. Finally, we show that c-Jun kinase (JNK) mediates ceramide-activated PKR inhibitory action on IRS1. Altogether, our data show that ceramide inhibits effi ciently insulin signalling by targeting in a time dependent manner two important actors, e.g. PKB/Akt and IRS1 in myotubes.

Supported By: Société Francophone du Diabète

1739-PDefective Autophosphorylation of the Insulin Receptor with a Novel 3-basepair In-frame Deletion (ΔArg1027 Glu1028) Found in a Patient with Severe Insulin ResistanceBOLORMAA ENKHTUVSHIN, HIROYUKI TAMEMOTO, SHUICHI NAGASHIMA, NAOKO SAITO, KENT SAKAI, MANABU TAKAHASHI, DAISUKE YAMAMURO, JUN-ICHI OSUGA, SHIN-ICHI TOMINAGA, SHUN ISHIBASHI, Shimotsuke, Japan

Type A insulin resistance (IR) syndrome is caused by genetic mutations in the insulin receptor (INSR) gene. We aimed to identify the molecular mechanism of severe IR in a 35-years-old nonobese woman who was diagnosed as having type 2 diabetes at age 31 during pregnancy. She presented acanthosis nigricans, hyperglycemia (fasting plasma glucose-173mg/dl, HbA1c-10.3%), severe hyperinsulinemia (fasting insulin-66µU/ml), high C-peptide (2.1ng/ml), normal lipid profi le and absence of polycystic ovary syndrome. Tests for insulin and INSR antibodies were both negative.

To explore the underlying molecular defect of IR, we sequenced the INSR gene. We found a 3-basepair in-frame deletion in exon 17 of one allele of the INSR, which is predicted to substitute Glutamine for Arginine and Glutamic acid (ΔArg1027 Glu1028) in the kinase domain. The same mutation was found in father’s INSR, who also showed severe IR with diabetes. To clarify how the mutation impairs the receptor function, we generated plasmid vectors expressing the wild-type (WT) and ΔArg1027 Glu1028 mutants under CMV promoter and transfected them to CHO-KI cells. Blasticidine was used for selecting stable transformants. Clones with comparable (expression levels) insulin binding levels (as demonstrated by Western blot analyses) revealed by Insulin binding assays were chosen for further experiments. Scatchard analyses of insulin binding assay using 125I-insulin showed that Bmax and Kd were comparable between the two receptors. Western blot analysis revealed that the expression levels and proteolytic processing of the INSR were comparable. Treatment with insulin robustly increased autophosphorylation of WT, but not of the mutant INSR.

In conclusion, we found defective autophosphorylation of the INSR with ΔArg1027 Glu1028 mutation, which might cause severe IR.

1740-PInsulin Increases the Expression of Vimentin and C-Myc, and Enhances Breast Cancer MetastasisZARA ZELENKO, MARILYN STASINOPOULOS, ROSALYN FERGUSON, RAN ROSTOKER, DEREK LEROITH, EMILY J. GALLAGHER, New York, NY, Haifa, Israel

Women with Type 2 diabetes (T2D) have a 49% increase in breast cancer related mortality compared with women without T2D. Epidemiological studies report that increased endogenous insulin levels and increased insulin receptor (IR) expression are associated with poor survival in breast cancer patients. We used the non-obese female MKR mouse to study the effects of hyperinsulinemia on breast cancer progression. In the MKR mice a signifi cant

increase in tumor size and pulmonary metastasis is observed, compared to wild type mice. In this study, we aimed to determine the mechanisms through which hyperinsulinemia and the canonical IR signaling pathway drive tumor growth and metastasis. 100,000 MVT-1 (c-myc/vegf overexpressing) cells were injected orthotopically into 8-10 week old MKR mice. MKR mice developed signifi cantly larger MVT-1 (353.29±44mm3) tumor volumes than control mice (183.21±47mm3), p<0.05 with more numerous pulmonary metastases. Western blot and immunofl uorescent staining of primary tumors showed an increase in vimentin, an intermediate fi lament, typically expressed in cells of mesenchymal origin, and c-myc, a known transcription factor. Both vimentin and c-myc are associated with cancer metastasis. To assess if insulin and IR signaling directly affects the expression these markers, in vitro studies were performed on MVT-1 and human MCF7 cells. 10nM of insulin signifi cantly increased the expression of c-myc at 60 minutes and vimentin at 48 hours in MVT-1 cells. Silencing the IR and inhibiting IR signaling decreased c-myc expression in both cell lines. These results imply that the hyperinsulinemia may drive tumor growth and metastasis through the IR by increasing vimentin and c-myc expression. These observations allow us to begin to understand the different key players that contribute to tumor progression in the setting of T2D. These downstream elements could open the fi eld to new targets for therapy to improve survival in women with breast cancer and hyperinsulinemia.

Supported By: ADA (1-13-BS-108)

INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

Guided Audio Tour: Dynamic Regulation of Insulin Secretion (Posters: 1741-P to 1748-P), see page 13.

& 1741-PA Novel Long-Acting Analogue of Xenin-25 with Promising Anti-diabetic PotentialNIGEL IRWIN, CHRISTINE MA MARTIN, PETER R. FLATT, VICTOR A. GAULT, Coler-aine, United Kingdom

Xenin-25 is a peptide co-secreted with glucose-dependent insulinotropic polypeptide (GIP) from intestinal K-cells following a meal. Xenin-25 is believed to play a role in glucose homeostasis and possibly potentiate the biological action of GIP. We have investigated the effects of sub-chronic administration of a longer-acting xenin-25 analogue, xenin-25[Lys13PAL], in mice fed a high fat (45%) diet. Initial acute studies confi rmed the superior persistent glucose lowering (p<0.05) and insulin releasing (p<0.05) actions of xenin-25[Lys13PAL] compared to native xenin-25. Twice daily intraperitoneal injection of xenin-25[Lys13PAL] for 14 days had no signifi cant effect on energy intake or body weight. Circulating plasma glucose and insulin levels were also unchanged. However, on day 14, overall plasma glucose levels during a glucose tolerance and oral nutrient challenge were signifi cantly (p<0.05) lowered by xenin-25[Lys13PAL] treatment. These changes were accompanied by signifi cant enhancement of intraperitoneal (p<0.05) and oral (p<0.001) nutrient-stimulated insulin concentrations when compared to controls. Moreover, xenin-25[Lys13PAL] treated high fat mice had markedly improved insulinotropic (p<0.01) and glucose-lowering (p<0.01) actions in response to exogenous GIP administration on day 14 when compared to saline controls. However, no appreciable change in insulin sensitivity was observed with xenin-25[Lys13PAL] treatment. Finally, ambulatory activity was signifi cantly (p<0.05 to p<0.001) increased during the dark phase in xenin-25[Lys13PAL] mice compared to controls. These data indicate that sustained administration of a stable analogue of xenin-25 exerts a spectrum of benefi cial metabolic effects in high fat fed mice. This demonstrates the utility of long-acting analogues of xenin-25 as novel treatments for type 2 diabetes.

Supported By: ERDF

& 1742-PInsulin-like Growth Factor Binding Protein (IGFBP1)-1: Favorable In Vivo Actions in Glucose RegulationNATALIE J. HAYWOOD, NADIRA Y. YULDASHEVA, AMIR AZIZ, JESSICA SMITH, PAUL A. CORDELL, MARK T. KEARNEY, STEPHEN B. WHEATCROFT, Leeds, United Kingdom

In humans, the circulating concentration of IGFBP1 has been proposed as a marker of insulin sensitivity. In prospective studies, low circulating levels of IGFBP1 predict the development of type 2 diabetes. IGFBP1 can impact on cellular functions via an RGD (α5β1 integrin binding) motif independent of IGF binding. However, whether IGFBP1 is causally implicated in glucose counter-regulation and could be exploited therapeutically remains unexplored.

WITHDRAWN

1665-p insulin action—adipocyte biology · a rabbit antiserum against pparγ pser492 was prepared...

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