1692-p 1694-p biopsy-proven insulitis of clinical...

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ADA-Supported Research

INSULIN ACTION—ADIPOCYTE BIOLOGY

1692-PStress in Beta Cells Obtained with Laser Capture Microdissection from Cadaver Pancreases of Brain Dead DonorsAREF EBRAHIMI, MIN-HO JUNG, JONATHAN M. DREYFUSS, HUI PAN, DENNIS C. SGROI, SUSAN BONNER-WEIR, GORDON C. WEIR, Boston, MA

Brain death of pancreas donors is thought to lead to the expression of infl ammatory, stress and apoptotic pathways in isolated islets resulting in poor clinical outcomes. To test this hypothesis we obtained cadaveric pan-creases from brain dead pancreatic donors (n=7, mean age 5011) and normal pancreatic tissue obtained at surgery done for pancreatic neoplasms (n=7, age 699). Frozen sections were subjected to laser capture microdissection to obtain beta-cell rich islet tissue, from which extracted RNA was analyzed with Affymetrix arrays. Gene expression of the two groups was evaluated with principle component analysis (PCA), and differential expression anal-ysis was performed for genes and pathways. The pathways expressed at the highest signifi cance included Glycolysis, Unfolded Protein Response, MTORC1 Signaling, and Pancreatic Beta Cells. Pathways of Apoptosis were not differentially expressed.

A striking fi nding was the unfolded protein response (UPR), which is a pro-tective component of the endoplasmic reticulum (ER) stress response that if severe can cause apoptosis. An important group of protective chaperone genes were upregulated in the cadaver donors (HSP90B1, HSPA5, PDIA6, DnaJB9 and DnaJC3). In addition the protective genes of ER-associated pro-tein degradation (ERAD) proteins (DERL 1, 2, 3) were upregulated. In con-trast the proapoptotic “executioner” genes CHOP and JNK were not upregu-lated. Other evidence of stress included upregulation of genes associated with NFkB activation and TNF pathways, including TNFRSF1A, IL17RB and IL13RA1. Other upregulated genes of interest included REG1B, LDHA and Tis-sue Factor.

In conclusion, while there was little evidence of active apoptosis in beta cells from cadaver donors, stress markers were found that could represent vulnerability resulting from brain death and/or the trauma of organ preser-vation. This could account for some of the early beta cell death found with islet transplantation.

Supported By: National Institutes of Health

1693-PAbnormal Responses to Oral and Intravenous Glucose Stimula-tion in Intestinal Transplant Recipients With or Without Pancreas AllograftDAHAE LEE, LAURENS CEULEMANS, DANIEL JACOBS-TULLENEERS-THEVISSEN, BART KEYMEULEN, ILSE WEETS, DIETHARD MONBALIU, JACQUES PIRENNE, CHANTAL MATHIEU, PIETER GILLARD, Leuven, Belgium, Jette, Belgium

Technique of liver-small intestinal transplantation (LITx) often includes a partial or whole pancreas allograft to preserve bile duct continuity. Aim of the study was to measure responses to oral and intravenous (IV) glucose stimulation in LITx recipients in comparison to recipients of isolated small intestinal transplant (ITx) and nondiabetic controls.

Plasma glucose, C-peptide and insulin were measured during 180-minute oral glucose tolerance test (OGTT) and 160-minute hyperglycemic clamp (HG clamp) in nondiabetic LITx (n = 3) and ITx (n = 3) recipients under immuno-suppression with azathioprine, tacrolimus and low-dose corticosteroids. Ten healthy volunteers served as controls.

All but 1 LITx and ITx recipients showed abnormal glucose profi le after OGTT despite higher basal C-peptide. 2/3 ITx recipients showed higher glu-cose peak during OGTT and 45% lower functional beta cell mass (FBM) than controls. In 1 LITx recipient with partial pancreas allograft, OGTT resulted in delayed but higher glucose peak in parallel with high C-peptide and insulin levels, leading to rapid decrease in glucose and severe hypoglycemia. Both LITx recipients with partial pancreas allograft had FBM above percentile 85 of controls. One LITx recipient with whole pancreas allograft showed very high baseline C-peptide, due to systemic venous drainage of recipient pan-creas and high insulin resistance (HOMA-R=3.60). Glucose peak during OGTT was higher and delayed but without late hypoglycemia. During all phases of the HG clamp, C-peptide and insulin were higher with FBM calculated at 150% of controls.

Intestinal transplant recipients show abnormal responses to both oral and IV glucose stimulation. Several factors may explain these observations: intestinal denervation, loss of normal anatomy, loss of FBM and insulin resis-tance. Analysis of these complex cases can help to understand regulation of glucose metabolism in normal and posttransplant conditions.

1694-PBiopsy-Proven Insulitis of Clinical Islet Transplantation Is Not Reversed by Steroid TherapyANNA LAM, BEHRUZ RAHIMI, SHARLEEN IMES, KIM SOLEZ, JAMES SHAPIRO, PETER A. SENIOR, Edmonton, AB, Canada

Gradual decline in islet function remains a challenge in clinical islet transplantation (CIT), but acute graft loss is relatively uncommon. Here we describe a case of acute decline in graft function with histology suggesting an immune mechanism. A 49 year old female (BMI 24.4 kg/m2, insulin 0.3 U/kg) with type 1 diabetes for 37 years underwent two CIT (6071 and 6827 islet equivalents/kg) following alemtuzumab induction with tacrolimus (TAC, mean 8 ug/L) and mycophenolate mofetil for maintenance. Initial engraft-ment was reasonable (β2 score of 12 at 1 week), but β2 score gradually declined (Figure 1) rising to 19 after the second CIT. Insulin independence was achieved only briefl y following each CIT. There was an acute decline in β2 score after day 120 (Figure 1) prompting liver biopsy. Three collections of insulin staining cells heavily infi ltrated with mononuclear infl ammatory cells and eosinophils were seen. TAC was increased (mean 14 ug/L) and prednisone 50 mg/day started, but graft function did not recover. Pre-CIT anti-insulin antibodies were positive and anti-GAD negative, while panel reactive antibodies were 0% pre- and post-CIT. This is the fi rst report of biopsy proven insulitis post-CIT, which may be due to acute rejection and/or recurrent autoimmunity. Unfortunately, this did not reverse with steroid therapy. Further study of allo- and auto-immunity in acute CIT failure may allow targeted immune therapy.Figure 1. Post-CIT Graft Function as Measured by BETA-2 Score, a Clinical Composite Score (Fasting C-peptide, Fasting Blood Glucose, Insulin Dose, and HbA1C) after Two Transplants.


Moderated Poster Discussion: Feel the Burn—Factors that Activate Brown Fat (Posters: 1695-P to 1701-P), see page 14.

& 1695-PDifferential Roles of Fox0 Proteins in White and Brown Adipose TissueERICA HOMAN, BRIAN T. O’NEILL, MASAJI SAKAGUCHI, CHRISTIE PENNIMAN, DOMENICO ACCILI, C. RONALD KAHN, Boston, MA, New York, NY

Insulin and IGF-1 are essential for mediating normal adipocyte differen-tiation and metabolism. In insulin/IGF responsive tissues, insulin and IGF-1 induce phosphorylation of the Forkhead Box O family of transcription factors (Fox0s) via action of Akt maintaining their cytoplasmic localization, reversing the activation of multiple Fox0-responsive genes involved in maintenance of energy homeostasis, glucose metabolism, cell cycle arrest, and cell death. We explored whether Fox0 activation controls the decrease in white and brown fat mass, glucose intolerance, and decrease in brown fat activation observed in mice with fat-specifi c knockout of both the insulin and IGF-1 receptors (FIGIRKO mice). To this end, we generated mice with fat-specifi c knockout of the insulin and IGF-1 receptors, as well as knock-out of the three Fox0s expressed in fat [Fox01, Fox03, and Fox04] (F-Quint KO) and compared these with control and FIGIRKO mice. As previously observed, FIGIRKO mice

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exhibited a marked loss of all white and brown fat depots, marked hypergly-cemia, insulin resistance, fatty liver and inability to maintain body tempera-ture during a cold challenge. By contrast, the F-Quint KO mice had similar glu-cose tolerance to control animals, had normalized brown fat mass and were able to maintain body temperature during cold exposure. However, like the FIGIRKO mice, the F-Quint KO mice failed to respond to insulin stimulation, had markedly decreased subcutaneous fat pad weights, absence of perigo-nadal fat pads, and hepatosteatosis. Thus, we demonstrate differential regulation of Fox0 proteins in adipose tissue such that suppression of Fox0s by insulin/IGF-1 action is critical for brown fat differentiation and systemic glucose metabolism, but not critical for the regulation of white adipose tis-sue mass and systemic insulin sensitivity.

Supported By: National Institutes of Health (5T32DK007260-39)

& 1696-PRegulatory Control of Adipose Mass: Insights from a Novel Model of LipodystrophyMASAJI SAKAGUCHI, SHIHO FUJISAKA, WEIKANG CAI, MASAHIRO KONISHI, BRIAN T. O’NEILL, HIROKAZU TAKAHASHI, C. RONALD KAHN, Boston, MA, Toyama, Japan

White and brown adipocytes are important for regulation of energy stor-age and expenditure and, under normal conditions, remain relatively stable, with very low turnover rates. Insulin and IGF1 signaling regulate development and function of both brown and white adipose tissue. To better understand these hormones and factors regulating adipose tissue mass in adult animals, we created inducible fat-specifi c knockouts of IR, IGF1R or both using fl oxed mice and mice carrying a tamoxifen-inducible Cre-ERT2 transgene on the adiponectin promoter. Within 3 days after tamoxifen treatment, mice with KO of IR or both IR and IGF1R displayed a >70% decrease in WAT and BAT mass, associated with marked decreases of serum adiponectin and leptin, and a metabolic syndrome including severe hyperglycemia, hyperinsuline-mia, β-cell proliferation and cold intolerance. However, within 10 to 30 days these phenotypes disappeared, and knockout mice displayed virtually com-plete recovery of WAT and BAT mass. Based on FACS analysis of the stro-movascular fraction and lineage-tracing with a Rosa26-Tomato/GFP (mTmG) reporter, this recovery was due to proliferation of preadipocytes and regen-eration of new adipocytes. Leptin administered by subcutaneous pump infu-sion during this 30 day period completely prevented development of hyperg-lycemia and fatty liver, but had no signifi cant effect on loss of WAT and BAT following IR/IGFR knockout. In vivo leptin also suppressed the increase in preadipocyte proliferation and recovery of fat mass, as monitored by FACS analysis and mTmG lineage tracing. In vitro, on the other hand, leptin had no effect on proliferation of brown or white preadipocytes and only minimal effects on their differentiation. Thus, in this new model, development of lip-odystrophy provokes a powerful stimulus for the regeneration of both white and brown adipose tissue. The factors that regulate regeneration of adipose mass are distinct from leptin, but are, at least in part, regulated by leptin or its effects on metabolism.

Supported By: Japan Society for the Promotion of Science

& 1697-PFat-specifi c Rheb Ablation Promotes Beige Fat Development and ThermogenesisWEN MENG, XIUCI LIANG, HONGZHI CHEN, SIJIA HE, FANG HU, FENG LIU, Chang -sha, China, San Antonio, TX

Beiging of white adipose tissue (WAT) has potential anti-obesity and antidiabetes effects, yet its underlying mechanisms remain elusive. We have recently demonstrated that fat-specifi c knockout of Grb10 in mice upregu-lates mTORC1 signaling, promotes thermogenesis, and increases the expres-sion of thermogenic and lipolytic genes. To directly elucidate role of mTORC1 in beige fat development, we generated mice in which the expression of the mTOR upstream activator, Rheb, is disrupted in adipose tissue. Adipose-specifi c Rheb ablation induces WAT beiging, enhances energy expenditure and cold tolerance, increases resistance to high-fat diet-induced obesity, and improves global insulin sensitivity in vivo. Protein kinase A (PKA) activ-ity and UCP1 expression are higher in subcutaneous WAT of RhebfKO mice compared to control mice. On the other hand, Rheb overexpression inhibited PKA activity and UCP1 expression in adipocytes. Our results identify adipose Rheb as a key regulator of WAT beiging and reveal a potential mechanism underlying the crosstalk between the mTORC1 and PKA signaling pathways in adipocytes.

Supported By: National Basic Research Program of China (2014CB910501 to F.L.); National Natural Science Foundation of China (81130015 to F.L.), (31471131 to F.H.)

& 1698-PRaptor Defi ciency Promotes Browning of White Adipose Tissue via Adiponectin-dependent MechanismsXIAOFENG DING, YAN LUO, XIN YANG, XIAOQING WANG, XIANYUN ZHENG, XUEXIAN YANG, MEILIAN LIU, Albuquerque, NM, Changsha, China

Beige adipocytes burn lipid by dissipating energy in the form of heat and offer a new way to battle obesity and its related disorders. However, what intracellular signaling pathways drive the browning effect remains largely unknown. Here, we show that inhibition of mammalian target of rapamycin complex 1 (mTORC1) by adipose specifi c-deletion of raptor (raptor fKO), a key component of mTORC1 promoted browning of inguinal WAT (iWAT) and enhanced basal and cold-induced energy expenditure, concurrently with an increase in population of beige cells in iWAT. However, disrupting raptor had no signifi cant effect on CL316,243-induced UCP1 expression and oxygen consumption in the primary brown adipocytes. On the other hand, raptor defi -ciency led to marked downregulation of expression and circulating level of adiponectin and upregulation of type 2 infl ammatory cytokines such as IL-4 and IL-13. Similar to raptor fKO mice, adiponectin-defi cient mice displayed increased basal and cold-induced energy expenditure, elevated browning effect and upregulation of IL-4/13 pathway. The effects of raptor defi ciency on browning effect and thermogenesis were diminished by administration of adiponectin receptor agonist adipoRon in vivo. Our study identifi ed mTORC1 signaling and adiponectin as the key regulators of recruitment and activation of beige adipocytes.

Supported By: American Diabetes Association (1-13-JF-37 to M.L.); American Heart Association

& 1699-PNFIA Controls the Brown Fat Gene Program by Co-Localizing with PPARgamma at Cell Type-Specifi c EnhancersYUTA HIRAIKE, HIRONORI WAKI, JING YU, MASAHIRO NAKAMURA, KANA MIYAKE, KEN SUZUKI, RYO NAKAKI, WEI SUN, TOMOHISA AOYAMA, YUSUKE HIROTA, GAKU NAGANO, HARUYA OHNO, KENJI OKI, MASAYASU YONEDA, SHUICHI TSUTSUMI, HIROYUKI ABURATANI, TOSHIMASA YAMAUCHI, TAKASHI KADOWAKI, Tokyo, Japan, Hiroshima, Japan

Brown fat dissipates energy in the form of heat, and is a promising target for treatment of obesity. However, global landscape of brown fat develop-ment is not entirely understood. Here we performed FAIRE-seq on murine brown and white fat tissues and found that the binding motif for Nuclear fac-tor I (NFI) transcription factor is enriched within brown-fat-specifi c open chro-matin regions. Of the four isoforms of NFI family, NFIA is highly expressed in brown fat compared to white fat or muscle. Introduction of NFIA into myoblasts results in lipid accumulation, activation of the brown-fat-specifi c gene program and suppression of muscle genes. Conversely, knockdown of NFIA in brown adipocytes suppressed the brown-fat-specifi c genes. NFIA selectively co-localize with PPARgamma at the brown-fat-specifi c enhanc-ers, and co-localization of NFIA facilitates binding of PPARgamma, leading to increased chromatin accessibility and active transcription. Brown fat of NFIA knockout mouse neonates show impaired expression of brown fat genes and reciprocal elevation of muscle genes. Finally, human perirenal brown fat of patients with pheochromocytoma show concurrent increase in NFIA and UCP1 expression. Collectively, these results indicate that NFIA is a novel key transcription factor that co-localizes with PPARgamma and activates the brown-fat-specifi c gene program.

& 1700-PRegulation of Brown Fat Activation and Development by Insulin’s Specifi c Actions on the EndotheliumKYOUNGMIN PARK, QIAN LI, CHRISTIAN RASK-MADSEN, ERNESTO MADD-ALONI, MOGHER KHAMAISI, MATTHEW D. LYNES, ALISON BURKART, MANOJ GUPTA, YU-HUA TSENG, GEORGE L. KING, Boston, MA

Dysfunctional white and brown adipose tissues (WAT and BAT) can con-tribute to insulin resistance and diabetes. Since angiogenesis in fat plays an important role in their function, we explored the possibility that enhanc-ing insulin actions in the endothelium can increase energy expenditure, decrease WAT and improve insulin sensitivity by enhancing BAT activation and development. Insulin’s actions on endothelium were enhanced by selec-tively overexpressing insulin receptor subtract 1 in endothelial cells to gen-erate the ECIRS1 transgenic mice which exhibited enhanced activation of Akt/eNOS pathway and NO production by >5 fold. When fed with high fat diet (HFD, 60% fat), ECIRS1 mice gained 12% less weight, had 15% reduc-tion in WAT mass and increased BAT by 2.3 fold vs. wild type mice (Wt). ECIRS1 mice attained lower blood glucose levels in glucose tolerance test.

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The anti-obese action, and improved insulin sensitivity in ECIRS1 mice were prevented by a general inhibitor of eNOS activity (L-NAME). CLAMS analysis showed that ECIRS1 mice had higher rates of respiratory exchange rate by 46% ±11% (p<0.05), but without differences in motor behaviors and food intake. The increase in energy expenditure and decrease in WAT suggested an improvement of mitochondrial activity in BAT from ECIRS1 mice. Bioen-ergetic analysis of isolated mitochondria from BAT of ECIRS1 mice showed higher oxygen consumption rate by 23% ±11% (p<0.05), which were pre-vented by L-NAME. The size and vascularity of BAT were also increased by 43% ± 13% and 37% ±16% respectively (p<0.05) which were also inhibited by L-NAME. The distribution of mitochondrial size in BAT, measured by elec-tron microscopy, exhibited increases in size by 63% ± 14% and 34% ±10% on RD and HFD respectively, vs. Wt mice. These fi ndings are the fi rst to dem-onstrate that enhancing insulin action specifi cally to the endothelium can regulate the development and activation of BAT leading to an improvement of systemic insulin sensitivity in diet-induced obesity.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases

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1702-PAdipose Mitochondrial Biogenesis in Browning and a Potential Role of miRNA-494MENGISTU LEMECHA SHUKARE, KATSUTARO MORINO, TAKESHI IMAMURA, HIROTAKA IWASAKI, NATSUKO OHASHI, HIROTAKA YAMAMOTO, SATOSHI UGI, HIROSHI MAEGAWA, Otsu, Japan

Cold exposure causes browning in adipose tissue. Mitochondrial bio-genesis is activated by various stimuli including beta-3 adrenergic recep-tor (ADRB3). However, molecular mechanisms underlying this phenomenon especially by micro-RNA remains unknown. In this study, we tested the mito-chondrial biogenesis during adipogenesis and browning in 3T3L1 adipocyte.

Protein expression of mitochondrial transcription factor A, succinate dehydrogenase, pyruvate dehydrogenase, and adenine nucleotide translo-cator proteins were increased during differentiation of 3T3L1 from day 0 to day 8. These proteins were further increased during browning stimulation for 6 days with triiodothyronine, 3-isobutyl-1-methylxanthine and rosigli-tazone. We also observed robust change in the expression of miRNA-494 which we have been reported the role in mitochondrial biogenesis in skel-etal muscle. Uncoupling protein 1 (UCP-1), peroxisome proliferator-activated receptor gamma, coactivator 1 alpha, and ADRB3 mRNAs were increased 1.2-, 1.2-, 1.3-fold, respectively by miRNA-494 overexpression compared to empty vector in adipocytes. Furthermore, mRNA of UCP-1 and ADRB3 were

reduced by miRNA494 inhibitor for 0.8- and 0.6- fold, respectively. More-over, expression of UCP-1 and miRNA-494 were strongly stimulated by iso-proterenol, beta adrenergic agonist, 20- and 13-fold, respectively. Finally, we tested the expression of UCP-1 and miRNA-494 in the adipose tissue from 3 hours of cold exposed mice, and found that these expression were signifi -cantly increased compare to the mice in room temperature.

Our result suggests that mitochondrial biogenesis enhanced by miRNA-494 under stimulation of ADRB3 maybe a potential mechanism of browning process.

1703-PEffect of Weight Loss on Adipose Senescent Cells in ObesityANA ESPINOSA DEYCAZA, BARBARA (GISELLA) CARRANZA-LEON, ESBEN SON-DERGAARD, DEBRA HARTENECK, MARIA MORGAN-BATHKE, DANAE A. DELI-VANIS, MICHAEL D. JENSEN, Rochester, MN, Nashville, TN

Senescent preadipocytes cannot replicate or differentiate into mature adipocytes. Senescent cells are more prevalent in adipose tissue (AT) of obe-sity and may play a role in AT dysfunction. Because weight loss improves AT dysfunction, we studied whether weight loss reduces the number of senes-cent cells in AT.

Seventeen overweight/obese participants (4 men) underwent body com-position studies (DXA) and femoral-abdominal subcutaneous AT biopsies, for senescent cell determination, before and after weight loss. Senescent cells in AT were identifi ed by senescence associated β-galactosidase staining and expressed as proportion of total number of nucleated cells (DAPI). Adipose dysfunction was estimated using AT insulin resistance (Adipo-IR) calculated as fasting palmitate concentration x fasting insulin concentration (N=14). We used a comprehensive lifestyle intervention weight loss program.

Median age was 39 years (range: 23-55) and BMI 33.1 kg/m2 (range: 29.6-36.5). At baseline, the percentage of AT senescent cells averaged 2.8% and 4.4% in the abdominal subcutaneous and femoral fat, respectively (p=0.001, abdomen vs. femoral). There was a positive correlation between % body fat and both abdominal AT senescent cells (rs= 0.58, p=0.02) and femoral AT senescent cells (rs= 0.49, p=0.052). The median loss of 11% of their initial body weight (IQR 7.4, 13.5%) resulted in a median Adipo-IR reduction of -1.3 mmol/l X pmol/l (IQR -0.6, -3.4) (p=0.007), but no signifi cant change in senes-cent cells proportion in the abdominal or femoral fat depots. The median change in senescent cells was 0.48 (IQR -0.38, 1.9) (p= 0.12) in the abdominal AT and 1 (IQR -0.5, 1.84) (p= 0.22) in the femoral AT.

Despite a positive relationship between senescent cells in AT and adipos-ity, the proportion of senescent cells in AT is not affected by weight loss. The improvement in AT insulin sensitivity after weight loss without reductions in senescent cells suggests that senescence might not play a role in obesity-associated AT dysfunction.


1704-P11β-Hydroxysteroid Dehydrogenase Type 1 (11β-HSD1) Mediates Insulin Resistance through JNK Activation in AdipocytesCHAO ZHENG, GUANG LIANG, Wenzhou, China

Inducing insulin resistance is the major side effect of glucocorticoids. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a key enzyme that catalyzes the conversion of the inactive glucocorticoid to active form. how-ever, the molecular mechanisms by which glucocorticoid induce insulin resis-tance remain limited. JNK plays an important role in insulin resistance. We hypothesize that JNK may mediate 11β-HSD1-induced insulin resistance. Our results found that JNK was activated in adipose tissue of HFD mice and in cultured adipocytes after glucocorticoids stimulation or overexpressing 11β-HSD1. Inhibition of 11β-HSD1 blocked the activation of JNK in adipose tissue of HFD mice as well as in cultured adipocytes stimulated with gluco-corticoids or overexpressing 11β-HSD1. Furthermore, prednisone stimulation or 11β-HSD1 overexpression signifi cantly impaired insulin signal pathway, while these effects were reversed by JNK inhibitor C66 or dominant nega-tive JNK, respectively. Finally, oral administration with either PF00195715 or C66 in obese mice remarkably mitigated insulin resistance. Taken together, glucocorticoids and 11β-HSD1 mediate insulin resistance through JNK acti-vation in adipocytes. Our fi ndings suggest that inhibition of JNK represents a valid strategy for treating insulin resistance induced by glucocorticoid excess and abuse.

Supported By: National Natural Science Foundation of China (81200630 to C.Z.), (81500291 to G.L.); Natural Science Fund Committee of Zhejiang Province (12H07001 to C.Z.); Wenzhou Science and Technology Bureau (H20150001 to C.Z.)

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1705-PA Human Mutation in PI 3-Kinase Creates Tissue Specifi c Insulin ResistanceMARIE H. SOLHEIM, JONATHON N. WINNAY, JASON K. KIM, ANDERS MOLVEN, PAL R. NJOLSTAD, C. RONALD KAHN, Boston, MA, Worcester, MA, Bergen, Nor-way

Phosphatidylinositol 3-kinase (PI3K) is a central component of insulin signaling in control of glucose metabolism, cell growth/differentiation, and apoptosis. We have previously identifi ed a heterozygous missense mutation (Arg649Trp) in the p85 regulatory subunit of PI3K (PIK3R1) in patients with a familial syndrome characterized by insulin resistance, partial lipodystro-phy and short stature. We have generated knock-in mice with this mutation to investigate the mutation’s role in insulin action and signaling. Similar to the patients, mutant mice showed a reduction in body weight and length, partial lipodystrophy and systemic insulin resistance. This was associated with a reduced capacity of insulin and other growth factors to activate the PI 3-kinase pathway and its downstream targets such as Akt in vivo in liver, muscle and fat, as well as in vitro in in hepatocytes and immortalized brown preadipocytes derived from these mice. Euglycemic-hyperinsuline-mic clamp studies revealed marked insulin resistance in the knock-in mice with a 58% reduction in the glucose infusion rate (46.4±6.6 vs. 73.3±3.0 mg/kg/min, p<0.01) and an associated decrease in the rate of glucose turn-over 51.8±3.4 mg/kg/min vs. 63.8±2.0 mg/kg/min, p<0.05) when compared to controls. In addition, hepatic glucose production was suppressed during the clamp by only 76% in mutant mice compared to the 100% suppression in controls (p<0.01). Surprisingly, no difference was observed in glucose uptake in skeletal muscle and brown adipose tissue between control and mutant mice, whereas white adipose tissue glucose uptake was marked reduced (70.8±12.2 vs. 32.6±4.2 nmol/g/min) (p<0.01). Thus, despite ubiquitous expression of the mutant allele, the mutation uncovers differences in insulin signaling and metabolic phenotype among insulin sensitive tissues with a clear reduction in adipose tissue glucose uptake and a relative failure of insulin to suppress gluconeogenesis, creating a novel form of tissue-specifi c insulin resistance.

1706-PRegulation of Filamin A Cleavage Prevents Adipocyte Lipid Accumu-la tionJYOTI RANJAN, SAIE MOGRE, KALYANI V. GUNTUR, STEPHANE GESTA, VIVEK K. VISHNUDAS, RANGAPRASAD SARANGARAJAN, NIVEN R. NARAIN, Framing-ham, MA

FlnA was identifi ed as a potential target for obesity from the Berg Inter-rogative Biology® platform using an adipocyte model generated by modu-lation of glucose and lipids in vitro. Filamin A (FlnA) is a 280 kDa scaffold-ing protein with reported function in regulating lipid droplet formation and in insulin signaling. We have previously demonstrated that reducing FlnA expression in human adipocytes leads to enhanced lipid accumulation and basal lipid mobilization. FlnA expression profi le in intra-abdominal white adipose tissue of high-fat diet mice indicated an increase in FlnA cleavage. In several cell types, FlnA cleavage is known to be regulated by calcium-dependent cysteine protease, calpain. As expected, treatment of immortal-ized human adipocytes with calpain resulted in FlnA cleavage, while treat-ment with calpastatin (a specifi c calpain inhibitor) reduced it. Furthermore, increased FlnA cleavage induced by calpain was associated with increased triglyceride accumulation in adipocytes subjected to conditions recapitulat-ing pathology of obesity. Interestingly, treatment with calpastatin, as well as other calpain inhibitors (acetyl calpastatin, PD150606 and ALLN peptide) prevented triglyceride accumulation induced by simulated conditions. This effect was also associated with increased adiponectin expression, secre-tion, and a reduction in basal lipolysis. Characteristic changes observed in response to calpastatin treatment could be prevented by knocking down expression of FlnA, suggestive of the requirement of FlnA for calpastatin to mediate these changes. Altogether, these data demonstrate that inhibition of FlnA cleavage by calpain inhibitors could effectively prevent increased adiposity. Therefore in adipose tissue, increasing FlnA levels by prevention of its cleavage could serve as an effective therapeutic strategy for treat-ment of obesity and associated metabolic disease.

1707-PIncreased Adipokine Production in the Chow-Fed Adipocyte-specifi c OSMR Knockout MouseCARRIE M. ELKS, JENNIFER L. BAILEY, HARDY HANG, RANDALL L. MYNATT, JACQUELINE M. STEPHENS, Baton Rouge, LA

Oncostatin M (OSM), an IL-6 family cytokine produced by adipose tissue macrophages, is highly up-regulated in obesity. Acting through its cognate receptor (OSMR), OSM signifi cantly up-regulates pro-infl ammatory and pro-fi brotic genes in cultured adipocytes, and OSMR knockdown prevents these changes. We hypothesized that adipocyte-specifi c OSMR knockdown would have similarly benefi cial effects in vivo. Adiponectin-Cre mice were crossed with fl oxed OSMR mice to create mice lacking OSMR in mature adipocytes (OSMR FKO). Chow-fed male OSMR FKO mice and littermate controls were examined at 7-8 months of age. No signifi cant differences in body weight or 4-hour fasting blood glucose were detected between genotypes. Akt phos-phorylation in response to an acute insulin bolus was blunted in epididymal adipose tissue of OSMR FKO mice, suggesting decreased adipose tissue insu-lin sensitivity. When assessed by protein array, epididymal fat of OSMR FKO mice also revealed signifi cant increases in several insulin resistance- and infl ammation-associated adipokines: IGF binding protein 3 (increased 3.5-fold), lipocalin 2 (increased 1.5-fold), C-reactive protein (increased 2-fold), and resistin (increased 2.5-fold). Collectively, these data suggest a possible requirement for intact adipocyte OSM signaling in maintenance of adipose tissue homeostasis and insulin sensitivity. Current efforts are focused on elucidating the molecular mechanisms responsible for altered adipose tissue homeostasis in the OSMR FKO mouse model.

Supported By: National Institutes of Health (P20 GM103528)

1708-PCB1 Antagonist Increased Natriuretic Peptide Receptors that Pro-mote Fat Browning in the Subcutaneous and Visceral Fat in the Fat-Fed DogMALINI S. IYER, RICHARD N. BERGMAN, JOYCE M. RICHEY, ISAAC ASARE BEDIAKO, ORISON O. WOOLCOTT, STELLA P. KIM, CATHRYN KOLKA, DEBORAH J. CLEGG, MORVARID KABIR, Los Angeles, CA

We have recently demonstrated that CB-1 receptor antagonism increased expression of genes involved in browning of adipose tissues, specifi cally in the subcutaneous (SC) and visceral (VIS) depots. The mechanism (s) by which the CB1 antagonist Rimonabant (RIM) promotes adipocyte browning is unknown. Natriuretic peptides (NPs) are a group of peptide-hormones mainly secreted from the heart which promote browning of the white adipose tis-sues. The current study examines the longitudinal changes of NPs receptors (NPR1, 2, 3) expression in the SC and VIS depots by CB1 antagonist RIM. Conscious dogs were fed a high fat diet (HFD, 52% fat) for 6 weeks followed by a continued 16 weeks of fat feeding with either HFD + placebo (PL) (n=9) or HFD + RIM (1.25 mg/kg per day; n=11). Biopsies from SC and VIS depots were obtained for gene expression: before HFD (Pre-fat), after 6 weeks of fat (HFD) and 16 weeks of HFD +/- RIM. RIM increased NPR1 expression in SC depot by 2.5 fold (P<0.05) and in VIS depot by 5 fold (P<0.001) com-pared to HFD groups. Similarly, RIM increased NPR2 by 4 times in the SC and VIS depots (P<0.001). There is a tendency for NPR3 to be increased by CB1 antagonist only in the VIS depot by 2 times (P=0.052). Our data suggests that one of the mechanisms by which the CB1-R antagonist increases browning of adipose tissue is through upregulation of the key factors, NPs. Increasing of the browning process in the SC and VIS depots is an important mechanism by which the CB1 antagonist regulates energy homeostasis.

Supported By: Sanofi

1709-PEffect of Low Carbohydrate Weight Loss Diet on Adipose Cell SizeTRACEY MCLAUGHLIN, LI-FEN LIU, ERIN AVERY, WEN-JUN SHEN, CORAAL COHEN, FREDERIC KRAEMER, SAMUEL CUSHMAN, CHRISTOPHER D. GARDNER, Stanford, CA, Bethesda, MD

Larger adipose cell size is associated with risk for insulin resistance and type 2 diabetes. Diets high in carbohydrate (CHO) are insulinogenic and may promote fat storage in adipocytes, thus contributing to cell enlargement. The goal of the current study was to test the hypothesis that weight loss via CHO vs. fat restriction would lower ambient insulin concentrations and reduce adipose cell size. To test this, 90 healthy obese participants were randomly assigned to low CHO or low fat diet for 6 mos. Intake of CHO or fat was limited to 20g/d for 8 weeks and then gradually increased. Abdominal adipose tissue biopsies were performed at baseline and 6 mos in diet-com-pliant participants who lost ≥5% initial body weight. Adipose cell size distri-bution was measured via Beckman Coulter Multisizer III (A), yielding adipose

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cell size (peak diameter), and distribution (nadir separates small vs. large cell populations). Ambient insulin and FFA concentrations were measured dur-ing diet-congruent 4-hr meal tolerance tests (MTT). 30 subjects completed. Mean age, BMI, and female sex were 40 yrs, 33 kg/m2, and 50%female. Subjects lost 9±3 kg. The low CHO diet decreased insulin but not FFA concen-trations, whereas the low fat diet consistently decreased FFA (B). Adipose cell size decreased signifi cantly on the low CHO but not the low fat diet (C). In conclusion, a low CHO vs. low fat hypocaloric diet reduces adipose cell size but yields greater circulating FFA.Figure.


1710-PProtective Role of High Adiponectin Levels on Metabolic Functions in a Mouse Model of PCOSBELEN CHANCLON GARCIA, ANNA BENRICK, YANLING WU, LAILA HADI, STE-PHEN FRANKS, ELISABET STENER-VICTORIN, INGRID WERNSTEDT ASTER-HOLM, Gothenburg, Sweden, London, United Kingdom

Polycystic ovary syndrome (PCOS) is a hormonal disorder that affects 5-10% of women in their reproductive age. PCOS is often related to meta-bolic disturbances, such as obesity and insulin resistance. Serum adiponec-tin and adipocyte size are the strongest factors linked to decreased insu-lin sensitivity in PCOS. Therefore, we hypothesize that adiponectin has a protective effect on the development of metabolic dysfunction in this PCOS model. To address this we have investigated the metabolic function in mice over-expressing adiponectin in the adipose tissue (tg) and in knockout (ko) mice with or without dihydrotestosterone (DHT)-induced PCOS. DHT-pellets were implanted subcutaneously in pre-pubertal female mice to induce PCOS while controls received placebo-pellets. Adiponectin tg, ko and wt mice were divided into placebo and DHT groups. Insulin/glucose tolerance, and body composition measurements were performed between 14-16 weeks of age. Wt-DHT mice displayed reduced serum adiponectin levels and became insulin resistant compared to controls, while tg-DHT mice were protected against this effect of DHT. Ko-DHT mice developed more severe insulin resistance than wt-DHT animals. Both wt-DHT and ko-DHT groups displayed impaired glucose tolerance compared to placebo but there was no signifi -cant difference between groups. Preliminary data shows that wt-DHT ani-mals have larger adipocytes than controls, and that tg-DHT mice are pro-tected against this effect. Gene expression analysis of the gonadal adipose depot shows decreased levels of genes involved in metabolic pathways like AdipoR2, IRS1, PPARγ and ChREBP in both wt-DHT and ko-DHT, which was not observed in tg-DHT mice. Moreover, pancreas of the tg-DHT mice show increased mRNA levels of insulin receptor, Glut2 and IGF1R, genes related to beta cell viability. There were small differences in body weight and body composition between genotypes. We conclude that adiponectin have a pro-tective role on metabolic functions in this PCOS mouse model.

1711-PManipulation of Filamin A Expression in Adipocytes as a Potential Modality to Limit Adipose Tissue ExpansionSYAMALA AKELLA, ISHITA DEB MAJUMDAR, KALYANI V. GUNTUR, STEPHANE GESTA, VIVEK K. VISHNUDAS, RANGAPRASAD SARANGARAJAN, NIVEN R. NARAIN, Framingham, MA

FlnA was identifi ed as a major modulator of obesity phenotype using the Berg Interrogative Biology® platform from an in vitro obesity model contain-ing adipocytes as one of the cellular model. Filamin A (FlnA) is a 280 kDa scaffolding protein that crosslinks actin to form fi lamentous network in cyto-plasm. Although it is reported to play a role in lipid droplet formation and in insulin signaling, the function of FlnA in regulating adipocyte biology is still poorly understood. To determine the role of FlnA in adipocyte functions, immortalized human preadipocyte cell lines stably expressing either a con-trol (shCont) or FlnA shRNA (shFlnA) were generated. FlnA protein expres-sion was reduced by 50 to 60% in shFLNA expressing adipocytes compared

to control. Triglyceride accumulation, as well as basal and isoproterenol stimulated lipolytic activity, were signifi cantly increased in shFlnA com-pared to shCont adipocytes. However, these changes were independent of an improved adipocyte differentiation, as expression of adipogenic markers (PPARg, Cebpa, FABP4 and GLUT4) was not affected. In addition, increased adiponectin expression and secretion was noted in shFlnA adipocytes, although insulin stimulated Akt phosphorylation was not changed. Interest-ingly, when adipocytes were subjected to a high nutrient environment in vitro, consisting of high levels of glucose, lipid and insulin, cells with reduced FlnA expression exhibited higher triglyceride storage capacity than control. Altogether, the data suggests that FlnA is involved in limiting triglyceride storage capacity of human adipocytes. Therefore, methods to increase FlnA expression in adipose tissue represent a potential strategy to limit adipose tissue expansion. In many cell types, proteases have been involved in regu-lating intracellular levels of FlnA and might therefore represent potential alternative targets to mitigate adipose tissue expansion.

1712-PCharacterization of Infl ammation during the Development of Obe-sity in CatsEMILY C. GRAFF, HAN FANG, ANNIE MAGUIRE, OLGA C. NORRIS, ALISON R. EMMERT, ROBERT L. JUDD, Auburn, AL, Columbia, MO

In humans, obesity is characterized by systemic and adipose tissue infl am-mation that contribute to secondary disease processes, such as type 2 dia-betes mellitus (T2DM) and atherosclerosis. Cats are a naturally occurring model of human obesity and T2DM. However, obese cats do not develop atherosclerosis and studies suggest that this may be due to a unique immune response during the development of feline obesity. Therefore, the goal of this study was to investigate systemic and adipose tissue infl ammation dur-ing the development of obesity and insulin resistance in cats. Adipose tis-sue, whole blood, serum and MRI data were collected from twelve, lean, male neutered, cats at baseline and following 18 months of ad libitum chow diet. Changes in insulin sensitivity, lipid metabolism, adipokine profi les, sys-temic and adipose tissue infl ammation, and distribution of fat mass were evaluated. The cats had a signifi cant increase in total fat mass, with pref-erential expansion of subcutaneous adipose depots. Increased adiposity in cats resulted in decreased insulin sensitivity, altered lipid metabolism and changes in adipokine profi les. During the development of obesity, the mean adipocyte area increased 138% in abdominal and 143% in subcutaneous adi-pocytes. No changes in circulating leukocytes were noted. However, serum concentrations of MCP-1 and TNF-α were signifi cantly increased. Crown-like structures (CSL) were rare to absent in all adipose tissue samples and numbers of CLS were independent of time (baseline vs. endpoint) and loca-tion (subcutaneous vs. abdominal). In conclusion, during the development of obesity and insulin resistance, cats have altered lipid metabolism, adipocyte hypertrophy, and changes in adipokines and circulating cytokines consistent with what is described in humans. However, unlike humans, cats preferen-tially expand subcutaneous adipose tissue depots rather than intra-abdom-inal and they do not develop classic adipose tissue infl ammation, character-ized by the development of CLS.

1713-PKBRPL2001, a Novel GPR120 Agonist, Improves Insulin Sensitivity and Glucose Tolerance and Decreases Hepatic Steatosis in Rodent Models of Type 2 DiabetesRAGHURAM ANUPINDI, RAJIV SHARMA, RAGHIB HUSAIN, Kalyani, India

GPR120 (FFAR4), a member of rhodopsin-like family of GPCRs, is widely expressed in many tissues and its activation improves many aspects of meta-bolic homeostasis. KBRPL2001 is a selective GPR120 agonist (hEC50: 51 nM) that showed improvement in these metabolic parameters. KBRPL2001 treat-ment displayed increase in GLUT-4 localization at the plasma membrane of stimulated cells when compared with LA treated 3T3 adipocytes. In STC1 cells, treatment with KBRPL2001 exhibited activation of MAPK pathway, as determined by increased phosphorylation of Erk at 3 and 10uM treatment. Chronic administration of KBRPL2001 in diet-induced obese (DIO, 60% fat) mice (30 mg/kg, p.o., b.i.d., 7 wk) caused a signifi cant reduction in the AUC of blood glucose levels in OGTT (20%, p<0.001), reduction in fasting blood glucose (192 to 148 mg/dL) and plasma insulin (2.3 to 1.2 ng/mL), demonstrat-ing its potential in improving insulin sensitivity. The treatment resulted in signifi cant reduction (p<0.05) in body weight gain compared to DIO vehicle. In epididymal fat pads of DIO mice treated with KBRPL2001, decrease in pro-infl ammatory Adipose Tissue Macrophages (ATMs) and increase in anti-infl ammatory ATMs was observed. Similarly, increased presence of anti-infl ammatory macrophages was seen in liver tissue of compound treated

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INSULIN ACTION—CELLULAR AND MOLECULAR METABOLISM

animals, indicating its potential to reduce infl ammation associated with insu-lin resistance. KBRPL treatment also showed signifi cant reduction in plasma triglycerides (p<0.001) and a corresponding, non-signifi cant decrease in liver triglycerides, indicating its potential in addressing hepatic steatosis/NAFLD. The preclinical data suggest that KBRPL2001, a novel GPR120 agonist, can be a potential therapy for type 2 diabetes and metabolic disorders.


1714-PVascular Endothelial Cell-specifi c PDK1 Knockout Mice Present Insulin Resistance and Deterioration of Mitochondrial Biogenesis in Muscle under STZ-induced HyperglycemiaATSUSHI OBATA, KAZUHITO TAWARAMOTO, SEIZO OKAUCHI, TOMOHIKO KIMURA, HIDENORI HIRUKAWA, MASASHI SHIMODA, KENJI KOHARA, AKIHITO TANABE, TOMOE KINOSHITA, SHINJI KAMEI, TOMOATSU MUNE, KOHEI KAKU, HIDEAKI KANETO, Kurashiki, Japan

The phosphatidylinositol 3-kinase signaling pathway in vascular endothe-lial cells is important for systemic angiogenesis and glucose metabolism. We previously reported that vascular endothelial cell-specifi c PDK1 knockout mice (VE-PDK1-KO mice) (Tie2 promoter-Cre/ PDK1fl ox/fl ox mice) presented the improvement of insulin resistance and glucose tolerance (Mol Endocrinol. 26, 95-109, 2012). In this study, we showed that VE-PDK1-KO mice presented insulin resistance under STZ-induced hyperglycemia. This opposed pheno-type suggests some uncovered pivotal roles of PDK1 in endothelial cells. We investigated the mechanism which induces insulin resistance in VE-PDK1-KO mice under STZ-induced hyperglycemia. STZ (150 µg/g) was intraperitoneally injected to VE-PDK1-KO and control fl ox/fl ox mice at 6 weeks of age. Ani-mals whose random fed glucose level was over 300 mg/dl were selected as diabetic mice. At 12 weeks of age, capillary blood volume in skeletal muscle was signifi cantly lower in VE-PDK1-KO mice than control mice accompanied with decrease of eNOS level. The protein level of p-Akt in skeletal muscle was also decreased in VE-PDK1-KO mice, suggesting that insulin resistance was induced in skeletal muscle. Immunohistological analysis revealed the decrease of endothelial cell number in skeletal muscle. TUNEL-positive endothelial cells were increased in skeletal muscle in VE-PDK1-KO mice. Furthermore, mRNA level of PGC1α was reduced. The downstream mRNA levels of PGC1α which regulate mitochondrial biogenesis such as Tfam, NRF1 and NRF2 were also signifi cantly decreased in VE-PDK1-KO mice. In fact, mRNA levels of Cpt1β and ACADM were reduced in skeletal muscle in VE-PDK1-KO mice. In conclusion, VE-PDK1-KO mice present insulin resis-tance due to decreased endothelial cell number and capillary blood volume and deterioration of mitochondrial biogenesis in muscle under STZ-induced hyperglycemia.

1715-PThe Role of β- and γ-actin in Insulin-stimulated Glucose TransportAGNETE B. MADSEN, JONAS R. KNUDSEN, YELIZ ANGIN, LYKKE SYLOW, ERIK A. RICHTER, THOMAS E. JENSEN, Copenhagen, Denmark

Insulin-stimulated glucose uptake into skeletal muscle accounts for the majority of whole body insulin-stimulated glucose disposal, but the under-lying basic molecular mechanisms governing this process remain unclear. Accumulating evidence in cell culture and rodents suggest that the cortical actin cytoskeleton plays a signifi cant role in the insulin-stimulated translo-cation of glucose transporter 4 (GLUT4) to the plasma membrane. Based on this it was hypothesized that muscle-specifi c knockout (KO) of either β- or γ-actin in adult mice would cause pronounced alterations in substrate utili-zation and glucose tolerance in vivo and reduce insulin-stimulated glucose transport ex vivo. No genotype differences were observed in in vivo mea-surements including body composition, fasting insulin and glucose levels, and indirect calorimetry, except for a decreased glucose tolerance in the γ-actin KO mice (19%, p=0.024). Ex vivo, maximal insulin-stimulated 2-deoxy-glucose transport was lower in soleus muscle in both β- and γ-actin KO mice (40%, p=0.001 and 33%, p=0.002 in β- and γ-actin KO mice, respectively). In the β-actin KO mice no genotype differences were observed in any of the measured protein expressions and phosphorylations in either muscle, nor was glucose transport affected in EDL from neither the β- or γ-actin KO mice. However, the γ-actin KO mice showed reduced insulin-stimulated Akt serine 473 and p70S6 kinase threonine 389 phosphorylation, although not signifi cant when related to total protein expression. These data imply that γ-actin may be more important for glucose transport into skeletal muscle, due to the more pronounced phenotype of γ-actin vs. β-actin KO. However,

altogether, the relatively mild and muscle type specifi c phenotypes of both the β- and γ-actin KO mice indicate that mature skeletal muscle does not rely on cortical remodeling of either β- or γ-actin for glucose transport to the extent suggested by cell culture studies, although compensation between the two actin isoforms cannot be excluded.

Supported By: Danish Diabetes Academy; Novo Nordisk Foundation

1716-PLipoprotein Lipase Is a Key Regulator of Energy Metabolism in the BrainKIMBERLEY D. BRUCE, ALENA RODRIGUEZ, SACHI GORKHALI, ANDREW LIBBY, ROBERT H. ECKEL, Aurora, CO

Lipoprotein Lipase (LPL) is a key regulator of lipid metabolism. Abnormal LPL activity and expression has been implicated in the pathogenesis of a plethora of metabolic diseases. While the nutritional and molecular regula-tion of LPL in peripheral tissues is fairly well established, the role and regu-lation of LPL in the brain is much less clear. Nonetheless, LPL is abundantly expressed in several regions of the brain, and a reduction in neuronal LPL expression is thought to be involved in hypothalamic lipid sensing, hyper-phagia and the development of obesity (Wang et al. 2011. Cell Metab). To gain novel insights into the function of LPL in the brain, immortalized hypo-thalamic neurons were genetically manipulated to either over, or under express LPL (Libby et al., 2015. BBRC). Using these cells we explored the effect of nutritional cues, and the dynamics of candidate molecular factors thought to be involved in LPL inhibition (Angptl4), and lipoprotein receptor lipid uptake (VLDLR, and ApoER2). Interestingly, LPL activity and expres-sion was lowest at high glucose conditions (20 mM). We also found that Angptl4 expression was profoundly increased in cells with reduced expres-sion of LPL (p < 0.001 vs. WT), consistent with a lack of Angptl4-mediated LPL inhibition. VLDLR gene expression was increased when LPL was knocked down (p < 0.01 vs. WT), suggesting that VLDLR expression may be compen-satorily increased following reduced LPL-mediated lipid uptake. In contrast, ApoER2 expression was markedly reduced when LPL was knocked down (p < 0.01 vs. WT), implying that ApoER2 may be involved in LPL-dependent lipid uptake. Taken together our fi ndings show for the fi rst time that neuronal LPL is responsive to hypothalamic glucose concentrations, and is intricately involved in neuronal lipoprotein receptor-mediated lipid uptake.


1717-PA Novel Factor POlDIP2 Is Suppressed in Livers of Type 2 Diabetic Mice and Dysregulates Hepatic Cholesterol HomeostasisKEYANG CHEN, CHENGWEI YANG, TAO LI, QUNAN WANG, KEVIN JON WIL-LIAMS, Hefei, China, Philadelphia, PA

The NADPH oxidase-4 (NOX4) has emerged as a major metabolic regulator that fails to function normally in type 2 diabetic liver (ATVB 2012;32:1236-1245). The molecular basis for its dysfunction has not been established. Polymerase-δ interacting protein-2 (POLDIP2) was identifi ed as a NOX4 partner for strengthening focal adhesions in vascular smooth muscle cells (Circ Res 2009;105:249-259). We hypothesized a role for POLDIP2 in diabetes and lipid metabolism.

Here, we found that hepatic POLDIP2 protein levels are substantially decreased in hyperphagic, obese, T2DM KKAy mice compared with nondi-abetic KK littermate controls. Moreover, hepatic cholesterol content was doubled in KKAy vs. KK mice, and it signifi cantly and inversely correlated with Poldip2 mRNA levels in liver. To identify mechanisms of POLDIP2 dys-regulation in T2DM, we found that high levels of glucose (25mmol/L) - alone or with insulin (10nM), leptin (8nM), or AGEs (200ug/ml) - sharply suppress POLDIP2 protein expression in cultured rat hepatocytes. Treatment of cul-tured hepatocytes with high glucose plus insulin doubled cellular cholesterol content, similar to our fi ndings in vivo. Importantly, siRNA-mediated knock-down of POLDIP2 in cultured hepatocytes signifi cantly decreased insulin-induced production of regulatory hydrogen peroxide, suggesting that POL-DIP2 is required for healthy NOX4 activity.

To address POLDIP2 function in vivo, we used adenoviral particles to restore hepatic levels of POLDIP2 protein to normal in T2DM KKAy mice. This intervention increased hepatic hydrogen peroxide production, decreased hepatic cholesterol content by nearly 50% to reach levels seen in livers of nondiabetic KK littermates, and lowered plasma LDL cholesterol levels.

Taken together, our fi ndings demonstrated that a novel factor, POLDIP2, regulates cholesterol homeostasis in liver, is defi cient in T2DM, and may therefore contribute to metabolic dysregulation in states of overnutrition.

Supported By: American Diabetes Association (1-13-BS-209 to K.J.W.); National Natural Science Foundation of China

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1718-PObesity Increased Mitochondrial DNA Damage and DAMPs which Regulate Infl ammatory Signaling and Insulin ResistanceLARYSA YUZEFOVYCH, VIKTOR PASTUKH, MYKHAILO RUCHKO, MICHELE SCHULER, GLENN WILSON, JON SIMMONS, WILLIAM RICHARDS, MARK GILLESPIE, LYUDMILA RACHEK, Mobile, AL

Mitochondrial DNA (mtDNA) damage has been implicated in the devel-opment of insulin resistance (IR), since mtDNA is highly specialized and encodes for proteins essential for energy metabolism and, also, mtDNA dam-age heightens mitochondrial oxidative stress, which is very critical for IR. Recently it has been shown that cells damaged by mechanical or infectious injury release pro-infl ammatory mtDNA Damage Associated Molecular Pat-terns (DAMPs) into the circulation (c-mtDNA). In this study, we demonstrate in obese type 2 diabetes (T2D) patients and mice fed a high fat diet (HFD) that elevated c-mtDNA correlate closely with IR. HFD-fed mice defi cient in the DNA repair enzyme, 8-oxoguanine DNA glycosylase (OGG1), displayed skeletal muscle mtDNA damage, elevated c-mtDNA, and an IR phenotype. Importantly, OGG1-defi cient mice reconstituted with human OGG1 (hOGG1) targeted specifi cally to mitochondria were protected against mtDNA dam-age and elevations in c-mtDNA fragments and have reduced obesity, both systemic and tissue infl ammation and IR. Importantly, administration of exog-enous mtDNA DAMPs induced infl ammatory signaling, hyperglycemia and IR in vivo, providing a direct causative role for mtDNA DAMPs in the develop-ment of IR. Furthermore, exogenous mtDNA fragments induced TLR9-medi-ated NF-κB activation, increased mitochondrial oxidative stress, reduced mitochondrial function and suppressed insulin-mediated glucose uptake in vitro. Collectively, these fi ndings show that mtDNA damage with attendant mitochondrial dysfunction and release of pro-infl ammatory mtDNA DAMPs contribute to the obesity-related IR phenotype and point to the prospect of developing new diagnostic and treatment strategies focused on assessment and protection of mtDNA integrity.

Supported By: American Diabetes Association (7-13-BS-139-BR to L.R.)

1719-P

1720-PRegulation of Basal and Insulin-stimulated Rates of Muscle ATP Synthesis by Plasma Phosphate Assessed by 31P NMR Spec tros-copyDOMINIK PESTA, DIMITRIOS TSIRIGOTIS, DOUGLAS E. BEFROY, DANIEL CABAL-LERO, MICHAEL JURCZAK, GARY W. CLINE, SYLVIE DUFOUR, ANDREAS L. BIRKENFELD, DOUGLAS ROTHMAN, THOMAS CARPENTER, CARL INSOGNA, KITT F. PETERSEN, CLEMENS BERGWITZ, GERALD I. SHULMAN, New Haven, CT, Toronto, ON, Canada, Dresden, Germany

Hypophosphatemia in intensive care patients leads to muscle weak-ness resulting in respiratory and heart failure. Similarly, mice with geneti-

cally induced hypophosphatemia due to ablation of the renal sodium phos-phate co-transporter NaPi2a, have reduced spontaneous activity and forced exercise capacity. We examined the potential role of muscle mitochondrial ATP synthetic rate (VATP) in this process using 31P magnetic resonance spectroscopy (MRS) saturation transfer (31P-ST) in vivo. Basal and insulin-stimulated VATP were reduced in mice on low phosphate diet or in NaPi2a knockout mice (NaPi2a-/-) using this non-invasive technique. Likewise, VATP was reduced in a patient with hypophosphatemia due to a mutation in the gene encoding the renal phosphate transporter NAPI2C. Restoration of nor-mophosphatemia normalized VATP in NaPi2a-/- mice and the patient with a mutation in the gene encoding the renal phosphate transporter NAPI2C Using L6 and RC13 rodent myocytes and isolated muscle mitochondria, we showed that VATP is directly related to cellular and mitochondrial phosphate uptake. Therefore, decreased muscle mitochondrial ATP synthetic rate may in part explain the muscle weakness seen in hypophosphatemia and may serve as a non-invasive marker for hypophosphatemic myopathy.

Supported By: Austrian Science Fund

1721-PTransgenic Mouse Model of Breast Cancer Develops Insulin Resis-tance in Skeletal MuscleHEE JOON KANG, HYE-LIM NOH, SUCHAORN SAENGNIPANTHKUL, JOSE MERCADO-MATOS, RANDALL H. FRIEDLINE, JONG HUN KIM, TAEKYOON KIM, KUNIKAZU INASHIMA, BORAM HAN, ALYCIA QUICK, XIAODI HU, KAREN KELLY, KI WON LEE, LESLIE M. SHAW, JASON K. KIM, Worcester, MA, Seoul, Republic of Korea

Epidemiologic evidence suggests a potential link between cancer and dia-betes that share multiple risk factors. Here we examined glucose metabo-lism in a transgenic mouse model of breast cancer expressing an oncogene, polyoma middle T antigen driven by the Mouse Mammary Tumor Virus pro-moter (MMTV-PyMT). A hyperinsulinemic-euglycemic clamp was performed in female MMTV-PyMT mice with palpable tumors at the mammary region and control mice at 8~9 weeks of age (n=9~12/group). Despite similar body weights, MMTV-PyMT mice developed insulin resistance with a signifi cant decrease in whole body glucose turnover (Figure 1; *P<0.05). This was largely due to a 20% decrease in insulin-stimulated glucose uptake in skeletal mus-cle (Figure 2). Insulin resistance was selective to muscle in MMTV-PyMT mice that showed normal glucose metabolism in white and brown fat as well as liver. MMTV-PyMT mice showed signifi cant increases in plasma IL-6, G-CSF, and MCP-1 levels (Figure 3), whereas adipokine levels did not differ between groups. Overall, these results indicate that tumor-bearing MMTV-PyMT mice develop insulin resistance in skeletal muscle. Also, our fi ndings suggest a novel paradigm in which cytokines and chemokines derived from the tumor microenvironment affect systemic glucose metabolism thereby providing a direct link between cancer and insulin resistance.Figure.

Supported By: National Institutes of Health (R01-DK080756, U24-DK093000, R24-DK090963)

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1722-PStructural and Functional Analysis of the Diabetes Risk Factor ZnT8/SLC30A8 Zinc Transporter ProteinMARK J. DANIELS, MACIEJ JAGIELNICKI, MARK YEAGER, Charlottesville, VA

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

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

We successfully purifi ed ZnT8 constructs from a Pichia pastoris overex-pression system. With negative-stain electron microscopy (EM) and RELION single particle image analysis, the molecular boundary of an EM density map at 20Å resolution was suffi ciently well defi ned that we could dock our homol-ogy model of ZnT8. Our V-shaped structure is consistent with a homodimer and the bipartite appearance of each monomer is interpreted as the 22 kD alpha-helical TM bundle and the 10 kD C-terminal domain.

Complementary to our structural analyses, we have developed an in vivo functional assay in our P. pastoris expression system and found that ZnT8 enhances Zn2+ effl ux. We also purifi ed ZnT8 constructs from insect cells and reconstituted the proteins into proteoliposomes. These vesicles showed pH-dependent uptake of Zn2+ consistent with a Zn2+/H+ antiport. The results of this research will be a starting point for drug design in targeting diabetes and its resulting complications.

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

1723-PEpithelial Sodium Channel Inhibition with Amiloride Prevents Vas-cular Stiffening in Response to a Western Diet in Female MiceANNAYYA AROOR, VINCENT G. DEMARCO, GUANGHONG JIA, JAVAD HABIBI, MONA GARRO, ZHE SUN, LUIS MARTINEZ-LEMUS, GERALD MEININGER, IRIS JAFFE, JAMES R. SOWERS, Columbia, MO, Boston, MA

Over-nutrition/obesity predisposes individuals, particularly women, to arterial stiffening, an independent predictor of future adverse cardiovascu-lar events. We have recently developed a clinically relevant murine model fed a high fat and high fructose diet (western diet, WD) which causes vas-cular stiffness. In this model very low dose administration of the mineralo-corticoid (MR) antagonist spironolactone prevented development vascular stiffness in females on a WD. One of the mechanisms by which MR acti-vation promotes endothelial stiffness is through increased expression and activation of epithelial sodium channel (ENaC) in endothelial cells (EC). In this study, we tested whether amiloride, an inhibitor of ENaC, decreases aortic stiffness in vivo and ex vivo aortic explants in WD fed female mice. Four week-old C57BL6/J female mice were fed a WD with excess fat (46%) and fructose (17.5%) with or without amiloride (1mg/kg/day) for 16 weeks. Compared to mice fed a control diet (CD), aortic stiffness, determined by in vivo PWV, was signifi cantly increased in females on a WD and this cor-responded to WD-induced increase in EC stiffness, measured ex vivo by atomic force microscopy. These increases in stiffness were prevented by administration of a very low dose of the ENaC inhibitor, amiloride. Moreover, incubation of aortic explants ex vivo with 1 µM amiloride, a dose which it is more specifi c for ENaC, resulted in decrease in aortic stiffness in aorta from WD fed female mice. Moreover, in another study ENaC expression was also increased in WD fed mice which was decreased in EC specifi c MR KO mice with concomitant decrease in endothelial stiffness. Taken together, these fi ndings support the notion that a WD promotes ECMR activation of ENaC in ECs and associated vascular stiffness which is a marker/predictor of cardiovascular disease.


1724-PInsulin Regulates Glucose Transporter 4 Traffi cking in Diabetic BrainRAHUL AGRAWAL, ADRIANA VIEIRA-DE-ABREU, SIMON J. FISHER, Salt Lake City, UT

Evidence indicates that the brain may be insulin sensitive, but the mecha-nism by which insulin may regulate glucose transport in critical brain regions remains uncharacterized, especially in diabetes. The objective of the current study was to test the hypothesis that systemic insulin crosses the blood brain barrier and acts both chronically and acutely in regulating glucose transporter 4 (GLUT4) content and translocation in the hypothalamus (HYP) and hippocampus (HIPP). C57Bl6 male mice (3 months old) were injected with streptozotocin (200 mg/kg; DIAB) or vehicle (nondiabetic controls; CON). After 2 weeks of hyperglycemia (glucose > 300 mg/dl), DIAB mice were injected intraperitoneally with saline or insulin (5U/mouse; DIAB+INS) and sacrifi ced after 40 min. Systemic insulin administration acutely stimulated brain insulin signaling as noted by increased Akt phosphorylation by 40 and 32% in HYP and HIPP regions. As compared to nondiabetic controls, chroni-cally insulin-defi cient DIAB mice demonstrated 55% and 40% decreased plasma membrane (PM) GLUT4 content in HYP and HIPP, respectively. Acute systemic insulin administration increased relative PM GLUT4 content 3-fold (in HYP) and 2-fold (in HIPP) in DIAB mice, indicating that peripheral insulin rapidly stimulates brain GLUT4 recruitment to the PM. These results indi-cate that the chronic diabetes-induced down-regulation of PM GLUT4 can be reversed by acute insulin therapy.Figure. Insulin Acutely Restores Low PM GLUT4 Content in Hypothalamus of Diabetic Mice.


1725-PZinc Finger Protein 407 Overexpression Improves Glucose Homeo-stasis in MiceALYSSA CHARRIER, Cleveland, OH

Peroxisome proliferator-activated receptor gamma (PPARg) controls insulin sensitivity by regulating the expression of genes involved in glucose homeo-stasis, adipogenesis, and lipid metabolism. We recently discovered that zinc fi nger protein 407 (Zfp407) defi ciency in cultured adipocytes decreased the expression of PPARg target genes, including glucose transporter 4 (Glut4), thereby reducing insulin-stimulated glucose uptake. Co-overexpression of Zfp407 and PPARg enhanced the expression of a luciferase reporter con-struct utilizing a canonical PPARg DNA binding site demonstrating a syn-ergistic effect of Zfp407 on PPARg target gene expression. Therefore, we hypothesized that Zfp407 overexpression would increase PPARγ activity and improve glucose homeostasis in vivo, thus representing a novel thera-peutic approach for treating type 2 diabetes. We generated a new trans-genic mouse strain (ZFP-TG) that specifi cally overexpressed Zfp407 in skel-etal muscle (19-fold) and heart (3-fold). Transcriptome analysis by RNA-Seq identifi ed 1,300 differentially expressed genes in the muscle of ZFP-TG mice, among which PPARγ target genes were signifi cantly enriched. PPARγ mRNA and protein levels did not differ between ZFP-TG and control mice, suggest-ing that Zfp407 post-translationally regulates PPARγ activity. Among PPARγ target genes, Glut4 mRNA and protein levels were increased in heart and muscle. The increase in Glut4 and other transcriptional effects of Zfp407 overexpression together decreased total body weight and lowered plasma glucose levels relative to control littermates. Additionally, ZFP-TG male mice had decreased plasma insulin levels and the HOMA-IR score was decreased in both male and female ZFP-TG mice compared to WT controls. Collectively, these results demonstrate that Zfp407 overexpression improved glucose homeostasis. Thus, Zfp407 represents a new drug target for treating meta-bolic disease.

Supported By: American Diabetes Association (1-16-PDF-018)

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INSULIN ACTION—GLUCOSE TRANSPORT AND INSULIN RESISTANCE IN VITRO

1726-PInterrogating the Signaling Mechanisms of GPR119: a Novel Target for Type 2 Diabetes Mellitus (T2DM) and ObesityPRAMISHA ADHIKARI, EMMANUEL STURCHLER, PATRICIA MCDONALD, Jupiter, FL

GPR119 has emerged as a promising new drug target for the treatment of T2DM and obesity. This GPCR is expressed on intestinal L cells and pan-creatic β-cells and as such, provides a unique opportunity to target a single receptor promoting insulin secretion, β-cell proliferation, and secretion of gut-derived hormones (incretins) that induce postprandial insulin secre-tion. Collectively, these responses contribute to maintain normoglycaemia. Agonist activation of GPR119 leads to an increase in intracellular cAMP via coupling to Gαs-protein. G-protein signaling is terminated by β-arrestin recruitment, however, it is now appreciated that β-arrestins can also act as signal transducers. Recently, the concept of biased signaling or functional selectivity, whereby, a GPCR preferentially activates G protein signaling over β-arrestin signaling, or vice versa, has expanded the range of ligand activ-ity to target GPCRs. Such ligands, termed “biased” ligands, have recently gained increasing attention due to their potential to provide therapeutic ben-efi t over existing therapies. However, whether pathway-selective GPR119 targeting can provide therapeutic advantages is yet to be determined. The aim of this study is to interrogate the contribution of G-protein and β-arrestin signaling in mediating the physiological functions of GPR119. Ideally, GPR119 biased ligands would facilitate such studies, however as no such ligands exist, we have taken a genetic approach. Recombinant cell lines have been generated that stably express mutant GPR119 that can selectively activate Gαs or β-arrestin mediated signaling. Our results demonstrate that modifi ca-tions of the carboxy-terminal tail of GPR119 generates a Gαs biased signaling receptor, activation of which results in an attenuation of both MAPK phos-phorylation and desensitization as compared to wildtype receptor. We are currently investigating the physiological consequences of selectively engag-ing Gs at the GPR119 receptor.

1727-PThe Relevance of Insulin Signaling in the Regulation of Pluri-potencyMANOJ K. GUPTA, DARIO F. DE JESUS, ROHIT N. KULKARNI, Boston, MA

Self-renewal of mouse induced pluripotent stem cells (miPSCs) is achieved by the modulation of dedicated transcription circuits that eliminate differen-tiation-inducing signals. Among these circuits the relevance of insulin recep-tor (IR)-mediated signaling in regulating the identity of miPSCs is not fully understood. Here we report the relevance of insulin receptor (IR) dependent and independent signaling pathways in the maintenance of pluripotency. We derived IR Knock-Out (IRKO) miPSCs from E14.5 mouse embryonic fi broblasts (MEFs) of global IRKO mice using a cocktail of four reprogramming factors, namely Oct4, Sox2, Klf4 and cMyc. iPSCs were maintained in a 2- inhibitor (2i) feeder-free system. All clones stained positive for alkaline-phosphatase and formed teratomas containing the three lineages. Gene expression analy-sis revealed an upregulation of several genes associated with pluripotency including Klf4, Lin28a, Tbx3 and cMyc (p<0.05, n=3 clones/group) in IRKO iPSCs as compared to controls (C). Oct4 and Nanog protein levels were 4-fold (p<0.05, n=3) and 3-fold (p<0.01, n=3) increased in IRKO iPSCs as compared to C respectively and were confi rmed by immunofl uorescence staining. Basal signaling analysis demonstrated downregulation (p<0.05, n=3) of phospho-STAT3, p-mTOR and p-ERK, despite an increase (p<0.05, n=3) in total protein levels of mTOR and ERK in IRKO iPSCs. Furthermore, stimulation of phospho-ERK by leukemia inhibitory factor (LIF) was decreased by 3-fold (p<0.05, n=3) in IRKO iPSCs as compared to C. Therefore, IRKO iPSCs provide a unique opportunity to explore the signifi cance of insulin receptor signaling in the maintenance of pluripotency and its crosstalk with key pluripotency related signaling pathways.


1728-PInsulin-induced βcyto-actin Dynamics in Live Skeletal MuscleJONAS R. KNUDSEN, KRISTIEN J. ZAAL, EVELYN RALSTON, THOMAS E. JENSEN, Copenhagen, Denmark, Bethesda, MD

Prediabetic insulin-resistance impedes insulin-stimulated translocation of the glucose transporter 4 (GLUT4) to the surface membrane in skeletal mus-cle. In muscle cell culture, accumulating evidence suggests that this translo-cation requires increased dynamics and remodeling of βcyto-actin, the domi-

nant cortical actin cytoskeleton component. Furthermore, actin remodeling is abolished in the insulin-resistant state. In mature skeletal muscle insulin-stimulated glucose uptake is blunted by actin depolymerizing agents, indi-cating that this could be a conserved function. However, further evidence is needed to support this hypothesis. Here we imaged isolated individual skel-etal muscle fi bers expressing GFP-tagged βcyto-actin and used Fluorescence Recovery After Photobleaching (FRAP) to evaluate whether insulin induced any changes in the mobility of βcyto-actin, as suggested in cell culture and heart muscle. We found that βcyto-actin co-localized with the Z-disks in a stri-ated pattern throughout the fi ber. In addition, βcyto-actin was located around the nuclei. After insulin treatment, we detected neither changes in the distri-bution of βcyto-actin in live fi bers nor changes in the pattern of F-actin in fi xed fi bers. When FRAP was measured in the well resolved regions surrounding the nuclei, GFP βcyto-actin exhibited limited dynamics that were not signifi -cantly changed by insulin. We verifi ed, by immunoblotting, that insulin did affect the muscle fi bers. Thus, our data do not support the hypothesis that the insulin-induced changes in actin dynamics observed in muscle cells are conserved into mature skeletal muscle.

Supported By: National Institute of Arthritis and Musculoskeletal and Skin Diseases

1729-PBoth Enhancers and Potent Inhibitors Were Identifi ed in a Screen for Small Molecule Modulators of Acute Insulin ActionCYNTHIA CORLEY MASTICK, PAUL DUFFIELD BREWER, IRINA ROMENSKAIA, Reno, NV

Using a 1° assay for acute insulin action that is sensitive, responsive, and highly reproducible, we identifi ed 12 novel enhancers and 26 novel inhibi-tors of acute insulin action in a pilot screen 1120 drugs. Using our 2° kinetics assays to characterize these compounds, novel mechanisms for enhancing and inhibiting acute insulin action were discovered. In addition, drugs that may have adverse effects on glucose homeostasis and be contraindicated in diabetes were identifi ed. Enhancers include 6 cardiac glycosides related to ouabain. All are Na+/K+-ATPase inhibitors. Ouabain accelerates both endo-cytosis and exocytosis of Glut4 (1.5- and 3.3-fold). These compounds increase cell surface Glut4 by inhibiting sorting into the highly regulated storage ves-icles (GSVs). Inhibitors include 11 “antibiotics.” Five affect protein synthesis, but are structurally and mechanistically distinct. A requirement for protein synthesis for acute insulin action has not been described. Hits also include 10 phenothiazines and dibenzazepines. These drugs cause acute insulin resis-tance (hyperinsulinemia/normoglycemia) in humans, through an unknown mechanism. Four different phenotypes were observed for the inhibitors, indi-cating distinct mechanisms of action. Membrane traffi cking: the nutraceu-tical resveratrol and the natural product piperlongumine decreased surface Glut4 to the same extent and as rapidly as the PI3-kinase inhibitor LY294002 (LYi; t½=5 min). LYi inhibits exocytosis. Protein synthesis: the natural product licorine had the same slow kinetics and effi cacy as puromycin (t½=30 min). Signal transduction: propranolol and the natural product parthenolide had kinetics similar to an Akt inhibitor (t½=10 min). Endosomal pH: the phenothi-azine maprotiline and the anti-malarial chloroquine had very fast transition kinetics (t½=3 min). These amines inhibit endocytic acidifi cation.

Supported By: American Diabetes Association (1-12-BS-132 to C.C.M.)

1730-PN-WASP-Cortactin Signaling Promotes Skeletal Muscle GLUT4 Vesicle TranslocationRAGADEEPTHI TUNDUGURU, JEFFREY S. ELMENDORF, DEBBIE C. THURMOND, Indianapolis, IN, Duarte, CA

Insulin maintains glucose homeostasis by mobilizing GLUT4 vesicles from intracellular compartments to the plasma membrane (PM) of muscle and adipose cells, facilitating glucose uptake into these cells. Defects in GLUT4 translocation are associated with peripheral insulin resistance, pre-clinical diabetes and eventual progression to type 2 diabetes. Recruitment of GLUT4 to the PM of skeletal muscle cells requires fi lamentous (F)-actin remodeling. Recent in vitro data implicates the neural Wiskott-Aldrich syndrome protein (N-WASP) in insulin-dependent cortical F-actin rearrangement. However, the mechanism of action of N-WASP in regulating this cortical actin net-work, and any relatedness to skeletal muscle function, is unexplored. Here we show that inactivation of N-WASP by its specifi c inhibitor, Wiskosta-tin, fully abrogates the insulin-stimulated increase in GLUT4 translocation to the plasma membrane in skeletal muscle cells. Toward interrogating the underlying mechanism, interactions between N-WASP and Cortactin were assessed, given that Cortactin is an actin binding protein and implicated in actin remodeling in clonal muscle cells. Indeed, a ~1.5 fold increased bind-

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ing of Cortactin to N-WASP with insulin stimulation was detected using mouse skeletal muscle lysates as well as L6-mycGLUT4 cell lysates. In sum, these results suggest that N-WASP facilitates Cortactin-mediated F-actin remodeling for insulin-stimulated GLUT4 vesicle translocation to the PM of skeletal muscle cells. Additional studies delineating the signaling elements surrounding N-WASP-Cortactin, with the ultimate goal of identifying thera-peutic targets in this pathway, are currently underway.

Supported By: American Diabetes Association (1-15-BS-053 to J.S.E.); National Institutes of Health; JDRF

1731-PThe Regulation of Glucose Transport Is Altered during Diabetes-induced Atrial FibrillationZAHRA MARIA, ALLISON CAMPELO, BRENDA SMITH, BENJAMIN SCHERLAG, VERONIQUE A. LACOMBE, Stillwater, OK, Oklahoma City, OK

Diabetes and obesity have been identifi ed as major risk factors for atrial fi brillation (AF). However, whether a metabolic substrate underlies AF is unknown. Glucose transport into the cell via Glucose Transporters (GLUTs) is the rate-limiting step of glucose utilization. Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac iso-form. However, its role in the heart is not well known. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be impaired during type 2 diabetes (T2Dx)-induced AF. AF was induced by transesopha-geal atrial pacing in healthy and long-term high-fat-diet (HFD)-induced T2Dx rodents. Expression of GLUTs and key proteins involved in the insulin signal-ing pathway was measured by Western blot in cardiac myocytes. Active cell surface GLUT content was measured using the state-of-the-art biotinylated photolabeled assay in the perfused heart. After 6 months on a HFD, mice were obese and hyperglycemic, and developed insulin resistance compared to mice on a control diet. T2Dx animals showed an increased susceptibil-ity and propensity for AF. In the T2Dx atria, active cell surface and total GLUT4 content was down-regulated (by 66% and 40%, respectively, P<0.05) under basal condition. Long-term HFD-induced T2Dx resulted in impairment in Akt and AS160 phosphorylation, which was signifi cantly correlated with GLUT4 protein content in the atria. These data suggest an impairment of the insulin signaling pathway, which was further confi rmed by altered traf-fi cking of both GLUT-4 and -8 to the cell surface upon insulin stimulation in the T2Dx atria. In conclusion, our data suggest that: 1.) T2Dx increases the vulnerability to AF; 2.) GLUT-4 and -8 traffi cking is altered in the T2Dx atria due to impairments in the insulin signaling pathway. Therefore, alterations in atrial glucose transport may induce perturbations in energy production and could provide a metabolic substrate for atrial fi brillation during diabetes and obesity.

Supported By: Harold Hamm Diabetes Center

1732-PEvidence for a Cholesterolgenic Response as a Basis of Insulin Resistance in Mice Fed a High-Fat DietJEFFREY S. ELMENDORF, LIXUAN TACKETT, BRENT A. PENQUE, NOLAN J. HOFF-MAN, WHITNEY J. SEALLS, JOSEPH T. BROZINICK, Indianapolis, IN, Stony Brook, NY, Sydney, Australia

Clonal cell studies demonstrate that excess hexosamine biosynthesis pathway (HBP) activity increases O-linked N-acetylglucosamine modifi cation of the transcription factor Sp1, leading to transcriptional activation of HMG-CoA reductase (HMGR), the rate-limiting enzyme in cholesterol biosynthe-sis. This HBP-induced cholesterolgenic transcriptional response increases cholesterol in the plasma membrane (PM), while reducing cortical fi lamen-tous actin (F-actin) that is essential for insulin-stimulated GLUT4-mediated glucose transport in 3T3-L1 adipocytes and L6 skeletal muscle myotubes. To gain in vivo understanding of cholesterol-associated insulin resistance, 4-wk old male C57BL/6J mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet with adaptations regarding type of fat (palm oil instead of lard) and carbohydrates, to better mimic the average human diet in Western societies. At 8 wks, both glucose and insulin tolerance were impaired in HF-fed mice. Consistent with these data, fed insulin levels were signifi cantly increased by HF-feeding compared to LF-fed mice. Mixed hindlimb skeletal muscle from these HF-fed mice showed a 34% increase in PM cholesterol compared to LF-fed mice. In line with cell culture fi ndings, demonstrating increased PM cholesterol causes a loss of both F-actin and insulin-stimu-lated glucose transport; both were decreased by 21% and 26%, respectively in muscle from HF-fed mice compared to LF-fed mice. Epididymal fat pads also displayed a HF-feeding induced accumulation of PM cholesterol, as well as O-GlcNAc modifi cation of Sp1 and higher binding affi nity of Sp1 to the promoter region of HMGR. Together, these data suggest a mechanism

whereby increased HBP activity increases Sp1 transcriptional activation of a cholesterolgenic program thereby elevating PM cholesterol and compromis-ing cytoskeletal structure essential for insulin action.

Supported By: American Diabetes Association (1-15-BS-053 to J.S.E.); Eli Lilly and Company

1733-PCarnitine Acetyltransferase: A New Player in Skeletal Muscle Insu-lin Resistance?SOFIA BERG, NILS FÆRGEMAN, MICHAEL GASTER, Odense, Denmark

Carnitine acetyltransferase (CRAT) defi ciency has previously been shown to result in muscle insulin resistance due to accumulation of long-chained acylcarnitines. Myotybes established from type 2 diabetes mellitus subjects express primary insulin resistance. The aim of this study was to examine whether myotubes established from obese persons with and without type 2 diabetes mellitus (T2DM), and lean controls express differences in CRAT and in acylcarnitine species precultured under physiological conditions. Primary myotubes obtained from obese persons with or without T2DM and lean con-trols (N=10 in each group) were established at normophysiological condition and harvested for LC-MS-based profi ling of acylcarnitines. The level of CRAT mRNA and protein levels were determined by quantitative PCR (qPCR) and Western Blotting. Our results show that the protein and mRNA levels of CRAT are unchanged in obese with and without T2DM persons compared to lean controls. We measured 14 different acylcarnitine species and show that the myotube levels of palmitoylcarnitine (C16) and octadecanoylcarni-tine (C18) were slightly reduced in T2DM patients. Moreover, the total level or the levels of the other individual acylcarnitine species were unaltered between the three groups. The present results indicate that CRAT is not important for primary insulin resistance. Long-chain acylcarnitines do not accumulate in obese persons with and without T2DM under physiological conditions, suggesting that the main factor that causes primary insulin resis-tance in T2DM persons still remains unclear.

1734-P

1735-PReceptor-mediated Glucose Regulation in Stem Cell-derived Hepato cytes, Cardiomyocytes, and Skeletal MyoblastsDAVID MANN, NATSUYO AOYAMA, COBY CARLSON, MIKE HANCOCK, BLAKE ANSON, Madison, WI

Glucose homeostasis is tightly regulated in vivo as dysregulation results in tissue damage to multiple organ systems, including hepatic, cardiac and skeletal muscle tissues. Owing to functional limitations, there is a dearth of in vitro models to study normal and diseased tissues. Many primary tis-

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INSULIN ACTION—SIGNAL TRANSDUCTION, INSULIN, AND OTHER HORMONES

sue isolates and immortalized cell lines alter their responsiveness to stimuli in culture over time and become insulin resistant. Induced pluripotent cells (iPSCs) and the differentiated cell types derived therefrom afford a limit-less supply of donor consistent tissue for glucose regulation research and therapeutic discovery.

The liver is the major metabolic regulatory organ potentiating serum glu-cose levels. In hepatocytes, expression of glucokinase is regulated by insulin and glucagon. Here we show in iPSC-derived hepatocytes the cell signaling events triggered by these hormones, as well as the ability to monitor gluco-neogenesis.

Skeletal muscle is a peripheral target for insulin action and thus is an important cell type in diabetes research for studying glucose transport. Here we show that iPSC-derived skeletal myoblasts can be differentiated to elon-gated myogenin-positive myotubes capable of glucose uptake in response to insulin in a dose-dependent manner. Importantly, this signal increase can be modulated by the GLUT4-specifi c inhibitor, Indinavir.

Cardiomyocytes preferentially consume fatty acids for ATP production. However, under particular circumstances, glucose uptake is increased to optimize energy production. We report here an assay to measure intracel-lular glucose levels as demonstrated by an increase in response to insulin and other related molecules. This assay not only enables metabolic studies in human iPSC-derived CMs, but it sets the stage for comparison of disease-specifi c samples to wild-type/normal control cells.

1736-PAn In Vitro Contraction Model that Replicates Exercise-mediated Effects on Glucose Metabolism in C2C12 Skeletal Muscle Cells also Protects against Lipid-induced Insulin ResistanceSTEPHAN NIEUWOUDT, ANNY MULYA, CIARÁN E. FEALY, ELIZABETH E. MAR-TELLI, SATHYAMANGLA V. NAGA PRASAD, JOHN P. KIRWAN, Cleveland, OH, Kent, OH

In vitro models to replicate the metabolic effects of exercise and probe the cellular and molecular mechanisms that mediate skeletal muscle insulin resistance are beginning to emerge. Herein we describe an in vitro contrac-tion model that allows isolation of the specifi c effects of exercise on skeletal muscle glucose metabolism that are typically seen in vivo. We validated the model by probing the effects of contraction on insulin-stimulated glucose uptake and lipid-induced insulin resistance. The model utilizes a customized electrical pulse stimulation (EPS) system to provide the excitation-contrac-tion stimulus to muscle cells. Following 16 hours of stimulation at 1 Hz (1.5 V/mm), fully differentiated C2C12 myotubes were challenged with 0.5 mM palmitate (saturated fatty acid) for 4 hours. EPS increased the basal glucose uptake to the same level as insulin stimulation alone (1 µM, 30 minutes) in non-EPS control cells (P=0.70). The palmitate challenge signifi cantly sup-pressed insulin-stimulated glucose uptake (P<0.05), a key effect of skele-tal muscle insulin resistance. Most importantly, EPS effectively protected against lipid-induced insulin resistance (P<0.05). Western blotting shows that the protective effect on the insulin signaling pathway is signifi cant for the Threonine 308 phosphorylation residue site of Akt/PKB. Insulin receptor substrate-1 (IRS-1) associated phosphoinositide 3-kinase (PI3K) basal activ-ity, upstream of Akt/PKB signaling, was affected by contraction as well, with whole cell insulin stimulated PI3Kα activity matching glucose uptake results. These data suggest that in vitro EPS of skeletal muscle cells pro-vides an experimental model that reproduces the effects of exercise that are observed in vivo, and show that the protection against lipid-induced insulin resistance is mediated through intrinsic regulation of insulin signaling.



Moderated Poster Discussion: Advances in FGF-21 and Insulin Action (Posters: 1737-P to 1743-P), see page 15.

& 1737-PThe Transcriptional Coregulator CITED2 Impairs Vascular Endothe-lial Cell Insulin Signaling by Suppressing IRS-2SAM M. LOCKHART, XUANCHUN WANG, DITTE SORENSEN, SALLY DUN-WOODIE, LARS M. RASMUSSEN, CHRISTIAN RASK-MADSEN, Boston, MA, Syd-ney, Austral ia, Odense, Denmark

Insulin signaling in endothelial cells regulates leukocyte adhesion, angio-genesis and vascular permeability. Hypoxia-inducible factor (HIF) regulates

insulin signaling. Therefore we studied the role of CBP/p300 interacting transactivator-2 (CITED2), a negative regulator of HIF activity, in endothelial cell insulin signaling. We generated an animal model with endothelial spe-cifi c loss of Cited2 using Cre-Lox recombination. In isolated primary endothe-lial cells, loss of Cited2 enhanced insulin stimulated Akt phosphorylation at Ser473 by 3.1±0.8 fold while ERK1/2 phosphorylation remained unchanged. Consistent with this observation, biological functions of insulin were potenti-ated by Cited2 deletion. Cited2 knockout enhanced the effect of insulin on endothelial cell proliferation by 72±9%, and potentiated the ability of insulin to increase Vegfa mRNA by 42±9% and phosphorylation of endothelial nitric oxide synthase by 2.1±0.3 fold. Cited2 deletion did not affect phosphorylation of the insulin receptor at Tyr1345 or insulin receptor substrate (IRS)-1 expres-sion. However, loss of Cited2 resulted in a 66±9% increase in IRS-2 mRNA and a 3.6 fold increase in IRS-2 protein. Therefore, loss of Cited2 enhances endothelial cell insulin signaling via derepression of IRS-2 expression. Con-sistent with a role for CITED2 as a novel mediator of endothelial insulin resis-tance, Cited2 mRNA was increased 10.2±0.3 fold (n=2) in endothelial cells freshly isolated from mice with diet-induced obesity vs. lean controls. Impor-tantly, our fi ndings extend to human disease as CITED2 was elevated 3.8 fold in mammary artery from 5 patients with type 2 diabetes vs. 4 nondiabetic controls. These data identify CITED2 as a novel modulator of endothelial insu-lin signaling that is dysregulated in patients with type 2 diabetes. Inhibition of CITED2 is a potential approach to selectively enhance IRS-2/Akt signaling in endothelial cells and prevent vascular complications.

Supported By: Danish Diabetes Academy; National Institutes of Health (R21CA185196); Diabetic Complications Consortium (U24 DK076169); Diabetes Research Wellness Foundation

& 1738-PDiabetes Remission Induced by the Central Action of Fibroblast Growth Factor 1: Role of InsulinJENNIFER M. ROJAS, JARRAD M. SCARLETT, MILES E. MATSEN, KARL J. KAI-YALA, GREGORY J. MORTON, MICHAEL W. SCHWARTZ, Seattle, WA

In rodent models of type 2 diabetes, hyperglycemia is transiently ame-liorated by either systemic or intracerebroventricular (icv) administration of fi broblast growth factor (FGF)-19 or FGF-21. Based on the relatively long duration (up to 42 h) of the antidiabetic action of FGF1 following systemic administration, we investigated its glucose-lowering potential following icv injection. As reported elsewhere at this meeting, we found that a single icv injection of recombinant murine FGF1 (mFGF1) at a dose (3 µg) 10-fold below that needed for systemic effi cacy induces weight-loss independent diabe-tes remission in both ob/ob (on C57Bl6J background) and db/db mice with moderate hyperglycemia. Specifi cally, blood glucose (BG) values declined from ~300 to 200 mg/dl for up to 4 wk in db/db and 18 wk in ob/ob mice following icv FGF1 (icv Veh vs. FGF1, P<0.0001 by linear mixed model analy-ses). However, icv mFGF1 was ineffective in mice with severe, uncontrolled hyperglycemia (BG >300 mg/dl, including db/db, wild-type C57BL6J (WT) receiving a high dose of the pancreatic β-cell toxin streptozotocin, and ob/ob crossed onto the diabetogenic BTBR background). To test the hypothesis that glucose lowering elicited by icv FGF1 requires an intact insulin signal, we administered the high-affi nity insulin receptor (IR) antagonist S961 to diet-induced obese WT mice as a continuous subcutaneous infusion at a dose (29 nmol/wk) designed to achieve hyperglycemia (~300 mg/dl) com-parable to that observed in moderately diabetic ob/ob mice that respond robustly to icv FGF1. Although transient anorexia induced by icv mFGF1 was not altered by systemic IR blockade, the antidiabetic effect of FGF1 was completely blocked. We conclude that 1) diabetes remission can be induced by the action of brain FGF1, and 2) an intact insulin signal is required for this effect.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases (DK083042, DK090320, DK101997 to M.W.S.), (DK089056 to G.J.M.), DK007247, DK103375; Nutrition Obesity Research Center (DK035816); University of Washing-ton Diabetes Research Center (DK017047)

& 1739-PA Novel Mechanism for Fibroblast Growth Factor-21 to Regulate Hepatic Insulin Sensitivity via Inhibiting Mammalian Target of Rapamycin Complex 1QI GONG, ZHIMIN HU, FEIFEI ZHANG, XIN CHEN, HAOYANG JIANG, JING GAO, XUQING CHEN, YAMEI HAN, QINGNING LIANG, LEI SHI, EUGENE CHIN, YU WANG, HUI XIAO, FEIFAN GUO, YONG LIU, MENGWEI ZANG, AIMIN XU, YU LI, Shanghai, China, Hong Kong, China, Boston, MA

The hepatokine FGF-21 has emerged as a novel metabolic regulator that has potential to treat diabetes and obesity. We recently demonstrated that

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SIRT1 and RARβ are key upstream regulators of FGF-21 to regulate hepatic lipid metabolism. Although pharmacological and physiological studies have demonstrated benefi cial functions of FGF-21 in the liver, the downstream signaling pathways mediating these activities remain largely unknown. We showed that administration of FGF-21 inhibited mTOR/S6K1 activity and increased phosphorylation of Akt and GSK3β to improve hepatic insulin sensitivity and systemic glucose homeostasis in HFHS diet-fed mice. Strik-ingly, these effects were abrogated by hepatic knockdown of FGF-21’s core-ceptor βKlotho using adenovirus-mediated short-hairpin RNA. FGF-21 defi -ciency potentiated hepatic mTORC1 activity and resulted in attenuated Akt phosphorylation in acute insulin-treated mice. Importantly, we showed that insulin- and nutrient-stimulated activation of mTORC1 activity was inhibited by FGF-21 overexpression in human HepG2 cells, leading to increased phos-phorylation of Akt. FGF-21 was suffi cient to increase glycogen synthesis in primary mouse hepatocytes. Moreover, hepatic overexpression of mTORC1’s downstream effector S6K1 abolished FGF-21’s augmentation of insulin sen-sitivity in HFHS diet-fed mice. Our fi ndings indicate that FGF-21-mediated inhibition of mTORC1 may represent a molecular mechanism by which phar-macologic and genetic manipulation of FGF-21 ameliorate hepatic insulin resistance, hyperglycemia, and type 2 diabetes.

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

& 1740-PIntracellular and Extracellular Domain-Dependent Effects of Insu-lin and IGF-1 ReceptorsWEIKANG CAI, MASAJI SAKAGUCHI, ANDRE KLEINRIDDERS, BRIAN T. O’NEILL, JONATHON N. WINNAY, JEREMIE BOUCHER, C. RONALD KAHN, Boston, MA

The insulin receptor (IR) and IGF-1 receptor (IGF1R) are highly homologous, but mediate distinct cellular and physiological functions. To defi ne how the different domains of these receptors contribute to their unique signaling and functions, we created preadipocyte cell lines in which both endogenous IR and IGF1R had been genetically deleted and then reconstituted them with normal IR, IGF1R, or two chimeric receptors: one with the IR extracellular domain (ECD) fused to intracellular domain (ICD) of the IGF1R [IR/IGF1R] and the other with the ECD of the IGF1R fused to the ICD of the IR [IGF1R/IR]. Both cells expressing receptors with the ICD of the IGF1R (i.e., IGF1R and IR/IGF1R) showed higher mitogenic activity, while cells expressing recep-tors with the ICD of the IR (i.e., IR and IGF1R/IR) had more robust glycolytic responses to ligand stimulation. These correlated with increased activation of Shc, Gab-1, ERK1/2 and p70-S6K1 pathways in cells expressing recep-tors with IGF1R ICD, while cells expressing receptors with IR ICD displayed higher phosphorylation on IRS1. Receptors with IGF1R ICD were also more potent in regulating gene expression in pathways involved in proliferation and cell surface protein expression, whereas receptors with IR ICD were more potent in regulating genes involved in metabolic pathways, especially glucose metabolism. Surprisingly, some differences in intracellular signaling and gene expression regulations also correlated with the unique extracellu-lar domains of the two receptors. Changing amino acid residue 961 adjacent in the intracellular NPxY motif from leucine (present in the IR) to phenylala-nine (found in the IGF1R) resulted in changing the signaling and gene expres-sion pattern for IR-like to IGF1R like. These studies demonstrate how domain structural differences between IR and IGF1R result in differential regulation of signaling, gene expression, and cellular functions between IR and IGF1R.

& 1741-PModeling Integrated Insulin and Glucagon Signaling in Human iPS Derived HepatocytesWILLIAM C. ROELL, SIMONE GUPTA, JIANNONG DAI, MELISSA K. THOMAS, Indianapolis, IN

Mechanisms by which integration of hepatic insulin and glucagon signal-ing is dysregulated in type 2 diabetes are incompletely understood, in part due to limitations in translating rodent hepatic physiology to humans and to variable fi delity of disease modeling from cadaveric tissues. Human induced pluripotent stem cell (iPS) differentiated models provide a unique opportunity to address gaps in understanding hepatic physiology and pathophysiology. By modeling culture conditions for hepatocytes differentiated from human iPS cells, we explored their capabilities to recapitulate physiologic counter-regulation of hepatic glucose production. In this model, glucagon stimulated hepatic glucose production with an approximate potency of EC50 ~ 5 nM. Specifi city of this regulation was demonstrated by suppression of glucagon-stimulated glucose production with glucagon receptor antagonist co-admin-istration. Treatment of human iPS derived hepatocytes with insulin resulted in physiologic, dose-dependent suppression of glucose output with marked

potency (IC50 ~ 10 pM). Glucagon-stimulated maximal glucose output was dose-dependently suppressed by insulin, indicating functional integration of insulin and glucagon signaling. Under modeled conditions, human iPS-derived hepatocytes expressed multiple markers of mature hepatocytes (TAT, TDO2, HPX, albumin), including modulators of glucose metabolism (G6PC, PEPCK, FBP), insulin signaling (IRS1, pI3K, AKT, mTOR) and lipid metabolism (PGC1α, CPT1a, LIPC). Cultured human iPS-derived hepatocytes exhibited morpho-logic features reminiscent of human liver, including cobblestone epithelial morphology, glycogen storage, and capacity for intracellular lipid accumu-lation, dependent on extracellular substrate availability. Development of iPS-derived hepatocellular culture models will enable new opportunities for advancing mechanistic understanding of human hepatocyte physiologic and pathophysiologic states.

& 1742-PInsulin Uptake by the Brain Endothelial Cell (BEC) Is Receptor-dependent and Blunted by High-Fat-Diet FeedingSARAH M. GRAY, KEVIN W. AYLOR, EUGENE J. BARRETT, Charlottesville, VA

Insulin access to the brain may be critical for appetite regulation, metabo-lism, and cognition. Prior work suggests insulin crosses the blood-brain bar-rier (BBB) to reach brain interstitial fl uid where it can act on neurons. Little is known about the transit process in the BEC and if insulin resistance affects it. We tested whether BECs have an insulin receptor (IR)-mediated vesicular transport system and if high-fat diet (HFD)-induced insulin resistance affects this. We fed rats HFD or normal chow (ND) for 4 weeks before isolating and culturing BECs. We examined BEC insulin uptake using radiolabeled insulin (125I-ins), insulin signaling, and mRNA and protein expression. 125I-ins uptake was decreased in BECs from HFD rats compared to ND (p<0.01). 10 nM insu-lin increased p-Akt (Ser473) and p-eNOS (Ser1177) similarly in BECs from HFD and ND rats. Insulin did not increase ERK (Thr202/Tyr204), Src (Tyr416), or caveolin-1 (Tyr14) phosphorylation in either group. IR-β mRNA and IR-β protein were not signifi cantly different between HFD and ND. We then used cell-surface biotinylation and western blotting to test whether insu-lin affected IR-β endocytosis. Plasma membrane sheets were isolated from rat microvascular BECs treated with 10 nM insulin or control. Compared to control, insulin treatment decreased plasma membrane-bound IR-β indicat-ing receptor-mediated endocytosis. Blocking PI3-kinase or MEK pathways decreased insulin signaling in whole-cell lysates, but did not prevent IR-β endocytosis. This is consistent with our previous fi ndings that blocking these pathways did not decrease 125I-ins uptake. In conclusion, insulin promotes BEC insulin uptake by stimulating IR-β endocytosis. HFD feeding reduced 125I-ins uptake despite preserved signaling to Akt and eNOS. These fi ndings underscore the need to unravel the precise mechanisms regulating insulin uptake in BECs.

Supported By: National Institutes of Health; American Heart Association

& 1743-PQuantitative Proteomics of Mouse Intestinal Mucosa Lacking Insu-lin ReceptorsSTINA JENSEN, SARAH WHEELER, HENNING HVID, ERWIN SCHOOF, THOMAS KISLINGER, BO F. HANSEN, ERICA NISHIMURA, GRITH S. OLSEN, PATRICIA L. BRUBAKER, Toronto, ON, Canada, Måløv, Denmark

Mice with targeted deletion of the insulin receptor (IR) in the intestinal epithelium (IE-irKO) have been used to study the role of the IR in the gastroin-testinal tract (GIT). In general, IE-irKO mice do not show a strong metabolic- or intestinal-physiological phenotype. Hence, we used an unbiased Mass Spectrometry approach to quantify proteins in jejunal and colonic mucosa from IE-irKO and the two control mice: IE-irKOfl /fl and VIL-Cre. Mice were fed a chow- or high-fat-sucrose western diet (WD) for 12 wk. By proteomic analy-sis, 7931 proteins were identifi ed. Principle component analysis (PCA) clearly separated proteins from chow- and WD-fed mice. Moreover, the PCA indi-cated a distinct differential protein expression in the colonic mucosa of WD-fed IE-irKO mice compared to WD-controls. A greater number of signifi cantly different proteins were found in IE-irKO as compared to control mice when animals were fed the WD instead of chow. Paneth cell products, including Lysozyme, CRIS1C-2/3, Angiogenin-4, and Interlectin-1a, were signifi cantly reduced in IE-irKO jejunal mucosa, as were both Glucose-dependent Insuli-notropic Peptide (GIP) and Neurotensin. Mucin-2 was decreased in both jeju-num and colon of IE-irKO mice. A reduction of the SLC amino acid transport-ers and members of the ATPase family was also found in the colonic mucosa of WD-fed IE-irKO mice. Finally, Apolipoprotein A-I and II were increased in WD-fed IE-irKO colon and jejunum, respectively. Tissue resistance studied by Ussing Chamber and in vivo permeability to FITC-dextran (4 kDa) did not demonstrate any alterations in intestinal barrier integrity in IE-irKO. Together

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the data demonstrate altered protein expression in the intestinal mucosa of IE-irKO mice which was enhanced when the animals were challenged with a WD. In jejunum, Paneth-, Goblet- and Enteroendocrine cell products were reduced, suggesting a role for the IR in the regulation of both host protective functions and gut hormone expression in the GIT.

1744-PEffect of Metformin on Phosphorylation Profi le of Insulin Resistant Primary Human Skeletal Muscle CellsABDULLAH MALLISHO, MICHAEL CARUSO, NISHIT SHAH, BERHANE SEYOUM, YUE QI, DIVYASRI DAMACHARLA, DANJUN MA, XIANGMIN ZHANG, ZHENG-PING YI, Detroit, MI

Metformin is the principle biguanides and the fi rst line drug therapy used for treatment of type 2 diabetes. However, the mechanism of action of Met-formin is still not fully understood. The mainstay of action has been for a long time attributed to its hepatic effect on decreasing hepatic glucose pro-duction. However, Metformin has also been shown to improve insulin sen-sitivity in skeletal muscle. Phosphatases and kinases are proteins required for dephosphorylation and phosphorylation of proteins, respectively, in cells during various signaling pathways. These phosphorylation events carry the potential to be used as drug targets. In the present work, we studied the effect of Metformin on phosphorylation changes of proteins from primary cell culture of human skeletal muscle tissue from obese/overweight insulin resistant participants using phosphoproteomics. We identifi ed 2930 phos-phorylation sites in 1085 proteins. Of particular interest, Metformin treat-ment signifi cantly changed phosphorylation levels of 23 sites, including 6 phosphorylation sites in 4 phosphatase subunits and 17 phosphorylation sites in 9 kinases and kinase subunits. These results provide new informa-tion on the mechanism of action of Metformin in human skeletal muscle cells derived from overweight/obese participants and can potentially be use to better understand how Metformin increase skeletal muscle insulin sensitiv-ity in vivo in humans.

1745-PGlargine and Detemir Do Not Increase Tumour Incidence or Multi-plicity Compared with Unmodifi ed Insulin in a Carcinogen-induced Rat Model of Breast CancerYUSAKU MORI, EUNHYOUNG KO, STUART C. WIBER, GEORGE I. FANTUS, ALAN MEDLINE, ADRIA GIACCA, Toronto, ON, Canada

Glargine and determir are commonly used insulin analogues for basal insu-lin replacement. Concern raised by several studies that glargine increases the risk for all-type cancer was not confi rmed by subsequent studies. How-ever, whether glargine increases the risk for breast cancer is not fully clari-fi ed. This is of particular importance now, with two new glargine prepara-tions, biosimilar and centrated glargine, approved. To gain insight into this issue from preclinical studies, four wk old female Sprague-Dawley rats were started on a high fat diet to induce insulin resistance (hyperinsulinemic clamp onfi rmed) and given the carcinogen N-Methyl-N-nitrosourea (50mg/kg) 1 wk later. At 9 wk of age, the rats were randomly assigned to 4 groups: vehicle, NPH (unmodifi ed human insulin), glargine, and detemir (n=30/group). After 6 weeks of treatment (15 U/kg/day, 5 days/wk), mammary tumours were counted and extracted. No insulin increased mammary tumour incidence. However, tumour multiplicity (number of tumours per rat or per tumour-bearing rat) was increased with NPH or glargine (p<0.05, respectively) and almost increased with detemir (p=0.2) with no difference among insulins. Mammary tumours (all carcinomas) expressed higher levels of insulin recep-tor (IR) and insulin-like growth factor-1 receptor (IGF-1R) than non-tumorous mammary gland. Compared to MCF-7 human breast cancer cells, IR was higher in mammary tumours while IGF-1R expression was much higher in MCF-7. Glargine and detemir increased Akt phosphorylation in tumours, but Erk 1/2 phosphorylation was not changed by any insulin. In summary, in this model of estrogen-dependent breast cancer in insulin resistant rats, insulin glargine and detemir did not increase tumor incidence and multiplicity to a greater extent than unmodifi ed human insulin.

1746-PThe RING Finger Domain of TRAF6 Interacts with Akt for Its Ubiquit-ination and Activation on Insulin StimulationGEETHA THANGIAH, CHEN ZHENG, VISHAL KOTHARI, ANDREA CARTER, JAKE SUSTARICH, RAMESH JEGANATHAN, Montgomery, AL, Auburn, AL

Akt, a serine/threonine kinase, also known as protein kinase B, is respon-sible for glucose metabolism upon insulin signaling. Binding of insulin to its receptor on cell membrane activates several downstream signaling com-ponents in sequence, which eventually leads to Akt phosphorylation and

activation. For its activation, the Akt is translocated from cytoplasm to cell membrane. This process is through Lys63-linked ubiquitination by TRAF6, an ubiquitin E3 ligase. However, it remains unclear where the interaction occurs. Here, we show that TRAF6 through its RING fi nger domain interacts with Akt, leading to Akt ubiquitination, which is essential for Akt activation upon insulin stimulation. Absence of TRAF6 or its RING fi nger domain resulted in impaired the Akt ubiquitination thereby prevented it from activation. These results suggest that TRAF6 induces Akt ubiquitination by interaction of its RING fi n-ger domain with Akt, which regulates Akt activity in insulin signaling.

Supported By: Auburn University at Montgomery (to G.T.); Alabama Agricultural Experimental Station (to R.J.)

1747-PHigher Postprandial GLP-1 Responses Are Associated with Improved Insulin Action in Liver and Muscle but Not in Adipose Tis-sue in Morbidly Obese Nondiabetic SubjectsPIM W. GILIJAMSE, KASPER W. TER HORST, MARIETTE T. ACKERMANS, JOHANNES A. ROMIJN, MAX NIEUWDORP, MIREILLE J. SERLIE, Amsterdam, Netherlands

Glucagon-like peptide 1 (GLP-1) is an incretin which stimulates insulin secretion and improves insulin sensitivity. It remains to be established to what extent meal-induced GLP-1 responses contribute to insulin-mediated glucose and lipolysis fl uxes in humans. We aimed to study these metabolic fl uxes in relation to the postprandial GLP-1 response.We studied 29 mor-bidly obese nondiabetic subjects (18 , 11 ; age 43 ± 2 y; BMI 43 ± 1 kg/m2). The GLP-1 response was assessed during an oral liquid mixed meal test (50 g carbohydrates, 40 g protein, 67 g fat) and basal and insulin-mediated glucose and lipolysis fl uxes were measured using a two-step euglycemic hyperinsu-linemic clamp with infusion of [6,6-2H2]glucose and [1,1,2,3,3-2H5]glycerol. Liver fat content was assessed by magnetic resonance spectroscopy. Following the mixed meal, plasma GLP-1 levels increased 7 ± 1-fold after 30 min and did not return to baseline within 4 h. There was a negative correlation between the GLP-1 and insulin AUCs (r = -0.45, p = 0.015), indicating that a larger GLP-1 response was associated with lower insulin secretion following the mixed meal. The postprandial GLP-1 responses correlated strongly with hepatic (r = 0.59, p < 0.01) and peripheral insulin sensitivity (r = 0.53, p < 0.01), but not with adipose tissue insulin sensitivity (r = -0.20, p = 0.33) or basal endogenous glucose production (r = -0.18, p = 0.41). Finally, postprandial GLP-1 responses did not correlate with gender, age, BMI, waist circumference, or liver fat (p > 0.05), indicating that differences in baseline characteristics did not infl uence the fi ndings. Postprandial GLP-1 responses correlated positively with insulin action in liver and skeletal muscle but not in adipose tissue in obese subjects. The inverse correlation between postprandial GLP-1 and postprandial insulin suggests that the relationship between GLP-1 levels and overall metabolic health is stronger than the incretin effect.

Supported By: European Union

1748-PCurcumin Increases the Expression of Carbohydrate Responsive Element-binding Protein in Male Mouse Hepatocytes via Stimulat-ing p21 Activated Protein KinaseKEJING ZENG, LILI TIAN, ADAM SIREK, WEIJUAN SHAO, LING LIU, YU-TING ALEX CHIANG, JONATHAN CHERNOFF, DOMINIC NG, JIANPING WENG, TIANRU JIN, Guangzhou, China, Toronto, ON, Canada, Philadelphia, PA

In response to hyperglycemia, carbohydrate-responsive element-binding protein (ChREBP) facilitates hepatic lipogenesis in order to reduce the load of plasma glucose. We found previously that insulin can stimulate ChREBP expression via inactivating a transcriptional repressor, the POU homeodo-main protein Oct-1. Here we demonstrated that the effect of insulin in atten-uating the chromosomal Oct-1 occupation on ChREBP promoter and stimulat-ing ChREBP expression is mediated by an Akt-independent ERK activation. Interestingly, the curry dietary compound curcumin was shown to stimulate ChREBP expression in mouse hepatocytes, and short-term curcumin gavage increased hepatic expression of ChREBP. We then demonstrated that cur-cumin can also stimulate ERK phosphorylation and Oct-1 nuclear exclusion in hepatocytes in the presence of Akt inhibition. Furthermore, like insulin, curcumin treatment was shown to stimulate Thr423 phosphorylation of p21-activated protein kinase (Pak-1). The inhibition of Pak-1 or genetic knockout of Pak-1 attenuated the effect of curcumin on stimulating ChREBP expres-sion and reducing Oct-1 levels. Together, our observations indicated a novel signaling cascade, Pak-1/ERK/Oct1, for curcumin in exerting its glucose low-ing effect via promoting hepatic lipogenesis.

Supported By: Canadian Institutes of Health Research

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1749-PHKDC1, a Novel Fifth Hexokinase, Is Involved in Whole-Body Glu-cose MetabolismCAROLINA M. PUSEC, EMILY D. SMITH, ANTON E. LUDVIK, WILLIAM L. LOWE, BRIAN T. LAYDEN, Chicago, IL

Hexokinases are the fi rst enzyme in the glycolysis pathway, catalyzing the phosphorylation of glucose to yield glucose-6-phosphate (G-6-P) as its product. Four well-characterized hexokinase isoforms exist. Recently, Irwin et al. suggested a novel hexokinase-like gene, called Hexokinase Domain Containing 1 (HKDC1) as another hexokinase. Interestingly, through a GWAS we identifi ed and reported a strong genetic association between variants in the HKDC1 gene and 2 hour plasma glucose levels in women that were 28 weeks pregnant. We also reported that HKDC1 is a fi fth hexokinase validated by hexokinase assays. However, these data showed that, despite high sequence homology to hexokinase 1, the enzymatic activity of HKDC1 is more similar to glucokinase. To explore the in vivo role of HKDC1, global HKDC1 mice (HKDC1tg/tg) were generated. Homozygous knockout was an embryonic lethal mutation. We therefore used heterozygous HKDC1tg/twt mice and found impaired glucose tolerance with aging, but no other overt metabolic phenotypes. To circumvent the in utero lethality, we have now generated adult HKDC1 global knock mice via a cross of HKDC1fl /fl with a tamoxifen-inducible Beta-Actin Cre mouse line. Thus far, we have observed that these mice have elevated insulin in the post-absorptive state, while maintaining normal body mass, fasting blood glucose and insulin levels, and post-absorptive glucose levels after tamoxifen treatment. We are currently examining the role of HKDC1 in this model through additional extensive phe-notyping studies. Altogether, our data indicate that HKDC1 is a novel fi fth hexokinase that contributes to whole-body glucose metabolism.

Supported By: National Institute of Diabetes and Digestive and Kidney Dis-eases; National Institutes of Health

1750-PUnraveling the Mechanism behind the Benefi cial Effects of Omega-3 Fatty Acids: The FFAR4-Beta Arrestin-2 InteractionEVELYN WALENTA, VIVIAN A. PASCHOAL, DA YOUNG OH, La Jolla, CA

Recent studies demonstrated that the free fatty acid receptor 4 (FFAR4, formerly GPR120) is the control point for the robust effects of omega-3 fatty acids (ω-3 FAs) to mediate anti-infl ammatory and insulin sensitizing actions. This has been an important discovery in our understanding of the interconnections between obesity, infl ammation, and insulin resistance. The detailed mechanism by which fi sh oil supplementation to dietary regimens exerts benefi cial effects is of high interest. Not only will the discovery of this mechanism help to improve the administration of fi sh oil in a better targeted way, this will also help to identify and develop ligands for FFAR4, which is a promising new drug target.

It has been shown that the ability of beta arrestin-2 (ARRB2) to physi-cally associate with FFAR4 is likely a key mechanism in the anti-infl amma-tory pathway induced by ω-3 FAs. Investigating downstream, we analyze the for the FFAR4-ARRB2 interaction required phosphorylation of FFAR4. We identify a single phosphorylation site (T242) to be necessary for the anti-infl ammatory effects of FFAR4 activation by ω-3 FAs as well as a new FFAR4 specifi c agonist, CpdA. Furthermore, we compare the metabolic phenotypes of ARRB2 whole body knockout mice to FFAR4 whole body knockout mice. We can show that ARRB2 is necessary for the positive effects of ω-3 FAs on reduced lipid accumulation as well as expression of infl ammatory markers. Consequently, the ARRB2 knockout mice do not display the improved insulin sensitivity of wild type mice when fed an ω-3 FA rich diet or the new FFAR4 specifi c agonist, CpdA. This is refl ected in impaired hepatic glucose produc-tion suppression and muscle glucose disposal.

Taken together, we show that the positive effects of ω-3 FAs or CpdA can only operate through the FFAR4-ARRB2 pathway, and that ARRB2 is required for the benefi cial effects of FFAR4 activation by ω-3 FAs or its specifi c ago-nist on lipid accumulation, infl ammation, and insulin sensitivity.

Supported By: Larry L. Hillblom Foundation; American Heart Association

1751-PInsulin Degrading Enzyme Is Upregulated in Type 2 Diabetes Patients Pancreatic Alpha CellsCRISTINA M. FERNADEZ-DIAZ, LUIS ESCOBAR-CURBELO, JOSE F. LOPEZ-ACOSTA, JULIAN SANZ-ORTEGA, GERMAN PERDOMO, IRENE COZAR-CASTEL-LANO, Valladolid, Spain, Madrid, Spain, Burgos, Spain

Insulin Degrading Enzyme (IDE) is an endopeptidase with a broad spec-trum of activity against proteins such as insulin, glucagon and beta-amyloid. Interestingly, the IDE gene has been associated with the risk to suffer type 2

diabetes (T2D) in humans. Consequently, IDE has awaken a great level of interest as a therapeutic target in the treatment of T2D patients. However, the physiological role of IDE on the regulation of glucose homeostasis, and its potential therapeutic benefi t, remains largely unknown. To shed light on the role of IDE in the regulation of glucose metabolism in T2D patients, we analyzed IDE levels in cadaveric human pancreata from T2D patients that underwent the classical treatments used for the control of diabetes, i.e., oral hypoglycemic agents (OHAs) or insulin therapy. In addition, we mea-sured IDE levels in pancreata of a pre-clinical mouse model of diabetes (db/db mouse).

Islets histomorphometry showed that the ratio beta-/alpha-cells was reduced by 50% in T2D patients vs. control subjects. In parallel, the percent-age of alpha-cells was two-fold higher in T2D patients than in control sub-jects. Furthermore, T2D patients showed a two-fold increase in IDE positive cells in comparation with control subjects. Similar results were found in db/db mice. Finally, isolated rodent islets treated with insulin exhibited upregu-lation (~50%) of IDE levels.

In conclusion, we demonstrated that IDE expression is upregulated in human pancreata of insulin treated T2D patients. Of note, the vast major-ity of IDE-positive cells were alpha-cells, suggesting that insulin therapy through upregulation of IDE levels may represent a new cellular mechanism to counteract glucagon production in alpha-cells, contributing to improved overall glucose homeostasis in T2D patients.

Supported By: Ministerio de Economia y Competitividad

1752-PGut Microbiome Produced N-Formyl Peptides Act through the Receptor FPR1 to Modulate Insulin Secretion and MetabolismJOSHUA WOLLAM, ANDREW M. JOHNSON, YONGJIANG XU, MOHIT JAIN, JER-ROLD M. OLEFSKY, La Jolla, CA

In western societies, the increasing prevalence of obesity has been accompanied by a dramatic increase in type 2 diabetes. Diet-induced obe-sity is associated with systemic infl ammation, which is recognized as a con-tributor to insulin resistance and diabetes. However, the mechanisms by which the diet infl uences infl ammation remain poorly understood. A key link between diet and metabolism is the microbiome of the gastrointestinal (GI) tract, producing metabolites that infl uence host physiology. Interestingly, the composition of commensal microbiota and associated metabolites changes dramatically in obesity. Among these, N-formyl peptides are bacterial meta-bolic byproducts and infl ammatory chemokines that activate the G-protein coupled N-formyl peptide receptor 1 (FPR1). We have found that levels of the N-formyl peptide fMLF are elevated in systemic circulation of wild-type (WT) obese mice fed a high fat diet (HFD) compared to lean mice. Knockout of the FPR1 receptor (KO) improves glucose tolerance and insulin secretion in HFD conditions, dependent upon the enteroendocrine-produced hormone glucagon-like peptide-1 (GLP-1), which promotes insulin secretion. Further-more, treatment of WT HFD mice with an FPR1 antagonist improves glucose tolerance and increases plasma GLP-1 and insulin levels. Obese HFD KO mice also display a dramatically altered microbiome composition compared to WT littermates, as well as reduced circulating fMLF levels, suggesting feedback mechanisms exist between the intestinal microbiome and FPR1 signaling to infl uence metabolism. Overall, the altered microbiome in obesity modulates metabolic status through production of N-formyl peptides and activation of FPR1 in the GI tract, leading to suppressed GLP-1 production and insu-lin secretion. FPR1 antagonists may prove to be valuable therapeutic treat-ments providing protection against systemic infl ammation, hyperglycemia, GI disorders, metabolic disease and insulin resistance.


1753-PBrain Insulin Signaling Is Increased in Insulin-Resistant States and May Abet Alzheimer’s Disease DevelopmentMINI P. SAJAN, ROBERT IVEY, MARGARET FARESE-HIGGS, CSILLA ARI, SHIJIE SONG, MICHAEL LEITGES, URSULA BRAUN, BARBARA C. HANSEN, ROBERT FARESE, Tampa, FL, Oslo, Norway, Gulfport, FL

Increased coexistence of Alzheimer’s disease (AD) and type 2 diabetes mellitus (T2DM) suggests that insulin resistance abets AD development, but mechanisms are obscure. Nevertheless, it is commonly assumed that the brain itself is insulin-resistant (IR) in states of systemic insulin resistance, and insulin treatment (Rx) for AD is in trials. Here, we examined insulin signaling in brains of IR high-fat-fed mice, ob/ob mice, mice with impaired glucose transport owing to muscle-specifi c aPKC-λ knockout (MλKO), and monkeys with long-standing obesity/T2DM. In normal mice, 15-min insulin Rx increased brain Akt and aPKC activity. In all IR models, however, basal

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Akt and aPKC activities were maximally increased by hyperinsulinemia as they were unaffected by insulin Rx. Moreover, Akt hyperactivation in all IR models led to hyperphosphorylation of Akt substrates, GSK3β, mTOR, Fox01, Fox03a, and Fox04. And, with decreased Fox0 activity, PGC-1α levels were decreased in all IR models. Akt hyperactivation was also confi rmed in indi-vidual neurons of anterocortical and hippocampal regions that house impor-tant cognition/memory centers. Most notably, Aβ1-40/42 peptide levels were increased by insulin in normal mice and maximally increased basally in all IR models. And, and, in long-standing IR in obese/T2D monkeys, increases in Aβ1-40/42 peptide levels were accompanied by diminished amyloid precursor protein levels, indicating continued conversion to Aβ1-40/42. Tau-PO4 was also increased in ob/ob mice and T2D monkeys. Importantly, with correction of hyperinsulinemia by liver-specifi c inhibition of aPKC and improved systemic insulin resistance, brain insulin signaling normalized. As Fox0s and PGC-1α are essential for memory and maintenance of neuronal structure, function and regeneration and, as Aβ1-40/42 and Tau-PO4 may accumulate in interneu-ronal plaques and intraneuronal tangles, the aforesaid aberrations may link hyperinsulinemia states obesity and T2DM to AD.

Supported By: National Institutes of Health; U.S. Department of Veterans Affairs

1754-PPKCε and CDKs Regulate Insulin Signaling in Response to a 3-Day High-Fat DietBRANDON M. GASSAWAY, MAX C. PETERSEN, HANS R. AERNI, YULIA V. SUROVTSEVA, KARL W. BARBER, JOSHUA B. SHEETZ, SVETLANA ROGULINA, JANIE MERKEL, VARMAN T. SAMUEL, GERALD I. SHULMAN, JESSE RINEHART, West Haven, CT, New Haven, CT

Insulin resistance is the main driving force behind type 2 diabetes mellitus (T2DM). In the liver, diacylglycerol (DAG) is a key mediator of lipid-induced hepatic insulin resistance by activating protein kinase C epsilon (PKCε), which in turn directly inhibits the insulin receptor kinase resulting in hepatic insulin resistance. Knockdown of PKCε in liver protects rats from lipid-induced hepatic insulin resistance in vivo by increasing InsR kinase activity. However, PKCε might not be the best therapeutic target for T2D since this kinase controls other important signaling networks unrelated to insulin sig-naling. We hypothesize that DAG-activated PKCε would act on a network of proteins that would be comprised of known and novel members of the insu-lin signaling pathway. This PKCε network is currently unknown and would potentially contain new candidates for antidiabetic therapeutics. Here we used quantitative phosphoproteomics combined with a systems-biology inspired functional screen to identify and characterize a novel hepatic PKCε network. We quantifi ed changes in protein phosphorylation in insulin-sen-sitive liver, lipid-induced insulin resistant liver, and lipid-loaded liver made insulin sensitive by knockdown of PKCε and uncovered over 550 changes in the phosphoproteome. We observed that Cyclin Dependent Kinases (CDKs) were the primary divers of changes in the phosphoproteome in response to the HFD. We also determined which of the observed phosphoproteins were direct PKCε or CDK substrates by employing a novel kinase substrate expression platform. Finally, we investigated our novel PKCε and CDK net-works with a high-content siRNA screen in cultured hepatocytes, where we examined the role of our liver phosphoproteins as novel inhibitors or activa-tors of canonical insulin signaling. The results of these experiments have identifi ed a more expanded network of proteins involved in insulin signaling, which includes PKCε and CDK substrates that may direct new therapeutic approaches for T2DM.

Supported By: National Institutes of Health; National Science Foundation; How-ard Hughes Medical Institute

1755-PGLP-1R Oral Positive Allosteric Modulators (PAM): Another Approach to Tackle T2DMKRISTIN BREITSCHOPF, ELISABETH DEFOBA, MATTHIAS LOEHN, HANS MAT-TER, MARÍA MÉNDEZ PÉREZ, MICHAEL PODESCHWA, NILS RACKELMANN, JENS RIEDEL, PAVEL SAFAR, Frankfurt, Germany, Tucson, AZ

The Glucagon-like peptide 1 (GLP-1) hormone is secreted from intestinal L-cells after food intake and plays a crucial role in blood glucose regula-tion, and in the control of appetite and body weight. Activation of the cor-responding GLP-1 receptor (GLP-1R) triggers glucose-dependent insulin secretion and leads to suppression of glucagon secretion in the pancreas. For the treatment of type 2 diabetes several pharmaceutical approaches, like GLP-1 mimetics (e.g., liraglutide (Victoza) or lixisenatide (Lyxumia)) and DPP-4 inhibitors (e.g., sitagliptin (Januvia)), are directed to enhance the acti-vation of the GLP-1R. We are following a new approach aiming at positive allosteric modulators (PAMs). These PAMs modulate the activity of GLP-1

(9-36), the inactive metabolite of GLP-1 (7-36) lacking the two N-terminal amino acids, by turning it into a ligand able to activate the GLP-1R in almost the same extent as GLP-1 (7-36) does. We designed small molecule PAMs, which highly activate the GLP-1R in the presence of inactive GLP-1 (9-36) metabolite. Using a recombinant cell line overexpressing the GLP-1R, we can show that the receptor is activated almost as effi ciently as with the active GLP-1 (7-36). In the presence of inactive GLP-1 (9-36) metabolite, our PAMs trigger glucose-stimulated insulin secretion in rodent as well as in human pancreatic islets. Oral glucose tolerance tests in diabetic mice models show that glucose levels are robustly reduced and insulin levels are increased fol-lowing administration of PAM and exogenous GLP-1 (9-36). Furthermore, a moderate reduction in blood glucose level is observed by exploiting the endogenous GLP-1 inactive metabolite levels only.

We conclude that the development of highly potent PAMs with good phar-macokinetic properties could be an attractive alternative to GLP-1 peptide mimetics.

1756-PAssociation between Low Body Mass Index (BMI) and Insulin Sen-sitivityAKANKASHA GOYAL, ANNEKA WICKRAMANAYAKE, SHANKAR VISWANA-THAN, RIDDHI DASGUPTA, MERCY INBAKUMARI, CHAITHANYA MURTHY KOCHER LAKOTA, PRAVEEN GANGADHARA, NIHAL THOMAS, MEREDITH HAWK-INS, New York, NY, Bronx, NY, Vellore, India

A few reports have suggested a J-shaped relationship between BMI and insulin sensitivity, with progressive insulin resistance as BMI falls below 18.5 kg/m2, suggesting a unique phenotype of “low BMI diabetes.” We there-fore studied insulin sensitivity using gold-standard methodologies across a spectrum of BMI ranging from 14 to 27 kg/m2 in subjects with and without diabetes. Euglycemic (90 mg/dl) hyperinsulinemic (80 mU/ m2/min) clamp studies were performed in n=35 ethnically homogeneous Indian males with non-ketogenic diabetes mellitus (DM) (avg age 36.5 y; HbA1c 10.5%; range: % body fat by DXA 7.1-31.8) compared with n=16 matched nondiabetic (ND) subjects (avg: age 33.4 y; HbA1c 5.3%; range: % body fat 10.6-26.5), using 6, 6-deuterated glucose to quantify glucose uptake. Therapeutic regimens were intensifi ed for two weeks to eliminate glucose toxicity in DM subjects. Plasma levels of fat-derived proteins including adiponectin were measured by chemiluminescence. A direct linear correlation was found between BMI and insulin resistance in both groups, with an estimated average 0.48 unit decrease in glucose uptake per one unit increase in BMI (p<0.0001, adjust-ing for diabetes status and adiponectin). Additionally, an average 2.85 unit decrease in glucose uptake was estimated in the DM subjects compared to ND (p=0.0001, adjusting for BMI and adiponectin). These studies are novel in their documentation of improved insulin sensitivity at lower BMIs in both DM and ND subjects, with a progressive linear decrease in glucose uptake with increasing BMI. Across this BMI range, DM subjects were more insulin resistant than ND subjects of comparable BMI and fat mass, underscoring the importance of “glucose toxicity.” When controlling for BMI, changes in adiponectin at low BMI’s had no signifi cant effect on glucose uptake. These are the fi rst studies to examine insulin resistance in DM subjects with low BMI using sophisticated methodologies and promote a shift in our under-standing of the phenotype of low BMI diabetes.

1757-PInsulin Regulation of TLR4 Signaling in Human LeukocytesLOUIS F. AMOROSA, ZHIYONG ZHANG, MARIE MACOR, SUSSETE COYLE, LEON-ARD LEE, BEATRICE HAIMOVICH, New Brunswick, NJ

Human peripheral blood leukocytes (PBL) are a readily available model for studying infl ammatory cellular signaling. We used these cells to investi-gate insulin signaling in patients with chronic and acute insulin resistance. Our recent studies demonstrated that MMP9 expression in PBL is involved in the cleavage of AMPKinase, in vivo and in vitro, following stimulation with lipopolysaccharides, a TLR4 ligand. MMP is also expressed in PBL of free living type 2 diabetic patients and patients who develop acute insulin resistance associated with cardiopulmonary bypass surgery. These stud-ies link MMP expression and AMP Kinase degradation with a cascade of transcription factor phosphorylations associated with clinical insulin resis-tance. (Zhang Z, et al, J Immunol, 2015). In a subset of these patients with type 2 diabetes, we have identifi ed additional differences in signaling inter-mediates including AKT phosphorylated on Ser473. Insulin, but not LPS, induced mTORC2-dependent phosphorylation of AKT on Ser473 and Fox01/O3 on Thr24/25. Furthermore, insulin and AS1842856, a Fox01 inhibitor, sup-pressed the expression of LPS-induced signaling intermediates in a dose- and time-dependent manner. We conclude that insulin is a homeostatic

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regulator of PBL response to TLR4 ligands through Fox01 phosphorylation. The infl ammatory signaling signature expressed in PBL of type 2 diabetic patients suggests TLR4 dominance and lack or defective insulin signaling. The model demonstrates that the mechanism PBL employ to react to infl am-matory stimuli, is associated with impaired transduction of the insulin signal as found chronically in PBL of patients with type 2 diabetes and acutely in nondiabetic patients following cardiopulmonary bypass.

1758-PPlasma SerpinB1 Levels Are Strongly Correlated with Circulating ANGPTL8 Levels in Patients with Type 2 DiabetesSHINSUKE TOKUMOTO, AKIHIRO HAMASAKI, YUKIKO KAWASAKI, SACHIKO HONJO, YOSHIYUKI HAMAMOTO, Osaka, Japan

Background and Aims: SerpinB1, a protease inhibitor secreted by the liver, has recently been described as a potent stimulator that increases beta cell proliferation in mice and humans. While we previously reported that ANGPTL8 correlated with insulin secretion capacity, the pathophysiological role of SerpinB1 in patients with type 2 diabetes mellitus (T2DM) remains poorly understood. The aim of the study was to evaluate the relationship between plasma SerpinB1 levels and other biomarkers including ANGPTL8.

Materials and Methods: Overnight fasting plasma samples from 8 healthy subjects and 29 T2DM patients were collected. HbA1c, fasting plasma glu-cose, total cholesterol, triacylglycerol, and creatinine clearance (CrCl) cal-culated by 24-hour urine collection were measured in all T2DM patients. Plasma levels of serpinB1 (Cusabio, Catalogue No.CSB-EL021065HU) and ANGPTL8 (Eiaab, Catalogue No.E11644h) were determined by enzyme-linked immunosorbent assay according to the manufacturer’s protocol. Correla-tions were evaluated by Spearman’s rank test. P values <0.05 were consid-ered statistically signifi cant.

Results: Plasma serpinB1 levels were signifi cantly higher in T2DM patients (0.37 ± 0.20 ng/ml) than in healthy subjects (0.21 ± 0.16 ng/ml; p < 0.05). In T2DM patients, plasma serpinB1 levels showed a signifi cant posi-tive association with ANGPTL8 (r = 0.40 p = 0.02), and, both serpinB1 and ANGPTL8 levels signifi cantly correlated only with age (serpinB1: r = 0.42 p = 0.02; ANGPTL8: r = 0.46 p = 0.01), duration of diabetes (serpinB1: r = 0.53 p = 0.003; ANGPTL8: r = 0.45 p = 0.01), and CrCl (serpinB1: r = −0.39 p = 0.03; ANGPTL8: r = −0.45 p = 0.01).

Conclusion: We showed that serpinB1 levels were higher in T2DM patients than in healthy subjects, suggesting that serpinB1 plays a role in regulation of beta cell mass in human. Since both serpinB1 and ANGPTL8 levels corre-lated with each other and with the same parameters, both proteins could be similarly regulated in T2DM patients.

1759-PModel of the Glucose-Insulin-Glucagon Dynamics after Subcutane-ous Administration of a Glucagon Rescue Bolus in Healthy HumansSABRINA L. WENDT, JAN K. MOELLER, AHMAD HAIDAR, BRITTA V. BYSTED, CARSTEN B. KNUDSEN, HENRIK MADSEN, JOHN B. JOERGENSEN, Glostrup, Denmark, Kongens Lyngby, Denmark, Montreal, QC, Canada

In healthy individuals, insulin and glucagon work in a complex fashion to maintain blood glucose levels within a narrow range. This regulation is dis-torted in patients with diabetes. The hepatic glucose response due to an elevated glucagon level depends on the current insulin concentration and thus endogenous glucose production (EGP) can not be modelled without knowledge of the concentration of both hormones in plasma. Furthermore, literature suggests an upper limit to EGP irrespective of glucagon levels. We build a simulation model of the glucose-insulin-glucagon dynamics in man including saturation effect of EGP. Ten healthy subjects received a 1 mg sub-cutaneous (SC) glucagon bolus (GlucaGen®). Plasma samples were collected until 300 minutes post dose and analyzed for glucagon, insulin, and glucose concentrations. All observations were used to fi t a physiological model of the glucose-insulin-glucagon dynamics using the Hovorka model with a novel multiplicative description of the effects of insulin and of glucagon on EGP. Bayesian estimation by Maximum a Posteriori using prior knowledge reported in literature was used to estimate the model parameters for each subject. Profi le likelihood plots were used to investigate parameter identifi -ability. Unidentifi able parameters were fi xed at their prior mean values. The new model enables simulations of the glucose-insulin-glucagon dynamics in humans at both low and high glucagon concentrations (180-8000 pg/mL) and physiologic insulin concentrations (1.2-81.9 mIU/L). The model can be used for simulation of glucagon bolus strategies for treatment of hypoglycemia and for in silico simulation of dual-hormone artifi cial pancreas algorithms.

Supported By: Zealand Pharma A/S

1760-PInsulin Sensitivity and Clearance Determine Time to Post-Clamp Glycemic Stabilization in Nondiabetic SubjectsIBIYE OWEI, NIDHI JAIN, NKIRU UMEKWU, DAVID JONES, SAMUEL DAGOGO-JACK, Memphis, TN

Measurement of insulin sensitivity with hyperinsulinemic euglycemic clamp involves constant infusion of insulin along with variable dextrose (D20) in overnight fasted subjects. There is individual variation in the time to post-clamp glycemic stabilization, the determinants of which are unclear. Here, we tested the hypothesis that post-clamp glycemic stabilization is dependent on insulin clearance and insulin sensitivity. We studied 220 healthy subjects (110 black {B} and 110 white {W}). Each subject underwent anthropometry, oral glucose tolerance test (OGTT), and measurement of plasma insulin and c-peptide levels. Insulin sensitivity was assessed using the hyperinsulinemic clamp (ISI) and HOMA-IR. Insulin clearance was calcu-lated using the molar ratio of fasting C-peptide and insulin concentrations. At the end of the clamp, insulin infusion was stopped and dextrose infusion continued. Plasma glucose (PG) was determined every 10 min, with gradual weaning of D20 infusion. Glucose stabilization time (GST) was defi ned as the interval between stopping insulin infusion and D20 infusion. The mean (±SD) age of our cohort was 46.3±9.96 yr and BMI was 30.7±8.43kg/m2. The fi nal PG (mg/dl) at discharge was 124.2±26.9 for the entire cohort, 123.9±25.8 (men), 124.3±27.4 (women), 122.1±26.2 (B) and 126.1±27.4 (W). There were no ethnic or gender differences in the fi nal PG level. The mean GST (min) was 72.2±31.4 for all subjects, 77.7±34.8 (men), 69.7±29.6 (women), 73.3±33.2 (B) and 71.1±29.8 (W). The GST did not differ signifi cantly by race, gender or age. Using linear regression models, the signifi cant predictors of post-clamp GST were 2 hr OGTT PG (p=0.0002), insulin clearance (p=0.02), ISI (p=0.03) and HOMA-IR (p=0.01). We conclude that glucose stabilization following hyper-insulinemic euglycemic clamp was not related to gender or ethnicity, but was signifi cantly delayed by lower glucose tolerance, insulin resistance, and decreased insulin clearance.

Supported By: American Diabetes Association (7-07-MN-13 to S.D-J.); R01 DK067269, R01 DK067269-04S1

1761-PCharacterization of the Cardiac Function of Human Insulin and the Long-Acting Insulin Analogs M1-glargine and Degludec in Adult Rat Ventricular CardiomyocytesPAULUS WOHLFART, THORSTEN M. HARTMANN, NINA WRONKOWITZ, JUER-GEN ECKEL, NORBERT TENNAGELS, Frankfurt, Germany, Düsseldorf, Germany

An important aspect for the long-term use of insulin analogues are car-diovascular effi cacy and safety data. Previously, we have shown that under steady state conditions, insulin glargine (IGla) and its active metabolite M1 (IGlaM1) as well as insulin degludec (IDeg) all elicit a comparable signaling and contractility response in different cardiomyocyte cell models Indeed, under steady state conditions all insulins displayed a similar AKT phospho-rylation and contractility of electrically paced ARVM after 15 min. However, for IDeg a delayed onset of action could be observed. In the current study, we investigated the early response of human insulin (HI), GlaM1 and IDeg in adult rat cardiomyocytes (ARVM). In the early phase (1-5 min), only HI and IGlaM1 were able to stimulate fractional shortening of total cardiomyocytes length in a comparable dose-dependent manner (1, 10, and 100 nM), with a maximal shortening at 100 nM of 8.8 ± 1.1% and 7.3 ± 0.3%, respectively. In contrast, IDeg displayed no clear dose-response up to 1000 nM and a maximum increase of only 3.6 ± 0.3% at 1000 nM. The difference might be explained by a different binding behavior of IDeg towards the cell mem-brane embedded insulin receptor (IR). The IC50 values observed in radioac-tive insulin displacement assays differs between detergent solubilized (S-IR) and membrane embedded IR (M-IR) preparations (IC50 values [nM] S-IR vs. M-IR: 0.4/ 0.6 (HI), 0.7/ 0.9 (GlaM1), 2.3/ 18.2 (IDeg)), which could result in a different association kinetic. In summary, the data demonstrated that IDeg, and IGlaM1 show a different onset of action in AVRM most probably due to a different IR kinetic. However, under steady-state-conditions, a similar effi -cacy can be observed. Whether those differences might translate to in vivo conditions needs further investigation.

Supported By: Sanofi

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1762-PImpaired Cardiomyocyte Autophagic Flux in Ischemic/Reperfused Diabetic Heart: Critical Role of HypoadiponectinemiaYAJING WANG, BING LIANG, WAYNE LAU, BERNARD LOPEZ, THEODORE CHRIS-TOPHER, XINLIANG MA, Philadelphia, PA

Autophagy is an important regulator of myocardial ischemia/reperfusion (MI/R) injury. However, whether and how autophagy is altered in I/R diabetic heart remains unknown. Adiponectin (APN) is a cardioprotective molecule. APN knockout (APNKO) markedly increases MI/R injury. However, whether and how hypoadiponectinemia may alter cardiac autophagy in I/R diabetic heart is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes (HFD), and APNKO mice, we demonstrated that autophagosome formation was signifi cantly inhibited (P<0.01 vs. NC) and autophagosome clearance was virtually abolished (P<0.01 vs. NC) in HFD heart subjected to MI/R. APNKO largely reproduced the phenotypic alterations occurring in HF-D heart, with both autophagosome formation and clearance inhibited after MI/R. Treat-ment of HFD and APKNO mice with AdipoRon (an APN receptor agonist) stimulated autophagosome formation, increased autophagosome clear-ance, reduced infarct size, and improved cardiac function (P<0.01). Mecha-nistically, AdipoRon caused signifi cant phosphorylation of AMPK, BeclinT119, and VPS34S164, and enhanced expression of lysosome protein LAMP2 both in vivo and in isolated adult cardiomyocytes. Pharmacologic (Compound C) or genetic (dominant negative AMPKα2 overexpression) AMPK inhibition abol-ished AdipoRon-induced BeclinT119/VPS34S164 phosphorylation and autopha-gosome formation. However, AdipoRon-induced LAMP2 expression and autophagosome clearance were not affected by AMPK inhibition (P>0.05 vs. vehicle or WT). Collectively, these results demonstrate for the fi rst time that hypoadiponectinemia impairs autophagic fl ux, contributing to enhanced MI/R injury in diabetic individuals. Restoring APN signaling systems by APN receptor agonist activates APMK-mediated autophagosome formation and AMPK-independent autophagosome clearance, representing a novel inter-vention against MI/R injury in diabetic conditions.

Supported By: American Diabetes Association (1-14-BS-218 to Y.W.)

1763-PElectrical Stimulation of Peripheral Sympathetic Nerve Fascicle Enhances Non-Insulin-mediated Glucose Uptake in RatsDAISUKE SATO, RIKU YAMAGUCHI, HIROYUKI SASAKI, MASATAKA KUSUNOKI, ZHONGGANG FENG, TAKAO NAKAMURA, Yamagata, Japan, Nagoya, Japan, Yonezawa, Japan

Regarding the possibility that the sympathetic nervous system could regulate peripheral glucose uptake, we reported that electrical stimulation of peripheral sympathetic nerve fascicle (microstimulation), eliciting small muscle contraction, induced transient blood glucose (BG) reduction within 30 s of start of the microstimulation while plasma insulin (PI) changed little in rats. In the present study, we evaluated the effects of microstimulation with no muscle contraction, and assessed the glucose uptake caused by the stimulation.

We microneurographically detected peripheral sympathetic nerve signal to locate the tip of microelectrode in a sympathetic fascicle in the rat sciatic nerve, and then electrically stimulated the fascicle via the micro electrode (n=7). Based on our preceding study, the stimulation intensity was set at 0.05-0.10 V lower than muscle contraction threshold to enhance sympathetic nerve activity. BG and PI levels were measured throughout the microstimula-tion. Glucose uptake was assessed as glucose infusion rate (GIR) measured before, during, and after the microstimulation with the euglycemic clamp method in additional rats (n=9).

As a result, the microstimulation transiently reduced BG (from 68 ± 4 to 64 ± 3 mg/dL, p<0.01, mean ± SD) 30 s after start of the microstimulation while PI changed little (from 2.0 ± 0.7 to 1.7 ± 0.7 ng/mL) as previously seen in the microstimulation accompanying muscle contraction. The microstimu-lation signifi cantly increased GIR from 12.9 ± 1.5 to 15.2 ± 1.5 mg/kg/min (p<0.01). After the termination of the microstimulation, GIR was remained higher (14.7 ± 1.8 mg/kg/min) than that observed before the stimulation (p<0.05) although GIR seemed to slightly decrease.

These results indicate that the transient BG reduction induced with the microstimulation might be attributed to enhancement of non-insulin-medi-ated glucose uptake, and that the effect could remain even after terminating the microstimulation.

Supported By: Japan Society for the Promotion of Science

1764-PCirculating Apolipoprotein J Is a Novel Marker of Insulin Resist-anceJI A. SEO, MIN-CHEOL KANG, SANG SOO KIM, SOO HYUN HONG, MICHELLE CHUNG, THEODORE P. CIARALDI, ROBERT R. HENRY, YOUNG-BUM KIM, Ansan, Republic of Korea, Boston, MA, La Jolla, CA, Del Mar, CA

Insulin resistance is a major causal factor for developing type 2 diabetes. Apolipoprotein J (ApoJ, also called clusterin), a secreted sulfated glycopro-tein, has been implicated in altered pathophysiologic states such as cardio-vascular diseases, Alzheimer’s Disease, and cancer. However, the metabolic function of ApoJ in glucose homeostasis remains unclear. This study sought to determine whether serum ApoJ levels correlate with insulin resistance and if they change after an intervention that improves insulin sensitivity. Serum ApoJ, insulin resistance status using HOMA-IR and QUICKI were measured in nondiabetic (ND) and type 2 diabetic (T2D) subjects. The impacts of rosigli-tazone or Metformin treatment for 4 months on serum ApoJ levels were also evaluated in T2D subjects. ApoJ protein was measured by immunoblotting. Fasting serum ApoJ levels were elevated in humans with type 2 diabetes (T2D vs. ND; 141.3±9.3 vs. 100±9.1 AU, p=0.002). In ND subjects, insulin-resistant subjects had higher levels of serum ApoJ than insulin-sensitive subjects. Circulating ApoJ levels strongly correlated with fasting insulin, HOMA-IR, QUICKI, and body mass index. Multiple regression analysis indi-cated that ApoJ levels were a signifi cant independent association factor for HOMA-IR even after adjustment for age, sex, and body mass index. Rosigli-tazone treatment of subjects with T2D resulted in a reduction in serum ApoJ levels (before vs. after treatment; 100.0±20.6 vs. 67.6±7.9 AU, p<0.001) but Metformin had no effect on ApoJ levels. In conclusion, serum ApoJ levels are closely correlated with the magnitude of insulin resistance regardless of obesity and decreases after insulin-sensitizer treatment in type 2 diabetes. Thus, circulating ApoJ could be a novel marker of insulin resistance.

Supported By: American Diabetes Association (7-12-BS-094 to Y-B.K.); U.S. Department of Veterans Affairs

1765-PUterus Remains Sensitive to Insulin Induced AKT Activation due to Impaired Inhibition through IRS-1 in a Lean, Hyperinsulinemic Transgenic MouseCLARE A. FLANNERY, CAITLIN RADFORD, FARRAH SALEH, DEREK LEROITH, EMILY J. GALLAGHER, HUGH S. TAYLOR, New Haven, CT, New York, NY

Type 2 diabetes is an independent risk factor for developing endometrial adenocarcinoma. We hypothesized that hyperinsulinemia promotes abnor-mal endometrial proliferation, and sought to examine uterine insulin sensi-tivity in vivo. We previously found endometrial hyperplasia occurring in the lean, euglycemic, but hyperinsulinemic MKR transgenic mouse, despite a 60% reduction in mitogenic insulin receptor (IR-A) mRNA, relative to WT.

To determine uterine insulin sensitivity, we semi-quantifi ed phosphory-lated and total signaling proteins using western blot and densitometry from extracted uteri of MKR (n=9) and WT (n=5) mice at 15 weeks of age. Each group was analyzed in the proliferative and secretory phases (PP, SP) by 2-tailed t-test after log transformation.

We found Akt-ser473 phosphorylation was increased by 188% in PP (p=0.02) and 244% in SP (p=0.05) of MKR, whereas p-MAPK-thr202/tyr204 was similar between MKR and WT. In all MKR, phosphorylation of inhibitory IRS-1 site ser636/639 was absent in both phases, while phosphorylated in WT. P-IRS-1-ser318 was reduced by 66% in SP, vs. WT (p<0.05). Mean PTEN were similar.

Here we isolated the endometrial effect of excess insulin from obesity and unopposed estrogen in an in vivo model. Hyperinsulinemia did not induce resistance to insulin signaling in the uterus, as reported in muscle and adi-pose. Despite a protective reduction in mitogenic IR-A, the endometrium remains sensitive to insulin induced AKT activation due to impaired inhi-bition through IRS-1. In this setting, PTEN remains a protective barrier to unopposed AKT activation. In women with type 2 diabetes, endogenous hyperinsulinemia may promote abnormal cellular proliferation, leaving the endometrium susceptible to unregulated growth if PTEN is disrupted through mutagenesis. Mutations of PTEN, rendering it inactive or defi cient, are commonly found in endometrial adenocarcinoma.

Supported By: Eunice Kennedy Shriver National Institute of Child Health and Human Development (K08HD071010)

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1766-PGenetic Ablation of Interleukin-10 Receptor in Skeletal Muscle Affects Glucose Metabolism in Diet-induced Obese MiceSEZIN DAGDEVIREN, HYE-LIM NOH, HEE JOON KANG, SUCHAORN SAENGNI-PANTHKUL, RANDALL H. FRIEDLINE, XIAODI HU, KUNIKAZU INASHIMA, CAIT-LYN C. KEARNS, MARILIA L. LOUBATO, CECILIA P. UONG, JASON K. KIM, Worces-ter, MA

Skeletal muscle insulin resistance is a major characteristic of obesity and type 2 diabetes and is causally associated with infl ammation. We have pre-viously shown that interleukin-10 (IL-10) improves muscle glucose metabo-lism partly by attenuating local infl ammation in obese mice. To further exam-ine the effects of IL-10, we generated mice with muscle specifi c deletion of IL-10 receptor (MCK-IL10r KO) that developed without obvious anomaly and showed normal glucose metabolism on chow diet. MCK-IL10r KO and wild-type (WT) mice were fed a high-fat diet (HFD) for 6 wks (n=6~11) and 16 wks (n=5~7), and insulin sensitivity was assessed using hyperinsulinemic-euglycemic clamp in awake mice. Both groups of mice became obese after 6 or 16 wks of HFD (Figure 1). After 6 wks of HFD, basal glucose levels tended to be higher in MCK-IL10r KO mice (211±12 vs. 176±12 in WT mice; P=0.08). MCK-IL10r KO mice became more insulin resistant than WT mice after 6 and 16 wks of HFD with signifi cant decreases in whole body glucose turnover in these mice (Figure 2; P<0.05). This was mostly due to ~25% reductions in skeletal muscle glucose metabolism in HFD-fed MCK-IL10r KO mice as com-pared to HFD-fed WT mice (Figures 3 and 4). These results indicate that IL-10 directly affects skeletal muscle insulin resistance, and our fi ndings identify a novel role of IL-10 receptor-mediated signaling in the regulation of muscle glucose metabolism.Figure.

Supported By: National Institutes of Health (R01-DK080756, U24-DK093000, R24-DK090963)

1767-PIncreased Expression of sFRP4 Associates with Insulin Resistance in Liver and Skeletal MuscleTINA HÖRBELT, DEIKE HESSE-WILTING, MARLIES BEKAERT, MAREN CARSTENSEN-KIRBERG, FREDERIQUE VAN DE VELDE, YVES VAN NIEUWENHOVE, BRUNO LAPAUW, ANNETTE SCHÜRMANN, CHRISTIAN HERDER, MICHAEL RODEN, D. MARGRIET OUWENS, Düsseldorf, Germany, Potsdam, Germany, Ghent, Belgium

Secreted frizzled-related protein 4 (sFRP4) is a regulator of the activity of the Wnt signaling pathway, which is released by multiple tissues including adipose tissue. Emerging evidence links increases in circulating sFRP4 levels to the development of β-cell dysfunction. Yet, it is currently unclear whether changes in the expression of sFRP4 in adipose tissue affect insulin sensitiv-ity in peripheral tissues, such as the liver and skeletal muscle. Therefore, we examined whether the expression of sFRP4 is changed in visceral adipose tissue (VAT) biopsies obtained from a cohort of morbidly obese men with (n=27) and without type 2 diabetes (n=39), and normal-weight men (n=15) undergoing abdominal surgery. Furthermore, we analyzed whether recom-binant sFRP4 affects insulin signaling in primary murine hepatocytes and primary human skeletal muscle cells (hSkMC). Levels of sFRP4 mRNA were 3.2-fold increased in VAT from morbidly obese men, independent of type 2 diabetes, vs. normal weight men (p<0.001), and associated positively with BMI (r=0.58, p<0.001) and HOMA-IR (r=0.38, p<0.001). A negative associa-tion was found with circulating adiponectin levels (r=-0.42, p<0.001). In vitro,

exposing hepatocytes to sFRP4 impaired the insulin-mediated phosphory-lation of Akt-Ser473 by 27% and its substrates glycogen synthase kinase 3β-Ser9 (GSK3β-Ser9) by 27%, and Forkhead box proteins O1/3a (Fox01/3a)-Thr24/32 by 34% (all p<0.05). Although sFRP4 did not affect insulin-mediated phosphorylation of Akt-Ser473 and GSK3β-Ser9 in hSkMC, insulin-mediated phosphorylation of Fox01/3a-Thr24/32 was reduced by 30% (p<0.05). In con-clusion, increased expression of sFRP4 associates with insulin resistance, which may result from defects in the phosphorylation of the Forkhead tran-scription factors.

Supported By: German Center for Diabetes Research

1768-PProteotoxicity Constrains the Structure of a Globular Protein: Insulin as a Case StudyNISCHAY K. REGE, FARAMARZ ISMAIL-BEIGI, NELSON B. PHILLIPS, JONATHAN WHITTAKER, MICHAEL A. WEISS, Cleveland, OH

Insulin is a peptide hormone that initiates the uptake of glucose from the blood by peripheral tissues. The absolute or relative lack of insulin is the cause of type 1 or type 2 diabetes mellitus, respectively. Insulin’s evolution shows interplay between optimizing its ability to bind to its target, insulin receptor (IR), and maintaining a tertiary structure that confers resistance to proteotoxic aggregation: although insulin forms amyloid fi brils in phar-macological formulations, insulin derived amyloids are not seen in human pathophysiology. Among insulin’s highly conserved structural motifs is a type I β-turn spanning residues 20-23 of its B chain. We hypothesize that this structural motif is conserved as a result of its role in protecting against amy-loid fi bril formation rather than its necessity for IR engagement. Our results indicate that although elimination of this turn through mutation of GlyB23 attenuates insulin’s biological activity, the substitution of PheB24 for glycine restores activity through the creation of a putative non-canonical turn. How-ever, these double mutant analogs show increased susceptibility to amyloid formation compared to wild type insulin. Double mutants also show dimin-ished ability to assemble into oligomers that protect the molecule from non-native aggregation. These results underscore potential proteotoxicity’s role as a driving force for protein evolution and its contribution to amyloid-based diseases and diseases of protein misfolding.

Supported By: National Institutes of Health; National Institute of Diabetes and Digestive and Kidney Diseases

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INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

1770-PDNA Repair Enzyme OGG1 Regulates Obesity and Insulin Sensitiv-ity with AgeLARYSA YUZEFOVYCH, MYKHAILO RUCHKO, MICHELE SCHULER, LYUDMILA RACHEK, Mobile, AL

Oxidative stress and mitochondrial dysfunction in skeletal muscle are important factors leading to insulin resistance (IR) during the aging process but the underlying mechanisms are still unknown. Among the potential tar-gets is mitochondrial DNA (mtDNA), since mtDNA is highly specialized and encodes for proteins essential for energy metabolism and, also, damage to mtDNA heightens mitochondrial ROS (mtROS) production that is very criti-cal for IR. Despite extensive age-related studies on mtDNA mutations, until recently, the integrity of mtDNA and repair mechanisms have received little attention in diabetes research. We hypothesized that progressive oxidative mtDNA damage triggers development of age-associated oxidative stress in skeletal muscle, and thus, potentiates development of IR with age. To vali-date this hypothesis, we performed studies in vivo using DNA repair enzyme, 8-oxoguanine (8-OxoG) DNA glycosylase (Ogg1) knockout and overexpressing models: 1) Ogg1 -/- (KO); 2) Tg MTS-hOGG1 (Tg, mice overexpressing human OGG1 (hOGG1, subunit 1-α) in mitochondria; 3) wild type (WT, ogg1 +/+). We evaluated obesity and IR phenotypes, oxidative mtDNA damage, oxidative stress and expression of several mitochondrial proteins in skeletal muscle from young (~5 month old) and aging (~15 month old) males from all three OGG1 groups. Our results showed that mitochondrial hOGG1 overexpression amelio-rated age-associated obesity and IR phenotypes and protected against oxi-dative mtDNA damage and oxidative stress. Additionally, protein content for transcriptional coactivator peroxisome proliferator activated receptor alpha (PGC-1α) and mitochondrial protein porin was signifi cantly increased in skel-etal muscle from aging Tg mice. Collectively, these proof-of-concept results provide fi rst direct evidence that oxidative mtDNA damage triggers oxidative stress and development of age-associated IR in mice, thus making mtDNA damage and repair a novel target for treatment of IR and type 2 diabetes.

INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

Moderated Poster Discussion: Insulin Secretion in Animals and Humans (Posters: 1771-P to 1777-P), see page 14.

& 1771-PInsulin Receptor Knockout Using Two Beta-Cell-Specifi c Cre Mouse Lines Transiently Improves Glucose HomeostasisSØS SKOVSØ, LYNDA ELGHAZI, DEREK A. DIONNE, MELISSA M. PAGE, HONG LI, DARIA HUTCHINSON, XIAOKE HU, FARNAZ TAGHIZADEH, ERNESTO BERNAL-MIZRACHI, JAMES D. JOHNSON, Vancouver, BC, Canada, Miami, FL

Insulin signaling is important for glucose homeostasis and is disrupted in type 2 diabetes. The insulin receptor is highly expressed in beta cells, where its function remains to be fully established. Previous studies on mice gener-ated to delete insulin receptors (InsR f/f) from beta-cells reported impaired glu-cose tolerance, reduced insulin secretion, and reduced beta-cell mass. How-ever, the so-called BIRKO model employed Cre recombinase under the control of a short fragment of the RIP, which led to deletion of the insulin receptor in both beta-cells and the brain. We generated two new mouse models to re-address the function of insulin receptors specifi cally in beta-cells. First, we crossed InsR f/f mice with a mouse line expressing Cre within the endogenous Ins1 gene locus (Ins1 Cre), as our group and others have shown little or no Ins1 gene expression in the brain. At 25 weeks of age, female InsR f/f:Ins1 Cre mice exhibited improved glucose tolerance relative to InsR wt/wt:Ins1 Cre littermate controls. Interestingly, InsR f/f:Ins1 Cre mice were also signifi cantly heavier from 7-16 weeks of age. Second, we employed the tamoxifen-inducible Ins1-CreERT transgenic mouse model, which has been shown to have virtually no recombination in the brain. Similarly, InsR f/f:Ins1-CreERT mice had signifi cantly improved glucose tolerance 4 weeks after tamoxifen injection relative to both InsR wt/wt:Ins1-CreERT and InsR f/f littermate controls, although this reversed to impaired glucose homeostasis later in life. There were no signifi cant dif-ferences in body weight in the tamoxifen-inducible model, suggesting that effects of beta-cell InsR deletion were restricted to developing and/or young mice. Collectively, these data are consistent with the concept that insulin inhibits its own secretion, at least initially. Together, these data provide new information about the function of beta-cell insulin receptors and will provide more insight into the pathogenesis of type 2 diabetes.

Supported By: Canadian Institutes of Health Research

& 1772-PAssociation of a Type 1 Diabetes Genetic Risk Score with C-Peptide and Age of Diagnosis in Type 1 DiabetesDELNAZ ROSHANDEL, RICHARD A. ORAM, ANDREW T. HATTERSLEY, MICHAEL N. WEEDON, ROSE GUBITOSI-KLUG, SHELLEY B. BULL, ANDREW D. PATERSON, DCCT/EDIC RESEARCH GROUP, Toronto, ON, Canada, Exeter, United Kingdom, Cleveland, OH

A proportion of people with type 1 diabetes (T1D) have detectable C-pep-tide years after diagnosis: they maintain better glycemic control and experi-ence lower risk for diabetic complications. Multiple loci have been identifi ed for risk of T1D, but it is unknown whether they or a T1D genetic risk score (GRS) are associated with C-peptide.

The study population included 1303 Caucasian subjects from the Diabetes Control and Complications Trial (DCCT), recruited in 2 cohorts: primary and secondary with 1-5, and 1-15 yrs of diabetes, respectively. We calculated a weighted T1D GRS of 30 SNPs of which 5 were in HLA. T1D GRS, a DR3/DR4 GRS (2 SNPs) and individual SNPs (coded additively) were tested for association with stimulated C-peptide after a standard meal measured at DCCT baseline. Separate analyses of primary cohort (N = 651), secondary cohort with duration 1-5 yrs (N = 135), and secondary cohort with duration 5-15 yrs (N = 517) were combined through meta-analysis using Tobit models for c-peptide adjusting for sex, age at diagnosis and duration. Similarly, T1D GRS, DR3/DR4 GRS and individual SNPs were tested for association with age of diagnosis using linear regression.

T1D GRS and DR3/DR4 GRS were not associated with stimulated C-pep-tide. The risk alleles (T) of SNPs in HLA-A24 region (rs1264813, β (SE) = -0.3 (0.1), p = 3E-5) and Insulin gene (rs689, β (SE) = -0.2 (0.1), p = 0.01) were associated with lower stimulated C-peptide. T1D GRS (β (SE) = -0.8 (0.1), p = 3E-8), DR3/DR4 GRS (β (SE) = -0.5 (0.2), p = 6E-4) and the risk allele (T) of the non-HLA SNP rs5753037 (HORMAD2, β (SE) = -1.0 (0.3), p = 4E-4) were all negatively associated with age of diagnosis, and exceeded Bonferroni correction (< 0.002). At all loci, there was no signifi cant heterogeneity of the effect among the 3 subgroups.

We identifi ed risk alleles of 2 T1D susceptibility loci, HLA-A24 and INS, associated with lower C-peptide levels. T1D GRS, DR3/DR4 GRS and rs5753037 were associated with age of diagnosis.

Supported By: National Institutes of Health (U01 DK094157, U01 DK094176, DP3 DK104438)

& 1773-PRacial Differences in Early GIP Contribute to Postprandial Hyperin-sulinemia in Black Women: The Federal Women StudyPAOLA C. ALDANA, AMBER COURVILLE, MARY WALTER, MICHELLE T. DUONG, BRIANNA A. BINGHAM, LILIAN S. MABUNDO, MADIA RICKS, JOON HA, ARTHUR S. SHERMAN, ANNE E. SUMNER, STEPHANIE T. CHUNG, Bethesda, MD

Black women are more hyperinsulinemic than whites after exposure to intravenous glucose but the post-meal insulin response by race is unclear. Since incretins mediate 50% of postprandial insulin secretion, we hypothe-sized that black women would have higher insulin, active glucagon-like pep-tide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) compared to whites during a 2 h mixed meal test (MMT). To quantify insulin and incretin response, early (0-30 min) and total (0-120 min) area under the curves were calculated for the MMT (52% carbohydrate, 15% protein, 33% fat) in 36 fed-erally employed women without diabetes (17 African-American, 6 African immigrants, 13 whites; age 43±9 (mean±SD), range 25- 62 y; BMI 30.8±5.8, range 20.5-45.3 kg/m2). Glucose tolerance status was previously determined by OGTT and percent (%) body fat by DXA. Analyses of covariance were used to examine insulin and incretin response by race accounting for glucose and % fat. Glucose tolerance status did not differ by race. During the MMT, glucose concentrations did not differ by race (P≥0.50). After adjusting for % fat, early insulin response was 50% higher in black vs. whites (insulin0-30min 1100±770 vs. 719±371, P=0.01) and total insulin response was 20% higher (insulin0-120min 8012±5645 vs. 6598±3305 µU·min/mL, P=0.10) but this did not reach signifi cance. Postprandial early and total GIP was higher in blacks vs. whites (GIP0-30min: 3468±3053 vs.1778±1003, GIP0-120min: 54159±22792 vs. 39849±15582 pg·min/mL, both P<0.03). There were no differences by race in GLP-1 or C-peptide concentrations. Early GIP (P<0.001) and % fat (P<0.001) mediated 46% of the racial differences in early insulin response. Greater early GIP was related to black race (P=0.03), % fat (P=0.04), and glucose0-30min (P<0.01). Overall, racial differences in postprandial insulin response were identifi ed during a meal and may be partially explained by higher early GIP, not GLP-1, in black women relative to whites.

Supported By: National Institute of Diabetes and Digestive and Kidney Diseases

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