INSULIN ACTION—ADIPOCYTE BIOLOGY 1745-P …...A455 For author disclosure information, see page...

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CATEGORY

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INSULIN ACTION—ADIPOCYTE BIOLOGY


Guided Audio Tour: Adipocyte Biology (Posters: 1743-P to 1748-P), see page 19.

& 1743-PDifferentially Expressed Micro RNA and Co-Regulated Target Tran-scripts in Adipose Tissue of Insulin-Resistant SubjectsNEERAJ K. SHARMA, ASHIS K. MONDAL, LIJUN MA, SANDRA J. HASSTEDT, SWAPAN K. DAS, Winston-Salem, NC, Salt Lake City, UT

Micro RNAs (miRNAs) are a class of non-coding RNAs that regulate transcription at the cellular level. Recent animal studies suggest that miRNAs may modulate systemic glucose homeostasis via expression of target coding transcripts. In humans, obesity measures are strongly correlated with altered glucose homeostasis. Thus, identifi cation of miRNAs and co-regulated target transcripts that cause insulin resistance independent of obesity is challenging. We selected 20 insulin-resistant (IR) and 20 insulin-sensitive (IS) subjects from a cohort of 99 non-diabetic Caucasians who were characterized by FSIVGT; selected for extremes of insulin sensitivity (SI) (IR, SI = 2.01 ± 0.74; IS, SI = 7.19 ± 2.26); and matched for BMI, age, and gender. Global profi ling of subcutaneous adipose tissue miRNAs was done by Exiqon miRCURY LNA miRNA 7th gen array and mRNA was measured using an Illumina HumanHT-12 v4 Expression BeadChip array. Five of the 539 expressed miRNAs were differentially expressed (p<0.01 and FDR=0) in IR vs IS subjects. Real-time quantitative PCR using Qiagen miScript primer assay validated upregulation of let-7c (fold change =1.4, p=0.027) and miR-422a (fold change =1.6, p=0.04) in IR subjects. Signifi cant (p<0.05, FC≥1.3) upregulation and down regulation of 260 and 55 mRNA transcripts, respectively, occurred in IR subjects. The Gene Set Enrichment Analysis identifi ed signifi cant downregulation of genes involved in the TCA cycle, oxidative phosphorylation, fatty acid and lipid metabolism; genes involved in immune and infl ammatory response were upregulated. MiRNA target fi ltering and miRNA-mRNA expression pairing analysis identifi ed downregulated target genes, e.g. AGPAT9 for let-7c and ADORA1 for miR-422a. Our data suggest that a subset of miRNAs may play a causal role in insulin resistance by modulating target transcripts in adipose tissue.

Supported by: NIH (R01DK039311)

& 1744-PGlucocorticoid-Induced Expression of the FKBP5 Gene in Human Adipose Tissue—A Potential Mechanism for Dysregulation of Me-tabolism and Immune FunctionMARIA J. PEREIRA, JENNY PALMING, MARIA K. SVENSSON, MAGNUS RIZELL, JAN DALENBÄCK, MÅRTEN HAMMAR, PER-ARNE SVENSSON, JAN W. ERIKS-SON, Gothenburg, Sweden, Mölndal, Sweden

Glucocorticoid excess promotes central obesity, insulin resistance and type 2 diabetes. The aim was to explore effects of dexamethasone on gene expression in human adipose tissue, in order to identify potential novel mechanisms for glucocorticoid-induced insulin resistance.

Human subcutaneous and omental adipose tissue (10 M/15 F non-diabetic donors; age 28-60 yrs; BMI 20.7-30.6 kg/m2), was incubated with or without dexamethasone (0.003-3 µM) for 24 h. Adipose tissue was used for RNA extraction and microarray analysis (Affymetrix, Human Exon 1.0 ST Array, n=4) and subsequently, key genes affected by dexamethasone were analysed by real time-PCR (n=23).

Dexamethasone (3 µM) changed the expression of 527 genes in both subcutaneous and omental adipose tissue. FKBP5 (FK506-binding protein 5) and CNR1 (cannabinoid receptor 1) were the most responsive genes in both depots (~7-fold increase). Dexamethasone increased FKBP5 gene and protein expression in a dose-dependent manner in both depots. However, FKBP5 protein levels were 10-fold higher in omental than subcutaneous adipose tissue, whereas gene expression was similar. FKBP5 gene expression was positively correlated with markers of insulin resistance such as serum insulin, HOMA-IR and subcutaneous adipocyte diameter. Dexamethasone increased the expression of the secreted proteins leptin and TIMP4 (metallopeptidase inhibitor 4) in both fat depots. No genes responded in opposite directions to dexamethasone in the two depots, but, SERTM1 (serine-rich and transmembrane domain containing 1) was clearly down-regulated in omental, but unchanged in the subcutaneous depot.

In conclusion, dexamethasone infl uences gene expression similarly in subcutaneous and omental adipose tissue and markedly promotes gene expression of FKBP5. This gene which is known to modulate immune function, may potentially also be an important mechanism in glucocorticoid-induced insulin resistance.

& 1745-PReduction of Wilms’ Tumor 1-Associating Protein Ameliorates the Development of Obesity and Subsequent Insulin Resistance via GATA-3 and Cyclin A2 Mediated PathwaysMASATOSHI KOBAYASHI, MITSURU OHSUGI, TAKAYOSHI SASAKO, KAZUYUKI TOBE, KOHJIRO UEKI, TAKASHI KADOWAKI, Tokyo, Japan, Toyama, Japan

To understand the pathogenesis of obesity, it is important to explore the mechanisms of adipocyte differentiation. With this regard, GATA-2/3 transcription factors have been reported to be one of the negative regulators of adipocyte differentiation by inhibiting the function of PPARγ, while some other molecules have shown to interact with GATA in vascular endothelial cells, suggesting that the protein complex composed of these proteins may play a role in adipocyte differentiation. Of these, we focused on Wilms’ tumor 1-associating protein (WTAP), and hypothesized it might have some functions in adipocytes differentiation incorporation with GATA. We have found that WTAP physically interacts with GATA-3 and positively regulates PPARγ expression by suppressing GATA-3 function in vitro. Indeed, knockdown of WTAP in 3T3-L1 cells results in impaired differentiation into adipocytes. On the other hand, WTAP has been reported to modulate the function of Cyclin A2 in cell cycle transition of G2/M period in vascular endothelial cells. In adipocytes, we have found that knocking down of Cyclin A2 in 3T3-L1 cells leads to impaired differentiation into adipocytes with cell cycle arrest at G2/M. Similarly, the cell cycle in WTAP knocked-down 3T3-L1 cells is arrested at G2/M. Finally, we have analyzed Wtap+/- mice and found that they are protected from diet-induced obesity with smaller adipocytes, resulting in better insulin sensitivity than wild type (WT) mice. Moreover, embryonic fi broblasts from Wtap+/- mice also show lower ability to differentiate into adipocytes with the suppression of Cyclin A2 mRNA and the cell cycle arrest at G2/M in mitotic clonal expansion compared to those from WT mice. These results suggest that WTAP controls adipocyte differentiation through GATA-3 and Cyclin A2 mediated pathways, and could be a novel therapeutic target for obesity-induced diabetes.

& 1746-PTargeting Chemerin/CMKLR1 Pathway as a Potential Treatment for Type 2 DiabetesPING JIN, RACHEL STANTON, CALVIN VU, KELSEY RETTING, MOORTHY S.S. PALANKI, KATHLEEN OGILVIE, NANCY LEVIN, San Diego, CA

Chemerin is a recently discovered adipokine that regulates adipogenesis, infl ammation, and glucose metabolism through interactions with its cognate cell surface receptor chemokine-like receptor 1 (CMKLR1). Although elevated chemerin levels are clearly associated with high BMI, insulin resistance and metabolic dysfunction, confl icting results have been reported on the role of this hormone in glucose homeostasis. We performed oral glucose tolerance tests (OGTT) in diet-induced obese mice in which we manipulated chemerin levels. Acute administration of recombinant chemerin (40 µg/kg, IP) signifi cantly elevated blood glucose levels at 15 (44%, n=8, P<0.001) and 30 (38%, n=8, P<0.01) minutes during OGTT. Using antisense RNA targeted knockdown (30mg/kg, SC single administration), chemerin expression was reduced in liver (96%, n=4, P<0.0001) and white adipose tissue (60%, n=8, P<0.001) by day 7, circulating chemerin levels were also decreased (Chemerin levels in ng/ml: Vehicle 135.3±22.7, Antisense 30.9±5.6, n=8, P<0.002) 24 hours after antisense administration. Systemic chemerin depletion resulted in signifi cantly lowered blood glucose during OGTT at day 7 (Glucose AUC in hr*mg/dL: Vehicle 631±10, Antisense 443±53; Mean±SE, n=8, P<0.01). In addition, mice treated with chemerin antisense RNA also showed decreased body weight (6 %) and cumulative food intake (20%) over 7 days. Gene expression analysis revealed signifi cantly decreased serum amyloid A level and slightly increased IL-1β and IL-6 mRNA in the liver, consistent with reported literature that chemerin plays both pro- and anti- infl ammatory roles. A detailed understanding of the mechanistic involvement of chemerin in immune response and how this may affect metabolic processes requires further investigation. In summary, we have demonstrated that abrogating chemerin/CMKR1 signaling using antisense RNA improves glucose metabolism in mice, suggesting therapeutic potential for this pathway as a novel treatment for type 2 diabetes.

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& 1747-PIL-1 Receptor Associated Kinase-1 (IRAK-1) Null Mice have In-creased Glucose Tolerance and Insulin SensitivityXIAO JIAN SUN, SOOHYUN P. KIM, DIANALY AU, LIWU LI, MICHAEL J. QUON, Baltimore, MD, Blacksburg, VA, Bethesda, MD

IRAK-1 mediates pro-infl ammatory signaling by Toll-Like-Receptors that may contribute to insulin resistance (IR) by promoting cross-talk between innate immune signaling and insulin signaling. IRAK-1 phosphorylates IRS-1 at Ser24 resulting in decreased tyrosine phosphorylation of IRS-1 and impaired insulin-stimulated GLUT4 translocation. Therefore, we hypothesized that IRAK-1 contributes to IR mediated by pro-infl ammatory signaling. To test this in vivo, we compared glucose tolerance and insulin sensitivity in male IRAK-1 null mice (KO) vs. C57BL/6 wild-type (WT) control mice on normal chow diet. 6 week old male KO and WT were followed for 13 wk. At baseline (6 wk old) and after 13 wk chow diet (19 wk old) mice were fasted 5 h prior to glucose tolerance test (1 g glucose/kg, i.p.; IPGTT) or insulin tolerance test (0.75 IU insulin/kg, i.p.; ITT). No detectable difference was noted in body weight or food intake among KO vs WT over 13 wk. At baseline (6 wk) no detectable difference was noted in IPGTT or ITT. However, after 13 wk chow diet, KO had substantially improved glucose tolerance when compared with WT (IPGTT AUCglucose = 6850 ± 1100 vs. 10890 ± 840 (mg/dl)·min), p < 0.035; ANOVA for repeated measures, p < 0.004). No detectable difference in ITT (primarily muscle insulin sensitivity; ANOVA for repeated measures, p < 0.68) or fasting insulin (27 ± 5 vs 34 ± 3 µU/ml, p < 0.19). However, fasting glucose was lower in KO vs WT (169 ± 21 vs 223 ± 14, p < 0.01) that determined higher liver insulin sensitivity assessed by the surrogate index QUICKI (0.278 ± 0.008 vs 0.259 ± 0.005, p < 0.04). Circulating ET-1 was decreased in KO vs WT (82 ± 6 vs 104 ± 8 pg/ml, p < 0.03). Taken together, our results suggest IRAK-1 null mice have improved glucose tolerance due, in part, to increased hepatic insulin sensitivity. We conclude IRAK-1 contributes to cross-talk between pro-infl ammatory signaling and glucose homeostasis in vivo that may have therapeutic implications for treatment of diabetes.

& 1748-PSuccination of Protein Disulfi de Isomerase (PDI) Contributes to Re-duced Protein Folding in the Adipocyte Endoplasmic ReticulumALLISON MANUEL, JOHN W. BAYNES, NORMA FRIZZELL, Columbia, SC

S-(2-succino)cysteine (2SC)is a novel post-translational modifi cation of cysteine residues formed when fumarate reacts with available thiol groups. We have described the increase in 2SC, also termed protein succination, in adipocytes cultured in high glucose (HG) medium and in the adipose tissue of db/db mice. The increase in succinated proteins occurs in parallel with an increase in endoplasmic reticulum (ER) stress, and we have previously shown that the ER foldase Protein Disulfi de Isomerase (PDI) is succinated in the diabetic adipocyte. PDI is responsible for the correct folding of the majority of proteins that move through the ER. In the current study we determined the sites of succination in PDI and investigated the effect of succination on PDI activity.

3T3-L1 adipocytes were matured in either normal glucose (NG, 5 mM) or HG (30 mM) with 0.3 nM insulin (NG) or 3 nM insulin (HG). PDI was immunoprecipitated from cell lysates was and was separated by SDS-PAGE. Peptides from the digested gel band and total protein lysates were analyzed on an Orbitrap mass spectrometer. PDI reductase activity was measured spectrophotometrically using the insulin turbidity assay.

PDI contains two thioredoxin domains with the motif -CXXC- that are essential for its foldase activity. Mass spectrometry analysis of PDI from HG adipocytes demonstrated that PDI is succinated at Cys58 and Cys402, the second cysteine in both -CXXC- motifs. Analysis of total protein from HG cell lysates demonstrated succination of the -CXXC- motif in thioredoxin at Cys73. When recombinant PDI was incubated in 1-100 mM fumarate for up to 24 h the reductase activity was signifi cantly decreased (p<0.001, 100 mM, p< 0.05, 5mM).

In summary, the low pKa thiol in the -CXXC- motif of both PDI and thioredoxin is susceptible to succination during adipocyte glucotoxicity. Succination inhibits the foldase activity of PDI, providing a novel link between glucotoxicity and increased ER stress in the diabetic adipocyte.

1749-PThe Role of Plasminogen Activator Inhibitor-1 in the Regulation of Adipocyte FunctionINJI JUNG, JOO YOUNG HUH, INAH HWANG, HUNJOO HA, Seoul, Republic of Korea, Boston, MA

Dysfunctional adipocytes reach their limits to store fat and release free fatty acids into circulation, along with dysregulated adipokine and chronic infl ammation, and this causes insulin resistance in peripheral tissues including liver and muscle as well as adipocytes itself. The 50 kDa glycoprotein plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of tissue-type and urokinase-type plasminogen activator. Recent studies suggest that PAI-1 may play a role in the regulation of adipocyte function, since inhibition of PAI-1 ameliorates high fat-induced body weight gain, adipocyte hypertrophy, and hyperinsulinemia. However, the mechanisms by which PAI-1 controls the function of adipocytes are not fully elucidated. The present study examined the preventive and interventional effect of PAI-1 inhibitor, TM5441, on diet-induced obesity and molecular mechanisms involved in it. TM5441 attenuated obesity, hypertriglyceridemia, and insulin resistance in mice on high-fat diet (HFD). HFD-induced upregulation of proinfl ammatory cytokine expression such as MCP-1 and macrophage infi ltration in epididymal WAT were also prevented by TM5441. Altered HSL and ATGL were normalized with TM5441 treatment. Reduced JNK phosphorylation in WAT appears to be an underlying mechanism, since TM5441 signifi cantly downregulated JNK phosphorylation in HFD mice. In 3T3-L1 adipocytes, exogenously administered PAI-1 reduced adiponectin expression but increased PAI-1 expression as much as those by tumor necrosis factor α (TNFα),a positive control. TM5441 pretreatment protected TNFα-induced dysregulation of adipokine expression and insulin signaling through JNK inactivation. These data demonstrate that PAI-1 may directly affect adipokine expression, infl ammation, lipid metabolism, and insulin signaling via JNK activation, and suggest that TM5441 may become a new therapeutic agent targeting obesity-related metabolic disorders.

Supported by: Korea Science and Engineering Foundation (2012R1A2A1A0300692), (R31-2008-000-10010-0)

1750-PA Novel Function of Adipocytes in Lipid Antigen Presentation to iNKT CellsJIN YOUNG HUH, JONG IN KIM, IN JAE HWANG, YOON JEONG PARK, JAE BUM KIM, Seoul, Republic of Korea

Systemic low-grade chronic infl ammation has been intensively investi-gated in obese subjects. Recently, various immune cell types, such as macro-phages, granulocytes, helper T cells, cytotoxic T cells, and B cells, have been implicated in the pathogenesis of adipose tissue infl ammation. However, the roles of invariant natural killer T cells (iNKT cells) and the regulation of iNKT cell activity in adipose tissue are not thoroughly understood. Here, we demonstrated that iNKT cells were decreased in number in the adipose tissue of obese subjects. Interestingly, CD1d, a molecule involved in lipid antigen presentation to iNKT cells, was highly expressed in adipocytes, and CD1d-expressing adipocytes stimulated iNKT cell activity through physical interaction. iNKT cell population and CD1d expression were reduced in the adipose tissue of obese mice and humans compared to that of lean subjects. Moreover, iNKT cell-defi cient Jα18 knockout mice became more obese and exhibited increased adipose tissue infl ammation at the early stage of obesity. These data suggest that adipocytes regulate iNKT cell activity via CD1d, and that the interaction between adipocytes and iNKT cells may modulate adipose tissue infl ammation in obesity. *This work was supported by grants from the Korea Science and Engineering Foundation funded by the Korean government (Ministry of Education, Science and Technology, MEST: 2012-0001241).

Supported by: Ministry of Education, Science and Technology

1751-PRunx2 Regulates Insulin Signaling During Adipocyte Differentia-tionMIN DING, FARAH Y. GHORI-JAVED, MITRA ADHAMI, HAIYAN CHEN, AMJAD JAVED, Birmingham, AL

Alterations in insulin pathway are associated with enhanced fat synthesis and a concomitant decrease in bone formation. Fat producing adipocytes and bone forming osteoblasts are derived from a common mesenchymal progenitor. Runx2 is essential for commitment and differentiation of mesenchymal cells (MC) to mineralizing cell types but its role in regulating glucose homeostasis and insulin signaling is not known. We investigated this by isolating MC from Runx2 null mice. These cells failed to undergo osteoblast differentiation even when cultured in osteo-inductive media. To

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our surprise, Runx2 defi cient mesenchymal cells exhibited a preferential commitment to the adipocyte lineage. Robust activation of both early and terminal marker genes and adipocyte formation was observed upon stimulation with low dose of PPARγ agonist rosiglitazone. Surprisingly, when Runx2 null cells were treated with insulin, a marked suppression of adipocyte differentiation occurred. These fi ndings are in sharp contrast to the well-established requirement of insulin for the 3T3L1 adipocyte differentiation. Reconstitution of these cells with Runx2 restored insulin action in adipocytes. These results suggest that Runx2 defi cient MC bypass the critical requirement of insulin for adipogenesis. Gene microarray analysis revealed that more than 95% of the genes associated with insulin signaling were expressed at higher levels in Runx2 null MC compared to 3T3L1 pre-adipocytes. Insulin evoked a differential response, with 85% genes induced in 3T3L1 but only 3% in Runx2 null MC. A strong suppression of all components of the insulin signaling pathway was evident in insulin treated Runx2 null MC. We observed a selective utilization of exon 11 splice variant of the insulin receptor in the Runx2 null MC. Finally we demonstrate that impaired internalization of the activated insulin receptor contributed to the paradoxical insulin response in Runx2 null MC. In conclusion, Runx2 regulates insulin actions and lineage commitment of MC to adipocytes.

Supported by: R01AR062091

1752-PThe Anti-Adipogenic Action of Macrophages on Human Preadipo-cytes Does Not Require Interleukin-1βANNEMARIE GAGNON, CHARLIE FOSTER, ANNE LANDRY, ALEXANDER SORISKY, Ottawa, ON, Canada

When adipose tissue accumulates in obesity, the ability of preadipocytes to differentiate permits a hyperplastic expansion of functional adipocytes that preserves insulin sensitivity. Adipose infi ltration by macrophages is associated with an adipogenic defi cit and the appearance of infl amed, insulin-resistant hypertrophied adipocytes. Interleukin (IL)-1β has been reported to account for the anti-adipogenic action of macrophages in a mouse model. Using the THP-1 human macrophage cell line and human primary preadipocytes, our objective was to determine if IL-1β was necessary for the ability of conditioned medium from THP-1 macrophages (THP-1-MacCM) to 1) stimulate human preadipocyte inhibitor of κB kinase (IKK) β and 2) inhibit human adipocyte differentiation. IL-1β is secreted by THP-1 macrophages, and THP-1-MacCM or IL-1β (500 pg/ml; its concentration in THP-1-MacCM) acutely stimulated IKKβ phosphorylation and inhibitor of kB (IκB) degradation in preadipocytes. IL-1β (500 pg/ml) blocked adipocyte differentiation signifi cantly, reducing triacylglycerol (TG) accumulation by 73%, fatty acid synthase levels by 81%, and aP2 levels by 80%. In the presence of sc-514 (100 µM; an inhibitor of IKKβ), the inhibition of these adipogenic markers was completely prevented, as has been reported for THP-1-MacCM. IL-1β-immunodepleted THP-1-MacCM still increased IKKβ phosphorylation, but the previously observed degradation of IκB was attenuated. However, despite this alteration in the signaling response, there was no change in the anti-adipogenic action of IL-1β-depleted THP-1-MacCM on human adipocyte differentiation, assessed by accumulation of TG as well as by the levels of aP2 and peroxisome proliferator activated receptor (PPAR) γ. Therefore, in contrast to what has been suggested for mouse cell models, IL-1β is not required for the ability of MacCM to inhibit adipogenesis in human cell models.

Supported by: Heart and Stroke Foundation of Canada

1753-PRBP4: PEDF Ratio Predicts Insulin Sensitivity, Glycemia and Adi-posity More Robustly than the Individual AdipokinesCHIMAROKE EDEOGA, SOTONTE EBENIBO, SAMUEL DAGOGO-JACK, Memphis, TN

Retinol binding protein (RBP4) and pigment epithelium derived factor (PEDF) are adipocyte-derived molecules with metabolic implications. We tested the hypothesis that the ratio of RBP4:PEDF predicts insulin sensitivity more robustly than either adipokine alone. We measured RBP4 and PEDF levels in fasting plasma obtained from nondiabetic subjects who subsequently underwent hyperinsulinemic euglycemic clamp for assessment of insulin sensitivity. The study subjects included equal numbers of men and women and Whites and Blacks, and all had normal OGTT. Body fat was measured with DXA. The mean (+ SD) age of the subjects was 50.4 + 5.97 yr, BMI was 32.5 + 5.86 kg/m2 and fasting glucose was 96.1 + 5.21 mg/dl. RBP4 levels were similar Blacks vs. Whites (12.2 + 1.03 ug/ml vs. 11.3 + 1.13 ug/ml), and men vs. women (12.7 + 0.83 vs. 10.7 + 1.23), and correlated weakly with fasting (r=0.17) and 2-hr (r= 0.14) OGTT glucose levels. RBP4 levels were correlated

inversely with total fat (r= -0.49, P=0.03) and trunk fat (r=0.48, P=0.03), and weakly with insulin sensitivity (r= 0.32, P=0.06). PEDF levels were similar in Blacks (5.45 + 0.72 ug/ml) and Whites (5.66 + 0.74 ug/ml), and in men and women ( 5.21 + 0.72 ug/ml vs. 5.47 + 0.74 ug/ml), and correlated signifi cantly with fasting (r= 0.51, P= 0.03) and 2-hr (r=0.23) glucose levels during OGTT, and weakly with total fat (r=0.21), trunk fat (r=0.25) and insulin sensitivity (r=0.15). The RBP4:PEDF ratio showed stronger correlations with fasting glucose (r=0.42, P= 0.04), 2-hr glucose (r=0.42, P=0.04), total fat (r= -0.51, P=0.02), trunk fat (r=0.52, P=0.01) and insulin sensitivity (r= 0.45, P= 0.04) compared to either RBP4 or PEDF alone. Our results show that circulating RBP4 and PEDF levels do not display major ethnic or gender disparities, and individually show variable associations with adiposity, glycemia and insulin sensitivity. However, the RBP4:PEDF ratio is a robust predictor of insulin sensitivity, glycemia, and total as well as truncal fat mass.


1754-PInhibition of TSP1-Mediated TGF-β Activation Reverses Adipose Tissue Fibrosis but Does Not Affect Metabolic Dysfunction in High-Fat Fed MiceR. GRACE WALTON, LATHA MUNIAPPAN, MICHAEL SPENCER, AKOSUA ADU, BRIAN S. FINLIN, CHARLOTTE A. PETERSON, PHILIP A. KERN, Lexington, KY

Our group and others have shown that adipose tissue fi brosis is associated with metabolic dysfunction. The expression of thrombospondin (TSP1) is increased with insulin resistance and TSP1 activates TGF-β. We hypothesized that inhibition of TSP1mediated TGF-β activation would ameliorate high fat diet-induced metabolic dysfunction in BL/6 mice. LSKL peptide specifi cally inhibits TGF- β activation by TSP-1. Mice were placed on high fat diet for four-weeks and then given LSKL (1 mg/kg body weight, osmotic mini-pump, 5-6 weeks) or scramble peptide and maintained on a high fat diet. LSKL treatment decreased TGF- β signaling since phospho-SMAD2/3 immunostaining was reduced by 65% (n=6/group; p<0.05) in inguinal subcutaneous fat (SQF). SQF infl ammation was also reduced following LSKL treatment, evidenced by a 34% and 41% decrease in F4/80 mRNA and F4/80 immunostaining, respectively (p<0.05).

Importantly, SQF fi brosis determined by Sirius red staining was strikingly decreased by 67% in LSKL treated mice (p<0.01), with a concomitant 24% decrease in CollV mRNA (p<0.05). However, no differences were observed in food intake, body weight, weight gain, body composition, fasting glucose, or glucose tolerance using an IP GTT. In a follow-up study, we delivered a higher dose of LSKL (45 mg/kg body weight, i.p. injection) or scramble peptide for 14 weeks, beginning at onset of high fat feeding. No differences were observed in activity, food intake, body weight, weight gain, fasting glucose, glucose tolerance, or insulin tolerance. However, we observed a trend toward increased lean mass in LSKL-treated mice. These results indicate that TSP1 is a signifi cant activator of TGF-β in WAT in diet-induced obesity and that blocking TSP1-mediated TGF-β activation reversed high fat diet-induced adipose tissue fi brosis. However, improvement in adipose infl ammation and fi brosis did not affect insulin or glucose tolerance.

Supported by: DK71349

1755-PSweet Taste Receptor Regulates Adipogenesis Through Gs-Mediat-ed Microtubule Disassembly and Rho Activation in 3T3-L1 CellsYOSUKE MASUBUCHI, YUKO NAKAGAWA, JINHUI MA, MASAHIRO NAGASA-WA, ITARU KOJIMA, HIROSHI SHIBATA, Maebashi, Japan

3T3-L1 cells express a functional sweet taste receptor possibly as a T1R3 homomer that is coupled with Gs and mediates cAMP-irrelevant anti-adipogenic signals. Among recently found non-canonical roles of heterotrimeric G proteins, Gs-alpha-mediated tubulin GTPase activation and microtubules disassembly would be a possible mechanism of sweet taste receptor-mediated inhibition of adipogenesis, since a certain type of Rho-GEF is activated by microtubules disassembly and Rho is a negative regulator in adipogenesis. As expected, stimulation of 3T3-L1 preadipocytes with sucralose or saccharin evoked microtubules disassembly and Rho activation. These effects were attenuated by overexpression of the dominant-negative mutant of Gs-alpha (Gs-alpha-G226A), and were reproduced with cholera toxin but not forskolin. Microtubules disruption with nocodazole also activated both Rho and Rho-associated kinase (ROCK) 2. Finally, Y-27632, a specifi c inhibitor of ROCK, interfered with sweetener-induced inhibition of PPARgamma and C/EBPalpha expression. These results suggested that the anti-adipogenic signals downstream of the sweet taste receptor would be transmitted via Gs-alpha-mediated microtubule disassembly and consequent activation of the Rho-ROCK signaling pathway.

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1756-PActin-Severing Protein Cofi lin-1 Is a Critical Regulator in Adipocyte DevelopmentWULIN YANG, SHERMAINE THEIN, WEIPING HAN, Singapore, Singapore

Cytoskeletal remodeling is a prerequisite step for the morphological transition from fi broblast-like pre-adipocytes to mature adipocytes. We previously showed that acetylation of α-tubulin and the ensuing microtubule remodeling were essential in adipogenesis. Here we investigated the involvement of actin remodeling in adipocyte development, specifi cally on the role of actin-severing protein cofi lin-1 in regulating adipogenesis. Actin network in 3T3-L1 cells was extensively reorganized from stress fi bers across cell body to cortical localization underneath plasma membrane when the cells were treated with adipogenic cocktail to induce adipogenesis, but remained almost unaltered when oleic acid was applied to 3T3-L1 preadipocytes to promote lipogenesis and lipid droplet formation. Cofi lin-1 expression was upregulated during adipogenesis, coinciding with the initial actin remodeling, and the onset of morphological transition from pre-adipocytes to adipocytes. Adipocyte development, including lipid droplet formation and lipid accumulation, was signifi cantly impaired in 3T3-L1 adipocytes with stable cofi lin-1 knockdown. Moreover, actin network remained nearly intact in cofi lin-1 knockdown cells. These results suggest that actin cytoskeleton remodeling and actin-severing protein cofi lin-1 are essential for adipocyte development.

1757-PCrosstalk between ChREBP and PPARalpha in Brown Adipocytes Lipid MetabolismKATSUMI IIZUKA, WUDELEHU WU, YUKIO HORIKAWA, MASAYUKI SAITO, JUN TAKEDA, Gifu, Japan, Sapporo, Japan

We have reported that ChREBP plays an important role in the regulation of lipogenesis in the liver. Chrebp mRNA levels in brown adipose tissue are as abundant as those in the liver, however the role of ChREBP in brown adipose tissue is still unclear. In this study, we clarifi ed the role of ChREBP and PPARα in brown adipocytes. In liver and brown adipose tissue, Chrebpb mRNA levels in fasting mice were much lower than those in mice fed ad libitum, while Ppara mRNA levels in fasting mice were much higher than those in fed mice. During brown adipogenesis, levels of Chrebpa and Ppara mRNA were induced at day 4. Subsequently, the mRNA levels of ChREBP target genes, such as Chrebpb and Fatty acid synthase (Fasn), were induced. In differentiated HB2 cells, glucose increased the mRNA levels of ChREBP target genes in a dose-dependent manner, but decreased both Chrebpa and Ppara mRNA levels. Moreover, adenoviral overexpression of ChREBP and reporter assays showed that ChREBP suppressed PPARα transactivity in HB2 cells. Fasn and Chrebpb mRNA in brown adipose tissue from ChREBP-/- mice were much lower than those in wild-type mice. Similarly, Ppara mRNA levels in ChREBP-/- mice were much higher than those in wild-type mice. Finally, Wy14643, a selective PPARα agonist, partially suppressed glucose induction of Chrebpb and Fasn mRNA in HB2 cells. In conclusion, we have identifi ed that the crosstalk between ChREBP and PPARα plays an important role in the regulation of lipogenesis in brown adipocytes.

1758-PTSH Inhibits Insulin-Stimulated Human Adipocyte Lipogenesis in a BMI-Dependent MannerDAVID FELSKE, ANNEMARIE GAGNON, ALEXANDER SORISKY, Ottawa, ON, Canada

In subclinical hypothyroidism (SH), high levels of circulating thyroid stimulating hormone (TSH) maintain normal thyroid hormone levels despite mild thyroid failure. SH is associated with cardiovascular disease and insulin resistance, although the underlying pathophysiology is not fully understood. Functional TSH receptors are expressed on adipocytes, and may interfere with insulin action. We hypothesized that TSH may inhibit insulin-regulated adipocyte lipogenesis. Abdominal subcutaneous adipose tissue samples were obtained (approved by the Ottawa Hospital Research Ethics Board) from 8 weight-stable patients (7 female), undergoing elective abdominal surgery. Mean age was 49±12, and mean body mass index (BMI) was 32.9±11 kg/m2 (±SD). Stromal preadipocytes were isolated, placed in culture, and differentiated into adipocytes (~60%) over 14 days. To determine if TSH could inhibit insulin-induced lipogenesis, we stimulated the adipocytes with 5 mU/ml TSH and/or 100 nM insulin for 4 h and measured lipogenesis based on incorporation of 14C-glucose into cellular triacylglycerol. Our data indicate that TSH inhibits insulin-stimulated lipogenesis (up to 36.6%), but depends on BMI. A signifi cant negative correlation (r2=0.835; p < 0.05) between BMI and the inhibition of insulin-stimulated lipogenesis by TSH was observed

(n=5). To understand the molecular mechanism, we examined the effect of TSH on insulin-stimulated ser-473 Akt phosphorylation. Differentiated adipocytes were acutely stimulated with 5 mU/ml TSH and/or 100 nM insulin for 0-30 min. Immunoblot analysis revealed a trend towards insulin-stimulated Akt phosphorylation being inhibited by TSH at 15 min (49% decrease, n=4, p < 0.1, 2-way ANOVA). Our data show that TSH inhibits insulin-induced lipogenesis in human differentiated adipocytes. This raises the possibility that other insulin-regulated adipocyte responses may also be susceptible to TSH, which could help explain how TSH may play a role in insulin resistance.

Supported by: CIHR

1759-PAdipose Tissue INSR Splicing Is Regulated by Weight Loss and As-sociates With Insulin LevelsDOROTA KAMINSKA, MAIJA HÄMÄLÄINEN, PIRJO KÄKELÄ, SARI VENESMAA, LEILA KARHUNEN, HELENA GYLLING, MARKKU LAAKSO, JUSSI PIHLAJAMÄKI, Kuopio, Finland

Insulin receptor, INSR, has two isoforms based on alternative splicing of exon 11. INSR-A promotes cell growth whereas INSR-B is predominantly regulating insulin action and glucose homeostasis. We have previously demonstrated that expression of factors regulating alternative splicing is decreased in liver and skeletal muscle of obese and insulin resistant individuals. In this study we investigated if weight loss alters INSR alternative splicing and expression of splicing factors in adipose tissue and whether these changes relate to insulin action and type 2 diabetes.

To determine the relative ratio of INSR gene splice variants, we implemented the PCR-capillary electrophoresis method on adipose tissue samples from three independent studies (n=191): two follow-up weight loss studies (gastric bypass surgery study (n=110) and dietary intervention study(n=32)) and a population based study (n=49).

Expression of INSR-B mRNA variant increased in response to weight loss induced by both bariatric surgery (p=1×10 -5) and very low calorie diet (p=1×10 -4). The expression of INSR-B was negatively correlated with the levels of fasting insulin and associated with type 2 diabetes. Finally, expression of several splicing factors correlated negatively with the expression of INSR-B variant (p<0.05). The strongest correlation was with HNRNP A1, a known regulator of INSR exon 11 splicing.

In conclusion, we showed that INSR splicing is regulated by weight loss and associates with insulin levels and type 2 diabetes. Our results suggest that the change in adipose tissue INSR splicing in response to weight loss associates with insulin action and is most likely mediated by splicing factors.

Supported by: Academy of Finland, Emil Aaltonen Foundation; TEKES

1760-PCDK5 Regulatory Subunit-Associated Protein 1-Like 1 (Cdkal1) Negatively Regulates Adipocyte Differentiation through Activation of the Wnt PathwayKAZUMI TAKE, HIRONORI WAKI, TOSHIMASA YAMAUCHI, WEI SUN, JING YU, MASATO IWABU, MIKI OKADA-IWABU, KOHJIRO UEKI, TAKASHI KADOWAKI, Tokyo, Japan

Cdkal1 was identifi ed as a susceptibility gene for both type 2 diabetes and obesity. Although Cdkal1 in pancreatic β cells was shown to modulate insulin secretion, little is known about its role in adipocytes. Here, we show that Cdkal1 expression is induced during differentiation of 3T3-L1 adipocytes. Retroviral overexpression of Cdkal1 in 3T3-L1 cells strongly suppresses adipocyte differentiation, which is rescued by forced expression of PPARγ but not by PPARγ agonist treatment, implying the critical role of downregulation of PPARγ by Cdkal1. On the other hand, knockdown of Cdkal1 by shRNA promotes adipocyte differentiation. Experiments using deletion and site-directed mutagenesis of Cdkal1 constructs indicate that two CXXXCXXC motifs located in the N-terminal radical SAM domain is critical for the anti-adipogenic activity of Cdkal1. Molecular studies on the pathways that regulate PPARγ expression indicate that Cdkal1 suppresses PPARγ expression through activation of the Wnt pathway as is indicated by increased protein levels of β-catenin and Wnt target genes such as Wisp2 in Cdkal1-overexperssing cells in the early phase of adipogenesis. Our data suggested that Cdkal1 acts as a negative regulator of adipocyte differentiation.

Supported by: Grant-in-Aid for Scientifi c Research on Innovative Areas (23126101), (23126506)

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1761-PTissue Compression Initiates Proinfl ammatory Response in Adipo-cytesLIKANG CHIN, YUKIKO BANDO, BOBBY MONKS, YOSUKE SHIKAMA, MORRIS J. BIRNBAUM, PAUL A. JANMEY, MAKOTO FUNAKI, Philadelphia, PA, Tokushima, Japan

We previously reported that the elastic modulus of adipose tissue from ob/ob mice is more than twice that of adipose tissue from lean littermate controls, suggesting that stiffness increases in obesity. Furthermore, adipocytes seeded on 2-D gels with an elastic modulus representative of obese adipose tissue exhibited features of a saturated fatty acid-induced proinfl ammatory response, such as NFκB activation and increased monocyte chemotactic protein-1 (MCP-1) secretion. In contrast, adipocytes on 2-D gels with the stiffness of normal adipose tissue exhibited little, if any, proinfl ammatory response, even in the presence of saturated fatty acids. We hypothesize that the mechanical environment of the adipocyte changes during obesity and may contribute to cellular dysfunction. To determine how adipocytes respond to mechanical stresses likely to occur in vivo, we investigated the effect of compression on adipose tissue viscoelasticity and adipocyte function. The shear modulus of adipose tissue increased with increasing compression, and when highly compressed, the shear modulus of lean adipose tissue approached or exceeded the modulus of obese adipose tissue. When adipose tissue from lean mice was statically compressed, adipocytes exhibited a proinfl ammatory response marked by upregulated MCP-1, which is characteristic of obese adipose tissue. 3T3-L1 adipocytes compressed between two polyacrylamide gels with the shear modulus of normal adipose tissue also exhibited a proinfl ammatory response. These results suggest that compression of adipose tissue from gravitational and other forces may contribute to the development of adipocyte infl ammation in obesity.

1762-PRole of DNA Methylation for Leptin Gene Promoter in 3T3-L1 Adi-pocytesMASASHI KURODA, HIROSHI SAKAUE, AYAKO TOMINAGA, AKIKO FUKUTA, NA-GAKATSU HARADA, YUTAKA NAKAYA, Tokushima, Japan

Leptin plays a major role in the regulation of energy intake and expenditure. The peptide hormone is expressed in mouse white adipose tissue (WAT) but hardly expressed in 3T3-L1 adipocytes. We investigated the mechanisms regulating leptin expression in 3T3-L1 cells. With bisulfi te sequencing, we found CpG islands in leptin promoter region were highly methylated in 3T3-L1 adipocytes compared with in WAT. To examine the effects of DNA demethylation on leptin expression, we exposed 3T3-L1 cells to 5-azacytidine (5-aza-C), a DNA methyltransferase inhibitor, and analyzed leptin mRNA level and methylation status of promoter region. 5-aza-C treatment in mature adipocytes did not affect methylation status of leptin promoter and its expression. Treatment of preadpocytes with 5-aza-C induced demethylation, but did not change leptin expression. However, after adipocyte differentiation, preadipocytes with 5-aza-C treatment showed markedly increased leptin mRNA with lowered methylation in CpG islands of leptin. We next checked the effect of insuin, glucocorticoid and thiazolidinedione on leptin expression in 5-aza-C-pretreated 3T3-L1 adipocytes (3T3-L1AZ adipocytes). Any stimuli did not affect the gene expression in 5-aza-C-untreated adipocytes. On contrary, 3T3-L1AZ adipo-cytes showed increased expression of leptin in response to insulin, but decreased expression to glucocorticoid and thiazolidinedione. Finally, FoxO1 knock down, but not C/EBP alpha over expression, induced leptin mRNA in 5-aza-C treated preadipocytes. These results suggest that leptin expression in 3T3-L1 adipocytes requires at least 2 steps; (1) DNA demethylation before adipocyte differentiation, (2) transcriptional activation during the differentiation of adipocytes.

1763-PHigh Glucose Promotes Triacylglycerol Accumulation During the Differentiation of Human Subcutaneous Abdominal Preadipocytes into AdipocytesVIAN PESHDARY, ANNEMARIE GAGNON, ALEXANDER SORISKY, Ottawa, ON, Canada

Adipose tissue remodeling is important to maintain healthy metabolic function. The ability to form new adipocytes (adipogenesis) during chronic positive energy imbalance favours hyperpastic expansion and permits individual adipocytes to remain metabolically responsive. However, insuffi cient adipogenic capacity leads to dysfunctional adipocyte hypertrophy, resulting in infl ammation and insulin resistance that raise the risk of type 2 diabetes.

Although high glucose (HG) concentrations reduce mouse 3T3-L1 adipocyte differentiation in culture compared to normal glucose (NG), this has not been carefully examined for human adipogenesis. We hypothesized that human preadipocytes would behave in a similar manner. Human subcutaneous abdominal adipose tissue samples were obtained from 5 (1 male, 4 female) patients undergoing elective abdominal surgery, where age was 43±15.5, and body mass index was 25±2.5(mean±SD). Stromal preadipocytes were isolated and induced to differentiate over 14 days in 5 mM glucose (NG), 25 mM glucose (HG), or 5mM glucose/20 mM mannitol (osmotic control). At day 14, triacylglycerol (TG) accumulation was assessed biochemically, and the protein expression levels of adipogenic genes were evaluated by immunoblot analysis. Differentiated adipocytes in HG exhibited 2.3±0.2 fold more TG accumulation (mean±SE) compared to NG (n=7, p<0.001). No effect of mannitol was observed. Moreover, HG elevated sterol regulatory element-binding protein 1 (SREBP-1) protein expression 1.9±0.4 fold more (mean±SE) than NG (n=6, p<0.001). HG did not alter the protein expression of peroxisome proliferator-activated receptor gamma (PPARγ), fatty acid binding protein (aP2), nor fatty acid synthase (FAS) compared to NG. Our data suggest that HG may enhance lipogenesis during human adipocyte differentiation, in contrast to the anti-adipogenic affects of HG on mouse 3T3-L1 adipocyte differentiation.

Supported by: Heart and Stroke Foundation of Canada

1764-PAdipose ChREBP-β Expression Correlates With Endogenous Glu-cose Production in Lean MenM. KILICARSLAN, M.A. HERMAN, K.E. KOOPMAN, M.C. VAN EIJK, M.T. ACKER-MANS, S.E. LA FLEUR, M.J. SERLIE, Amsterdam, Netherlands, Boston, MA

Carbohydrate Responsive Element Binding Protein (ChREBP) is a transcription factor activated by glucose metabolites that regulates glycolytic and lipogenic gene expression. Adipose tissue expression of ChREBP-β, a recently discovered novel, potent isoform, predicts insulin-stimulated glucose disposal. However, whether adipose tissue ChREBP predicts hepatic insulin sensitivity has not previously been examined. We performed a 2-step hyperinsulinemic-euglycemic clamp and subcutaneous adipose tissue (SAT) biopsies in 12 healthy lean men (BMI 22.6 [19.6-24.3] kg/m2), ages 19-27. Basal endogenous production (EGP) was 11.9 (9.7-13.8) µmol/kgFFM/min. Insulin-mediated suppression of EGP was 75 (47-93) %. Peripheral glucose uptake was 58.5 (46.3-78.8) µmol/kg/min. We observed a trend towards a correlation between adipose tissue mRNA expression of ChREBP-β and insulin-stimulated glucose disposal (r=0.526; p=0.079). No correlation was observed between ChREBP-α and glucose disposal (r=-0.024; p=0.940). Neither ChREBP-α nor ChREBP-β correlated with insulin-stimulated suppression of EGP. Interestingly, while no correlation between ChREBP-α and basal EGP was observed (r=-0.369; p=0.238), ChREBP-β expression strongly correlated with EGP (r=0.646; p=0.023). These results suggest for the fi rst time that adipose tissue ChREBP-β regulates basal glucose turnover in addition to its previously reported role in insulin-stimulated glucose disposal.

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INSULIN ACTION—CELLULAR AND MOLECULAR METABOLISM

1765-PThe Complex P2X7 Receptor/Infl ammasome in Perivascular Fat of SmokersANNA SOLINI, CHIARA ROSSI, ELEONORA SANTINI, MASSIMO CHIARUGI, MA-RIO COMASSI, ANTONIO SALVATI, EDOARDO VITOLO, ELE FERRANNINI, Pisa, Italy

Smoking promotes atherosclerosis (ATH) and tissue infl ammation. Perivascular visceral adipocytes (PVA) release infl ammatory mediators related to insulin resistance (IR) and aggressive ATH. IL1β and IL18 are released in the mature form through the NLRP3 infl ammasome (INFL), whose activation is triggered by extracellular ATP via the P2X7 receptor (P2X7R). The adipocyte P2X7R-INFL complex is increased in patients with metabolic syndrome. We evaluated whether in healthy subjects smoking affects the PVA infl ammatory phenotype, compared IR-related cytokines to other infl ammatory cytokines, and explored the role of the P2X7R-INFL axis. In 25 smokers (S) and 15 non-smokers (NS), TNFα, IL6, RBP4, MCP1, P2X7R, and INFL components NLRP3, ASC, caspase1, IL1β, and IL18 gene and protein expression (exp) were determined in PVA by RT-PCR and Western Blot. Plasma and culture medium PVA (CMed) cytokines were also measured. S had higher PVA P2X7R gene (2.5±0.8 vs 1.2±0.4 target/reference, p<0.0001) and protein exp; similarly, NLRP3 (3.2±1.0 vs 1.9±0.6 of NS, p<0.0001) and caspase1 (1.6±0.5 vs 1.3±0.5, p=0.04) were more abundant in S. This was paralleled by higher MCP1 exp, strictly dependent upon INFL activation (2.7±1.4 vs 1.6±0.6, p=0.006), IL1β (3.2±0.8 vs 2.2±0.9, p=0.0007), and IL18 (2.8±1.4 vs 1.8±0.6, p=0.008). IL1β and IL18 were higher in S in CMed (IL1β 2.7±1.0 vs 2.0±0.9, p=0.03; IL18 3.3±1.1 vs 2.5±1.1 pg/ml, p=0.02), and plasma (IL1β 2.0±1.1 vs 1.4±0.5, p=0.04; IL18 273±104 vs 176±60 pg/ml, p=0.02). In the whole group, NLRP3 was related to P2X7R exp (r=0.48), PVA IL18 exp (r=0.34), CMed IL18 release (r=0.32), and circulating IL18 (r=0.42). No difference was observed in PVA exp of genes more strictly related to IR (TNFα 1.8±0.7 vs 2.0±0.8, RBP4 1.5±0.6 vs 1.4±0.4, IL6 5.8±2.0 vs 5.6±2.7). Plasma TNFα and RBP4 levels did not differ between groups. PVA of S show an increased exp of the complex P2X7R-INFL, coupled with enhanced MCP1 exp and IL1β and IL18 release, while IR-related cytokines do not differentiate S from NS.

1766-PSerum Retinol Binding Protein 4 Is Related to Hepatic and Peripher-al Insulin Sensitivity but Not to Beta-Cell Function in Patients With Type 2 Diabetes MellitusZHANG MIN, CHEN SHU, CHEN PING, REN MIN, WANG JUNKANG, Chengdu, China

Abstract objective: This study examined the relationship of serum retinol binding protein 4(RBP4) with hepatic and peripheral insulin sensitivity and beta-cell secretory function in patients with Type 2 diabetes mellitus (DM).Subjects and methods: Fasting insulin, glucose and RBP4 were measured in 56 Type 2 diabetic patients (include 34 men, 22 women) who were on diet treatment only and terms for insulin sensitivity and beta-cell secretion obtained by modelling. Results: RBP4 was positively correlated (r = 0.38, p < 0.05) to hepatic insulin sensitivity derived from mathematical modelling of fasting glucose and insulin data using the homeostasis assessment model (HOMA). Fasting insulin correlated negatively with RBP4 (r = -0.39, p < 0.05). There were also signifi cant negative correlations between RBP4 and either plasma glucose (r = -0.32, p < 0.05) or body mass index (r = -0.33, p < 0.05). RBP4 did not correlate with HOMA-modelled beta-cell function. In a multiple regression analysis, RBP4 was independently correlated only with insulin sensitivity (p < 0.05). Further studies in 17 of the diabetic patients (13 men, 4 women), showed a signifi cant positive correlation (r = 0.48, p < 0.05) between RBP4 and peripheral insulin sensitivity derived by continuous infusion of glucose with model assessment (CIGMA) but not between RBP4 and CIGMA-modelled beta-cell function. Conclusions: These results indicate that serum retinol binding protein 4 is related to hepatic and peripheral insulin sensitivity but not to beta-cell function in patients with Type 2 diabetes mellitus.

1767-PFat-Induced Reduction in Irisin Gene Expression in Visceral and Subcutaneous Adipose Depots Is Moderated by CB1 AntagonismMORVARID KABIR, MALINI S. IYER, QIANG WU, DARKO STEFANOVSKI, ORISON O. WOOLCOTT, STELLA P. KIM, CATHRYN KOLKA, VIORICA IONUT, JOYCE M. RICHEY, RICHARD N. BERGMAN, Los Angeles, CA

The expression of Irisin is a novel hormone secreted by myocytes and genes related to energy expenditure has not been evaluated in high fat diet (HFD) and CB1 antagonist rimonabant (RIM) treatment in the subcutaneous (SC) and visceral (VIS) fat depots. We demonstrated in the dog that RIM

improved deleterious effects of HFD by reducing body fat and increasing insulin sensitivity. The current study examines longitudinal effects of HFD and RIM treatment on Irisin, uncoupling protein 1 (UCP1) and PPARγ coactivator 1 alpha (PGC1-α) in VIS and SC depots. Animals were fed a HFD (52% fat) for 6 weeks to increase fat deposition in VIS and SC adipose depots followed by an additional 16 weeks of fat feeding with either 1) HFD + placebo (PL) (n=9) or with HFD + RIM (1.25 mg/kg per day; n=11). Biopsies from SC and VIS depots were obtained for gene expression: before starting HFD (Pre-fat) and after 16 weeks of HFD and RIM. In both fat depots irisin-decreased in HFD+PL and to a lesser extent in the HFD+RIM group, in the SC and VIS depots. UCP1 and PGC1-α expression demonstrated similar results (see Table). Irisin and genes related to energy expenditure decrease with high fat diet in the VIS and SC depots. CB-1 antagonist can improve expression of Irisin, especially in the SC depot. These data support an important role of Irisin in the regulation of energy expenditure in fat depots.Note: *P<0.05 vs pre-fat, ** P<0.005 vs pre-fat, # P<0.05 HFD+PL vs HFD+RIM. Values are means ± SEM.

SC VISPre-fat HFD+PL HFD+RIM Pre-fat HFD+PL HFD+RIM

Irisin 1.70±0.59 0.13±0.03* 0.37±0.08# 1.05±0.16 0.33±0.10** 0.85±0.36UCP1 11.27±2.72 1.80±0.23** 3.71±0.62** 4.26±0.94 2.18±0.37* 4.53±1.53PGC1a 2.11±0.39 1.62±0.30 2.36±0.53 3.21±0.51 0.66±0.12** 1.45±0.33*#

Supported by: sanofi -aventis


Guided Audio Tour: Cellular and Molecular Metabolism (Posters: 1768-P to 1773-P), see page 15.

& 1768-PThe Mitochondrial Pyruvate Carrier Complex: A Potential Target for Novel Insulin-Sensitizing and Weight Loss DrugsPATRICK A. VIGUEIRA, GEORGE G. SCHWEITZER, WILLIAM G. MCDONALD, ROLF F. KLETZIEN, JERRY R. COLCA, BRIAN N. FINCK, St. Louis, MO, Kalamazoo, MI

We have hypothesized that the insulin-sensitizing effects of thiazoli-dinediones (TZDs) are separable from their activation of PPARγ and are mediated through binding to a mitochondrial target. The mitochondrial pyruvate carrier 2 (Mpc2) was identifi ed by proteomics as a 14 kDa protein that was specifi cally crosslinked by a TZD photoaffi nity probe. Consistent with this, a chemical inhibitor of pyruvate transport that binds the Mpc complex (UK-5099) blocked the binding of the 14 kDa protein by TZDs. To examine the involvement of Mpc2 in the response to TZDs, we attempted to generate Mpc2 null mice, but unexpectedly generated mice that expressed an Mpc2 protein missing 16 amino acids of the N-terminus. Crosslinking of TZDs to the major 14 kDa protein was abolished in liver mitochondrial membranes from homozygous Mpc2 mutant mice, while other crosslinked proteins were not affected. Mpc2 mutant mice were viable and gained weight normally on a high fat diet. Obese WT and Mpc2 mutant mice were then treated with pioglitazone (PIO) or a novel PPARγ-sparing TZD (MSDC-0602). Administration of PIO or MSDC-0602 improved glucose tolerance, but whereas PIO treatment tended to increase body weight gain and adiposity, MSDC-0602 treatment reduced body weight and adiposity without affecting food intake or locomotor activity. However, the effects of MSDC-0602 on insulin sensitivity and adiposity were maintained in Mpc2 mutant mice indicating that Mpc2 per se may not be required for these actions. These data suggest that Mpc2 binds TZDs, but that another mitochondrial protein, possibly associated with the Mpc complex, may mediate the insulin-sensitizing and weight loss effects of MSDC-0602.

& 1769-PVitamin D Receptor (VDR) Activation, H2S Formation, and ROS In-hibition Mediate the Effect of Vitamin D on Glucose Transporter 4 (GLUT4) Translocation and Glucose Utilization in High Glucose Treated 3T3L1 AdipocytesPRASENJIT MANNA, SUSHIL K. JAIN, Shreveport, LA

Circulating vitamin D relationship with insulin resistance has not been resolved yet. This study examined the biochemical mechanism by which vitamin D supplementation may regulate glucose metabolism in diabetes. 3T3L1 adipocytes were treated with high glucose (HG, 25 mM) in the presence

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or absence of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] (25 or 50 nM), the active form of vitamin D. 1,25(OH)2D3-treatment caused insulin receptor (IR) phosphorylation (tyr 1151/1150), sirtulin 1 (SIRT1) activation, protein tyrosine phosphatase 1B (PTP1B) inhibition, PIP3 formation, GLUT4 translocation, and glucose uptake as well as glucose utilization in HG-treated adipocytes. 1,25(OH)2D3 supplementation also reduced ROS production and caused vitamin D receptor (VDR) activation, CSE activation and H2S formation in HG treated cells. Interestingly the effect of 1,25(OH)2D3 on GLUT4 translocation and glucose utilization was prevented either in CSE-knockdown or VDR-knockdown adipocytes compared to controls. 1,25(OH)2D3 treatment along with insulin enhanced GLUT4 translocation and glucose utilization compared to either insulin or 1,25(OH)2D3 alone in HG-treated cells. These results demonstrate the essential role of VDR activation, CSE activation and H2S formation in mediating the effect of vitamin D on GLUT4 translocation, glucose utilization via the insulin dependent as well as independent signaling pathways in HG-treated adipocytes.

Supported by: Malcolm Feist Chair in Diabetes and Cardiovascular Fellowship

& 1770-PAltered Mitochondrial Dynamics in Cybrid Cells Harboring Diabe-tes-Susceptible Mitochondrial HaplogroupPEI-WEN WANG, SHAO-WEN WENG, HSIAO-MEI KUO, CHIA-WEI LIOU, TSU-KUNG LIN, Kaohsiung, Taiwan

Mitochondrial DNA (MtDNA) haplogroups have been linked to longevity and degenerative diseases. In the Chinese population, mtDNA haplogroup B4 lends susceptibility while D4 resistance to diabetes. Mitochondria (Mt) control their quality dynamically through continuous fusion or fi ssion. These dynamics related proteins have been found to regulate cellular metabolism and ROS production.

We used cybrid cells derived from 143B osteosarcoma cell line harboring the same nuclear DNA but different mtDNA to investigate the relationship between glucose metabolism, ROS production, and Mt dynamics. Cybrid cells were cultured in a medium containing 25mM glucose and stimulated with 0, 0.1, and 1uM insulin. Mt membrane potential and respiratory control indices were higher in cybrid D4 than B4. Both intracellular and Mt ROS production was higher in B4 cells. Cytoplasmic Glut1/Glut4 translocation to cell membrane in cybrid D4 increased notably after insulin treatment while that in B4 cells did not. Under a confocal microscope the Mt network extent in cybrid D4 was about twice that in B4, accompanied with increased expression of fi ssion-related proteins (Fis1, Drp1) in B4. Upon insulin stimulation, the response of dynamic proteins decreased in cybrid B4 but increased in D4. Upon antioxidant (NAC, 5mM, 1hr), cybrid B4 cells showed increased Glut1/Glut4 translocation to cell membrane, increased expression of fusion-related proteins (Mfn1, Mfn2, OPA1), and increased Mt network formation.

We concluded that Mt have an independent role in pathogenesis of diabetes. The mechanisms are probably through altered ROS production and Mt dynamics. However, the causal relationship between Mt ROS production and fi ssion/fusion dynamic changes need further investigation.

Supported by: Kaohsiung Chang Gung Memorial Hospital (CMRPG891112)

& 1771-PDiacylglcerol Activation of PKCθ Leads to Increased IRS-1 1101 Serine Phosphorylation and Skeletal Muscle Insulin Resistance in Obesity and Type 2 DiabetesJULIA SZENDROEDI, TORU YOSHIMURA, ESTHER PHIELIX, CHRYSI KOLIAKI, ME-LISSA MARCUCCI, DONGYAN ZHANG, TOMAS JELENIK, CHRISTIAN HERDER, PETER NOWOTNY, GERALD I. SHULMAN, MICHAEL RODEN, Düsseldorf, Germany, New Haven, CT

Skeletal muscle insulin resistance is strongly associated with increased intramyocellular lipid content but the cellular mechanisms remain unclear. We measured myocellular diacylglycerols (DAGs), ceramides, activities of novel protein kinase C isoforms (PKCβ, δ, ε, θ) in healthy controls (CON, n=16, 30±5 years, BMI: 24±2 kg/m2), obese insulin resistant glucose tolerant humans (OBE, n=10, 29±2 y, 42±2 kg/m2) and patients with type 2 diabetes (T2D, n=10, 59±3 y, 36±1 kg/m2). Hyperinsulinemic-euglycemic clamps combined with stable isotopes were performed to measure muscle insulin sensitivity. We also assessed insulin receptor substrate 1 serine-phosphorylation (IRS1-Ser1101), basal and insulin-stimulated IRS-1 associated phosphoinositide-3-kinase (PI3K) activation in muscle of CON subjects (n=7, 28±2 y, 23±3 kg/m2) before/after 4.5 h lipid (LIP) or glycerol infusion (GLY). Activation of PKCθ was increased in both OBE and T2D compared to CON subjects (both P<0.03 vs. CON). Ceramide content and activation of PKCβ, δ and ε were comparable between groups. Following lipid infusion we observed a

similar increase in PKCθ activity (P<0.005) associated with ~80% increased membrane and cytosolic DAG content and ~70% increased (P<0.005) IRS1-Ser1101phosphorylation. In contrast to muscle ceramides, increases in DAG species (C16/C20_4, C18/C20_4, C18_2/C18, C18_1/C18, C18/C16) in both cytosolic and membrane fractions correlated negatively with muscle glucose disposal (r=-0.5, P<0.01). These changes were related to ~70% reduced (P<0.05) insulin-stimulated PI3K activity. These data support the hypothesis that DAG activation of PKCθ promotes increased IRS-1Ser1101 phosphorylation and triggers lipid-mediated muscle insulin resistance in obesity and T2D.

Supported by: DFG; DZD; EFSD

& 1772-PHeat Shock Protein 90 Beta (hsp90beta) Is a Novel Diabetic Target Regulating Metabolism in Skeletal MuscleENXUAN JING, PRAGALATH SUNDARARAJAN, SUWAGMANI HAZARIKA, SA-MANTHA FOWLER, VIVEK VISHNUDAS, POORNIMA K. TEKUMALLA, VIATCHE-SLAV R. AKMAEV, RANGAPRASAD SARANGARAJAN, NIVEN R. NARAIN, Natick, MA

Hsp90β was identifi ed as a critical node regulating diabetes by the Berg Interrogative BiologyTM platform. Hsp90β is an ATP dependent protein chaperone that targets multiple clients including vital components of insulin signaling and mitochondrial membrane proteins. However the role of hsp90β in substrate metabolism and diabetes remains unclear. Preliminary investigation suggested that metabolic factors regulate Hsp90β mRNA expression and protein abundance in human skeletal muscle myotubes. These data suggest that Hsp90β may be involved in the regulation of responses stimulated by various metabolic factors and cytokines in diabetes. Primary cultures of human skeletal muscle myotubes were used as a model system, simulation of diabetic conditions was achieved by altering glucose concentrations in the presence/absence of infl ammatory cytokines, hyperlipidemia and other physiological perturbations. The RNAi mediated knockdown of Hsp90β was associated with signifi cant alterations in functional end-points of glycolysis, mitochondrial respiration, and free fatty acid metabolism consistent with a potential therapeutic benefi t for diabetes. In addition, knocking down of Hsp90β was associated with increased insulin stimulated glucose uptake demonstrated by an observed increase in Akt and MAPK phosphorylation signaling. Taken together, the data presented in this study provides support for the novel role of Hsp90β as a critical regulator of cellular metabolism and insulin sensitivity in skeletal muscle cells and represents a viable therapeutic target in diabetes.

& 1773-PThe Glycogen Targeting Subunit PPP1R3C: A Novel Link between Glycogen and Lipid StorageBINBIN LU, DAVE BRIDGES, YEMEN YANG, KALEIGH FISHER, ALAN CHENG, ALAN SALTIEL, Ann Arbor, MI

Glycogen and lipid are the two major energy storage forms that are tightly regulated by hormonal and metabolic signals. In this study, we present data supporting a role for the mechanistic target of rapamycin complex 1 (mTORC1) and the glycogen targeting subunit of protein phosphatase 1(PPP1R3C/ PTG) in energy homeostasis. We show that hepatic glycogen and PTG levels were increased by feeding mice high-fat diet (HFD), corresponding with an increase in mTORC1 activity. Both PTG and glycogen levels are increased upon constitutive activation of mTORC1 in muscle tissue. In vitro, PTG gene expression was specifi cally controlled by activation of both mTORC1 and SREBP1, and the mTORC1-dependent increase in glycogen was attenuated by knockdown of PTG. Moreover, the HFD-dependent increase in hepatic glycogen was prevented in PTG knockout mice. Interestingly, PTG knockout mice fed HFD also exhibited improved liver steatosis and decreased lipid levels in muscle, in coordination with decreased glycogen. Further investigation revealed that lipid oxidation is increased in PTG knockout mice, possibly mediated by activation of AMPK, implying a previously unappreciated crosstalk between pools of glycogen and upstream nutrient-sensing pathways that lead to reduced lipid accumulation. Together, these data suggest that dietary and nutritional changes in glycogen levels can be partially accounted for by changes in the transcription of the glycogen synthesis scaffolding protein PTG, and further that a novel glycogen-lipid interaction has profound effects on energy storage and metabolism.

Supported by: NIH (R01DK060591), (R24DK090962)

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1774-PFoxo1 Inhibits Hepatic Lipogenesis Involving Its Ser253 Dephos-phorylation and Activation of AMPKKEBIN ZHANG, ZIHUI XU, YAJUAN QI, LING LI, QINGLEI ZHU, SHAODONG GUO, Temple, TX

O class of forkhead/winged helix transcription factor Foxo1 inhibits hepatic lipogenesis and the mechanism is not completely understood (Endo crinology, 2012, 153(20): 549-551). Mouse Foxo1-Ser253, equivalent to human Foxo1-Ser256, is phosphorylated by insulin via activation of PI-3K and Akt signaling cascade, promoting Foxo1 nuclear export and ubiquitylation, mediating the effect of insulin on gene expression and metabolic regulation. To determine the role of Foxo1-Ser253 in regulating lipid metabolism, we co-transfected plasmid DNA expressing Foxo1-S256A (alanine mutation to block phosphorylation) or Foxo1-S256D (aspartate mutation to mimic phosphorylation) in human hepatoma HepG2 cells and found that Foxo1-S256A mutant increased AMPK phosphorylation at Thr172 which reduces fatty acid synthase gene expression, while Foxo1-S256D had not such an effect. To further assess the role of Foxo1 in mice, we generated Foxo1-S253A mice in which Ser253 was replaced with alanine (A) to block phosphorylation. The homozygous Foxo1-S253 A/A mice displayed reduced blood triglycerides and hepatic lipid concentrations with enhanced hepatic AMPK phosphorylation. Thus, we fi rstly demonstrated that a non-phosphorylation of Foxo1-Ser253 suppresses hepatic lipogenesis involving AMPK phosphorylation in vitro and in vivo.

Supported by: NIH/NIDDK

1775-PIncreased Lipid Availability Induces Insulin Resistance and Impairs Muscle Mitochondrial Function in Lean Healthy SubjectsMUHAMMAD A. ABDUL-GHANI, ROY ELDOR, LUKE NORTON, MARCEL FOURCAUDOT, CYNHTIA GALINDO, DIVJIT TRIPATHY, RALPH A. DEFRONZO, San Antonio, TX, Jerusalem, Israel

The role of impaired mitochondrial (MITO) function in the pathogenesis of insulin resistance is debated. It is well established that elevation of the plasma FFA conc causes insulin resistance. In the present study, we examined whether in vivo elevation of plasma FFA impairs MITO function and PGC-1 expression (master regulator of MITO biogenesis) in lean healthy subjects.

Insulin-mediated total body glucose disposal (TGD) was measured in 9 lean NGT subjects before and after 3 day lipid infusion to cause physiologic elevation in plasma FFA (380±80 to 720±125 ueq/L). MITO ATP synthesis was measured ex vivo in MITO isolated from vastus lateralis muscle biopsies.

Physiologic plasma FFA elevation caused a 24+5% decrease in insulin-stimulated TGD (12.8±1.0 to 9.3±1.1 mg/kg·min, p<0.01) and decreased MITO ATP synthesis rate with complex 1 (34+9%, p<0.05) and complex 2 (30+5%, p<0.05) substrates. The decrease in ATP synthesis correlated with increase in plasma FFA conc (r=0.65, P<0.05), but not with the decrease in TGD. Lipid infusion caused no change in MITO ROS generation.

In vitro studies have implicated increased methylation of PGC1 alpha gene promoter in the pathogenesis of lipid-induced insulin resistance. Therefore, we examined the effect of lipid infusion on PGC1α expression and the inducible DNA-methyltransferase3b (DNMT3b). Lipid infusion caused an increase in expression (0.84±0.09 to 1.58±0.21 fold over HMBS control, p=0.002) of DNMT3b with no change in the constitutively expressed DNMT1 and with no change in PGC1α mRNA expression, total PGC1α methylation or non CpG methylation. However, total methylation of the alternative PGC promoter was decreased after lipid infusion (1.26±0.29 to 0.86±0.14% methylated residues/(patient·strand; p<0.05).

Conclusion: insulin resistance caused by physiologic elevation of plasma FFA conc in lean NGT subjects is associated with impaired mitochondrial function, which is unrelated to changes in PGC1α expression.

Supported by: NIH

1776-PDeletion of Acetyl-CoA Carboxylase 2 (ACC2) Increases Fatty Acid Oxidation Without Altering Body Fat or Insulin Sensitivity in 9-12 Month Old MiceAMANDA E. BRANDON, NIGEL TURNER, SIMON J. LESLIE, ELLA STUART, KYLE L. HOEHN, DAVID JAMES, EDWARD W. KRAEGEN, GREGORY J. COONEY, Darlin-ghurst, Australia, Sydney, Australia

The rate of long chain fatty acid oxidation (FAO) is largely determined by CPT-1-mediated entry of long chain fatty acids into mitochondria. An important enzyme regulating this process is acetyl-CoA carboxylase 2 (ACC2), whose product malonyl-CoA is a potent allosteric inhibitor of CPT-1 activity. We have previously shown that global deletion of ACC2 increased

whole body and skeletal muscle FAO, as well as skeletal muscle glycogen content, without changing glucose tolerance, body weight or fat mass in young mice (4-5 months)1. The aim of the current study was to determine if this scenario persisted in older mice and to determine the effect of ACC2 gene deletion on insulin sensitivity using a euglycemic-hyperinsulinemic clamp. At 9-12 months of age, male wild type (WT) and ACC2-/- mice (n = 8-10 mice/group) were of similar body weight (33.5 ± 0.4g and 33.6 ± 0.8g respectively) and fat mass (16.2 ± 1.1% and 19.4± 2.0% respectively by DEXA) and displayed identical glucose tolerance. As shown previously in young mice, the older ACC2-/- mice showed increases in whole body FAO (24hr average RER = 0.95±0.02 and 0.92±0.02 for WT and ACC2-/- respectively, p<0.05) and skeletal muscle glycogen content (WT 10.2 ± 0.9µmol/g; ACC2-/- 16.5 ± 1.4µmol/g, p<0.05) without any signifi cant change in energy expenditure. In line with the glucose tolerance results, clamp studies (n = 6 per genotype) revealed no difference in insulin action between groups, with similar glucose infusion rates (WT 37.9 ± 3.8; ACC2-/- 35.9 ± 4.0 mg/min/kg) or glucose uptake into skeletal muscle (WT 8.8 ± 1.1; ACC2-/- 10.4 ± 1.9 µmol/min/100g) and epididymal fat (WT 5.9 ± 1.4; ACC2-/- 4.3 ± 0.7 µmol/min/100g). These results demonstrate that:- (1) deletion of ACC2 can increase whole body FAO without any measurable change in energy expenditure; (2) Over the long-term, this increase in FAO is not suffi cient to alter body composition or insulin sensitivity.

1. Hoehn KL, et al Cell Metab 11: 70-76, 2010.Supported by: NHMRC (Australia)

1777-PThe Association between Iron Accumulation and Insulin Resistance in Skeletal Muscle of Subjects With T2DMTAE HO KIM, EUN SUK HA, SO-YEON AN, MIN SUK LEE, JA YOUNG JEON, SE-UNG JIN HAN, HAE JIN KIM, DAE JUNG KIM, SUNG E. CHOI, YUP KANG, KWAN WOO LEE, Goyang, Republic of Korea, Suwon, Republic of Korea

Insulin resistance (IR) is a major characteristic of type 2 diabetes mellitus (T2DM). Despite numerous studies, the pathogenesis of IR has not been well established. Ferritin is known to be associated with IR, but little is known about the possible sites (e.g., muscle, liver, adipose tissue) where ferritin may induce IR. It has also been suggested ferritin may be an underlying factor in the development of oxidative stress in skeletal muscle. Thus we hypothesized that ferritin in human skeletal muscle may play a signifi cant role in insulin resistance.

We recruited 16 T2DM subjects and 12 subjects with normal glucose tolerance (NGT) for this study. Whole-body insulin-mediated glucose uptake was determined using a euglycemic hyperinsulinemic clamp test. We performed biopsies on the vastus lateralis muscle and used immunoblotting to determine levels of 3-nitrotyrosine, ferritin heavy chain, ferritin light chain, transferrin receptor-1(TfR-1), divalent metal transporter-1(DMT-1), and iron regulatory protein-1(IRP-1) in skeletal muscles from both T2DM subjects and NGT subjects.

Tests on T2DM subjects resulted in a higher protein oxidative damage as assessed by detecting 3-nitrotyrosine, when compared with muscles from the NGT group (p-value <0.05). An elevated expression of ferritin, (heavy chain, p-value <0.01; light chain, p-value < 0.001), an important iron storage protein, was associated with T2DM. The iron transport protein, TfR-1, demonstrated a tendency to increase (P =0.07) and the level of DMT-1 protein expression was signifi cantly increased in muscle from the T2DM group (P =0.01). Protein levels of IRP-1 were signifi cantly decreased (P =0.01) in the T2DM group.

In conclusion, altered iron metabolism and iron accumulation in skeletal muscle may be associated with insulin resistance in T2DM patients.

1778-PRegulation of CEACAM1 by PPARα-Dependent MechanismsSAJA KHUDER, Toledo, OH

Carcinoembryonic Antigen-related Cell Adhesion Molecule (CEACAM1),a transmembrane glycoprotein, regulates insulin action by promoting receptor-mediated insulin endocytosis and degradation,a key mechanism of insulin clearance, which occurs mostly in the liver and kidney to a lower extent. Mice with null deletion of Ceacam1 show impairment of insulin clearance and consequently, hyperinsulinemia, insulin resistance, hepatic steatosis and visceral obesity. Obese humans exhibit insulin resistance associated with decreased CEACAM1 levels. High-fat (HF) diet causes insulin resistance by reducing CEACAM1 before infl ammation develops. Findings that show CEACAM1 is metabolically regulated; reduced at fasting and elevated in parallel to acute rise of insulin in response to refeeding, provide the impetus to investigate the mechanisms leading to decreased Ceacam1 expression in

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obesity, at fasting and in response to HF feeding. One common biochemical mechanism between these conditions is elevated fatty acid (FA) entry to the liver for clearing via β-oxidation. This is attained by activating Peroxisome Proliferator Activated Receptor α (PPARα), a nuclear receptor that forms heterodimers with retinoid x receptor alpha (RXRα) to bind to DNA at sites of target genes. Our preliminary data suggests that FA-induced PPARα activation results in direct transcriptional inhibition of Ceacam1. We have shown that PPARα activation by WY-14,643 selective PPARα agonist causes PPARα binding to Ceacam1 promoter in rat and mice hepatoma cells and in the liver of wild-type, but not PPARα knockout mice, fed a Wy-supplemented diet for 3 days. Deletion and block mutation analyses identifi ed a main site of PPARα/RXRα binding to Ceacam1 promoter. Further studies underway investigate mechanisms involved in Ceacam1 regulation. With CEACAM1 playing a key role to maintain insulin action and fat metabolism, such studies can potentially identify CEACAM1 as a tractable drug target to combat metabolic insulin resistance, fatty liver disease and obesity.

Supported by: NIH

1779-PInsulin Resistance Stimulates and Insulin Sensitization Attenuates Vascular Smooth Muscle Cell Migration and ProliferationEUGENIO CERSOSIMO, XIAOJING XU, FNU RAJLAKSHMI, SIKARIN UPALA, NI-COLAS MUSI, San Antonio, TX

We have previously shown differential activation of insulin signal pathways PI-3K and MAP-K by palmitate and high glucose in cultured vascular smooth muscle cells (VSMC). In this study, we examined the VSMC migration & proliferation (M&P) patterns in similar conditions.

VSMC from healthy human coronary arteries were incubated in growth medium (C-22062 & C-39267, Promo Cell). M&P was analyzed in re-suspended cells during exposure to high glucose (25mM), ± palmitate (200 µM), ± PPAR-γ agonist pioglitazone (PIO, 8 µM) for 5h. M&P was assessed by: i) polycarbonate membrane barrier with chemo-attractants & extended cell protrusions quantifi ed by optical density (OD595 nm); and ii) inverted microscopy images & % change in radius area, [ϖr2(BEFORE)-ϖr2(AFTER)/ϖr2(BEFORE)]*100 (2D Assay, Cell BioLabs, San Diego, CA). Mean data from 4 experiments each are presented.

Exposure to 25mM glucose increased M&P by 25%, from 0.20±0.02 to 0.25±0.02 and the % chamber area closure rose from ~20 to ~30%. PIO addition restored M&P to 0.20±0.03 and % chamber area closure decreased to ~25%. In the presence of palmitate, M&P increased by ~15%, from 0.21±0.01 to 0.24±0.02 and the % area closure increased from ~20 to ~30%. PIO addition to the media reduced M&P to 0.22±0.02 and % area closure to ~15%. Culture media containing 25mM glucose plus palmitate showed a 35% rise M&P from 0.22±0.02 to 0.30±0.03 and an increase in % area closure from ~20 to ~28%, but PIO addition decreased M&P to 0.21±0.02 and % area closure to ~10%.

Conclusions: High glucose and palmitate stimulate human coronary VSMC migration & proliferation in vitro, which is attenuated by insulin sensitizer pioglitazone. These data complement our earlier fi ndings of preferential activation of the MAP-Kinase pathway by palmitate and high glucose, mitigated by pioglitazone. Our results suggest that the MAP-Kinase insulin signal is involved in VSMC migration & proliferation in atherogenesis in conditions of insulin resistance.

Supported by: Texas Diabetes Institute

1780-PTwo Weeks of Metformin Treatment Induces AMPK Dependent En-hancement of Insulin Stimulated Glucose Transport in Mouse Skel-etal MuscleJONAS M. KRISTENSEN, JONAS T. TREEBAK, LAURIE J. GOODYEAR, JØRGEN F.P. WOJTASZEWSKI, Odense, Denmark, Copenhagen, Denmark, Boston, MA

The antidiabetic drug metformin improves insulin sensitivity primarily by inhibition of gluconeogenesis and stimulation of muscle glucose uptake. Metformin-induced activation of the 5´AMP activated protein kinase (AMPK) has been associated with enhanced glucose uptake in skeletal muscle but so far no direct causality has been examined. We hypothesized that an effect of in vivo metformin treatment on glucose uptake in mouse skeletal muscles is dependent upon AMPK signaling. To investigate this, an oral dose of metformin (300 mg*kg body weight-1*day-1) or saline treatment was given muscle-specifi c kinase α2 dead AMPK mice (KD) and wild type (WT) littermates either once or chronically for 2 weeks. Isolated soleus and Extensor Digitorum Longus (EDL) muscles were used for ex-vivo measurements of glucose transport and Western blot analyzes. We found that chronic treatment with metformin enhanced insulin-stimulated glucose uptake ex

vivo in soleus muscles of WT (~45%, P<0.01), but not in AMPK KD mice. Insulin signaling at the level of Akt protein expression or Thr308 and Ser473 phosphorylation was not changed by metformin treatment. Downstream of Akt, TBC1D4 Thr642 and Ser711 phosphorylation and Rab4 and GLUT4 protein expressions were not affected by metformin treatment. Also AMPK catalytic subunit, TBC1D4 and hexokinase proteins were unaltered after treatment. The acute metformin treatment did not affect glucose uptake in muscle of either of the genotypes. In conclusion, we provide novel evidence for a role of AMPK in potentiating the effect of insulin on glucose uptake in skeletal muscle in response to chronic metformin treatment. The lack of changes in protein activation or expression related to insulin signaling at the level of Akt, TBC1D4, Rab4, GLUT4 or HK suggest that AMPK affects steps further distal related to fusion or docking of GLUT4.

1781-PAdipoRs Actions Not only in Metabolic Organs but also in He-matopoietic Cells Protect from Nutrient Stress-Induced Insulin Re-sistance via Reducing Lipotoxicity and Infl ammationTOSHIMASA YAMAUCHI, MIKI OKADA-IWABU, MASATO IWABU, TAKASHI KA-DOWAKI, Tokyo, Japan

Adiponectin plays pivotal roles in mediating inter-organ metabolic and anti-infl ammatory crosstalk, thereby preventing type 2 diabetes and atherosclerosis. In skeletal muscle, by using muscle-specifi c AdipoR1 KO mice, we showed that AMPK, NAD-dependent deacetylase SIRT and PGC-1a are principal modulators of pathways downstream of AdipoR1, which increase mitochondrial metabolic functions, and ameliorate insulin resistance, like exercise.

With regard to liver, we show that hepatocyte-specifi c disruption of AdipoR1 results in increased gluconeogenesis, while hepatocyte-specifi c disruption of AdipoR2 results in decreased PPARa pathways such as decreased molecules involved in fatty-acid combustion including ACO, both of which are associated with insulin resistance. We next try to identify and characterize orally active AdipoR agonists (ARA). One of these compounds, ARA-1 activates AMPK and increases PGC-1a and mitochondria in C2C12 myotubes and also in skeletal muscle of mice on a high-fat diet. At the same time, ARA-1 increased fatty-acid combustion and decreased oxidative stress, which are associated with increased insulin sensitivity and exercise endurance. In liver, ARA-1 activates AMPK and suppresses gluconeogenic genes and activates PPARa pathways such as increased fatty-acid combustion. In adipose tissues, ARA-1 suppresses pro-infl ammatory adipokines such as MCP-1. Importantly, in AdipoR1/R2 double knockout (DKO) mice, all these effects are almost completely abolished. Interestingly, bone marrow transplantation experiments between wild-type and DKO mice revealed that AdipoRs actions not only in metabolic organs but also in hematopoietic cells play pivotal roles in the regulation of insulin sensitivity.

These data suggest that orally active AdipoR agonists are promising new therapeutic approach for treating nutrient stress-induced diseases such as type 2 diabetes.

1782-PIdentifi cation of Metabolic Differences between Poor-Glycemic Controlled and Well-Glycemic Controlled Type 1 Diabetes using Comprehensive Metabolomics ApproachTUMPA DUTTA, YOGISH C. KUDVA, XUAN-MAI PERSSON, YING LI, JERRY DEWEY, G. CHARLES FORD, JEAN-PIERRE KOCHER, K. SREEKUMARAN NAIR, Rochester, MN

Poor glycemic control in type 1 diabetes (T1D) individuals is associated with both micro and macrovascular complications. Although most of the microvascular complications are prevented by good glycemic control, cardiovascular death continues to be high. Here we utilized UPLC-Time-of-Flight mass spectrometry based large-scale metabolomic analysis of fasting plasma refl ecting cellular events to identify the molecular pathways and metabolites in T1D who are chronically in good (HbA1c of<6.5%) or poor (HbA1c≥8%) glycemic control in comparison with BMI, age and gender matched non-diabetic individuals. We identifi ed 38 metabolic pathways (p<0.05) in poorly controlled T1D affecting cholesterol, vitamin D, tRNA biosynthesis, branch chain amino acid (BCAA), amino acid metabolism, bile acids, urea, Krebs cycle, immune response and eicosanoids biosynthesis. Good glycemic control in T1D corrected several canonical pathways in lipid and amino acid metabolism but >24 pathways (p<0.05) remained altered including glycolysis, gluconeogenesis, glycogen, bile acid metabolism, tRNA biosynthesis, BCAA, amino acid, retinol and vitamin D metabolisms. Selected altered metabolic pathways such as non-esterifi ed fatty acids (NEFA), amino acid metabolites and Krebs cycle metabolites were validated by targeted

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quantitative measurements thus confi rming that these important metabolic pathways are not completely normalized by good glycemic control (HbA1c of <6.5%) using peripheral insulin treatment in comparison with non-diabetes. Future research should allow therapies targeted on these identifi ed pathways in the current study that remain abnormal despite good glycemic control in order to prevent the persistent chronic complications of diabetes. The study also identifi ed several hitherto unknown metabolites which could potentially be used to identify biomarkers of cardiovascular complications in T1D.

Supported by: UL1RR024150-01

1783-PMetabolic and Infl ammatory Effects of Acute Hyperglycemia in Type 2 Diabetic SubjectsULLA KAMPMANN, BRITT CHRISTENSEN, PERNILLE HOEYEM, RONI NIELSEN, LOTTE OERSKOV, NIELS JESSEN, NIELS MOELLER, Aarhus, Denmark, Silkeborg, Denmark

Chronic hyperglycemia and metabolic dysregulation cause microvascular and macrovascular complications in diabetes and these effects are also associated with chronic low grade infl ammation. It remains uncertain to which extent acute hyperglycemia affects indices of low grade infl ammation in type 2 diabetes and it is not fully described how protein and glucose metabolism respond to hyperglycemia.

The aim of our study was to examine the effects of acute hyperglycemia on protein metabolism, glucose metabolism and infl ammatory markers. Nine patients (seven males, two females) with type 2 diabetes were examined on two occasions in a randomized crossover design after 16 h of 1) hyperglycemia/insulin withdrawal and 2) euglycemia/insulin infusion. Whole-body glucose and protein metabolism were measured with tracers, indirect calorimetry was performed, blood samples were drawn and muscle and adipose tissue biopsies were obtained. Mean plasma glucose ± SEM differed signifi cantly (6.3±0.2 vs. 10.7±0.7; p < 0.001) in the two situations.There was no signifi cant difference in protein turnover, protein synthesis, amino acid degradation, and urea nitrogen secretion rate. The rate of isotopically determined total glucose turnover tended to be increased during eu/hyperglycemia (1.7 vs 1.93; p = 0.07). Oxidation rates of lipid, glucose and protein did not change signifi cantly. When comparing biopsies a signifi cantly increased phosphorylation of both AS160 (p = 0.02) and Akt (p = 0.03) in skeletal muscle in the euglycemic situation was found. Circulating levels of IL-1β, IL-10, IL-6, GM-CSF, IL-5, IFN-γ, TNF-α, IL-2, IL-4 and IL-8 did not differ signifi cantly between the two situations.

Our fi ndings indicate that the low grade infl ammation associated with type 2 diabetes is not worsened signifi cantly by acute hyperglycemia, but is more likely to be the result of chronic hyperglycemia or of a primary abnormality. Moreover, short term hyperglycemia does not affect substrate metabolism signifi cantly.

1784-PLipid Overload Impairs Autophagic Flux in Cardiomyocytes Inde-pendently of mTOR SignalingBHARAT JAISHY, E. DALE ABEL, Salt Lake City, UT

The regulation of autophagy in obesity is incompletely understood. We examined the relationship between cardiac mTOR signaling and autophagy in high fat fed (HFF) mice and in palmitate treated H9C2 cells. Eight-week-old male mice were fed a high-fat diet (HFD, 45% fat) or control chow (CNC) for 12 wks. Autophagosome turnover in mice was blocked by injecting chloroquine (CQ, 50 mg/kg). H9C2 cells were treated with vehicle or 500 µM Palmitate (Pa) for 4 h ± 80 µM CQ for the last 1 h. Autophagosomes were quantifi ed by counting mCherry-positive puncta in heart sections of mCherry-LC3 transgenic mice or as GFP-positive puncta in Ad-GFP-LC3 expressing H9C2 cells. LC3-II levels were determined by immunoblot. Concentrations of branch chain amino acid (BCAA) in the heart were measured by GC-MS. HFF increased LC3-II levels (1.4 fold vs. CNC, p<0.05, n=4-5) and increased the number of mCherry puncta. In parallel, mTOR signaling was reduced as evidenced by reduced phosphorylation of mTOR (0.57 fold vs. CNC, p<0.05) and its targets Ulk1, S6, and 4E-BP1γ (0.6, 0.4, 0.6 fold lower respectively, p<0.05) in concert with decreased levels of BCAAs: valine, leucine and isoleucine (0.67, 0.57, 0.35 fold lower respectively, p<0.05). Administration of BCAA by I.P. injection increased cardiac mTOR signaling in HFF mice (pS6=2.5 fold vs. Saline-HFD) but did not reduce LC3-II levels. In H9C2 cells, Pa treatment signifi cantly increased LC3-II level (2.5 fold vs. veh, p<0.05, n=6) and the number of GFP puncta (4 fold vs. veh, p<0.05), without altering mTOR signaling. CQ treatment increased LC3II level in CNC by 2-fold versus only 28% in HFF mice. Likewise, CQ increased autophagosome abundance by 3.4 fold (LC3-II level) and 16 fold (GFP-puncta) in control cells but only by 1.8 fold and 2.3 fold, respectively, in Pa

treated cells. These data suggest that HFD or palmitate treatment increases autophagosome abundance in cardiomyocytes by impairing autophagosome turnover via mechanisms that are independent of mTOR signaling.

Supported by: NIH

1785-PTime-of-Day-Dependent Regulation of the Metabolic Modulator TRIB3: Infl uence of the Cardiac Circadian ClockWEI ZHANG, LING TIAN, DENNIS STEVERSON, MARTIN E. YOUNG, W. TIMOTHY GARVEY, Birmingham, AL

Background: We have reported that TRIB3 acts to diminish tissue glucose transport (Liu AJP 2010). Also, during fasting TRIB3 is decreased in skeletal muscle and increased in adipose tissue, while high fat diet had opposite effects. These data suggest that TRIB3 partitions fuel utilization from adipose tissue towards muscle during fasting, and away from muscle and towards adipose during nutrient excess (Zhang AJP 2011). Whether TRIB3 coordinates fuel homeostasis over the course of a day is unknown.

Hypothesis: TRIB3 expression is altered as a function of time-of-day in metabolically active tissues.

Results: We measured TRIB3 expression in heart, liver, muscle, and adipose isolated from mice at 6 hr intervals (12 hr light and dark cycles). In the heart, TRIB3 increased 3-4 fold during the light period (non-feeding, 6am-6pm), and decreased during the night (feeding, 6pm-6am). This pattern was similar in adipose tissue. In contrast, TRIB3 oscillations were phase shifted in liver and muscle, with peak expression during the dark-to-light (fed-to-fasted) transition. To determine whether time-of-day-dependent oscillations were mediated, at least in part, by the cell autonomous circadian clock, mice expressing a dominant negative CLOCK in cardiomyocytes (CCM mice) were studied. Hearts from CCM mice exhibited an altered time-of-day-dependent pattern of TRIB3 expression, with increased levels over 24 hrs, suggesting that TRIB3 is regulated by the cardiomyocyte circadian clock.

Conclusion: (i) TRIB3 diurnally regulates glucose uptake and substrate metabolism in metabolically active tissues; (ii) TRIB3 expression oscillates in a time-of-day-dependent manner with tissue-specifi c patterns; and (iii) TRIB3 oscillations in the heart are dependent on the cardiomyocyte circadian clock. These observations indicate that TRIB3 diurnally coordinates whole body metabolic homeostasis during normal feeding and fasting via tissue-specifi c regulation of fuel utilization.

Supported by: NIH; U.S. Dept. of Veterans Affairs

1786-PDistinct Roles of Saturated (SFA) and Mono-Unsaturated Fatty Ac-ids (MUFA) in Impairment of Insulin-Stimulated Akt Phosphoryla-tion in HepatocytesASHIS K. MONDAL, NEERAJ K. SHARMA, SWAPAN K. DAS, Winston-Salem, NC

Elevated plasma free fatty acid (FFA) concentrations correlate with obesity and Type 2 diabetes (T2D). Diets enriched in SFA are associated with increased risk of T2D. To identify the molecular mechanisms that may be directly involved in FFA-mediated insulin resistance in liver, we tested the effect of different concentrations and proportions of palmitic acid (PA, C16:0) and oleic acid (OA, C18:1), using the human hepatocyte cell line HepG2 in vitro.

PA exposure (0.5-0.7mM) triggered an endoplasmic reticulum (ER) stress response, as indicated by an increase in IRE1, spliced XBP1, CHOP, GADD34 and BiP transcripts and eIF2α-pSer52. Despite the strong induction of JNK and c-JUN phosphorylation and ER stress activation, short-term PA exposure (6 hrs) only marginally (12-22%) repressed the insulin-stimulated Akt-ser473 phosphorylation (ISAP), but longer (16 hrs) exposure caused a signifi cant dose-dependent repression (38-73%, P= 0.014-8.0X10-9). Exposures to 0.7mM PA and OA mixtures in 1:1 and 1:2 ratios did not affect ISAP or induce ER stress and JNK. Surprisingly, similar exposure to OA alone enhanced ISAP compared to controls (94%, P=0.000002). A PI3K inhibitor (LY294002, 50µM) abrogated the protective effect of OA on ISAP. Induction of the FA β-oxidation enzyme CPT1A was similar at 6 hrs but was higher for PA vs OA (4 vs. 2.4 fold) at 16 hrs. But two CPT1A inhibitors (etomoxir and Br-PA) failed to ameliorate PA-mediated stress. In contrast to published studies on adipocytes, JNK (SP600125, 50µM) or Src-kinase (PP2, 15µM) inhibitors did not rescue the PA-mediated repression of ISAP in HepG2 cells. In concordance with the insulin-resistant phenotype of liver-JNK1-/- mice, we observed a signifi cant reduction of ISAP by SP600125.

Our study indicates that (a) FFA-mediated cellular responses are distinct and cell type-specifi c for SFA, and (b) reductions of ISAP by SFA in human hepatocytes are not mediated by JNK activation, but may involve PI3K.

Supported by: NIH

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INSULIN ACTION—GLUCOSE TRANSPORT AND INSULIN RESISTANCE IN VITRO


1787-PNovel Agonists of the NR4A3 Orphan Nuclear Receptor Augment Glucose Transport System Activity in Skeletal Muscle Cells and Lower Blood Glucose in db/db MiceQINGLAN LIU, WEI ZHANG, YUCHANG FU, W. TIMOTHY GARVEY, Birmingham, AL

We have previously reported that the orphan nuclear receptor NR4A3 enhances insulin sensitivity and glucose transport activity. Thus, NR4A3 is a potential drug target for treatment of diseases characterized by insulin resistance, and our studies have provided rationale for screening combinatorial libraries to identify small molecule agonists. To this end, we have designed a mammalian cell-based “one-hybrid” luciferase assay system for high-throughput screening of potential NR4A3 activating ligands. We screened 130K compounds, received 1,600 specifi c hits (Av + 3SD) (Hit rate 1.28%), and identifi ed 430 compounds that met criteria for high activity. From these compounds, 33 top hits were selected through a cheminformatic analysis that identifi ed unique chemical scaffolds for secondary testing. We observed that: (i) In Vitro: three compounds in particular (hits #12, #14, #15) markedly increased glucose transport activity by 1.5-2.6 fold (P<0.05), GLUT4 translocation (P<0.05), and AKT phosphorylation in cultured L6 muscle cells in a NR4A3-dependent manner, (ii) In Vivo: treatment of hyperglycemic db/db mice using single IP injections of these 3 compounds substantially reduced blood glucose for >24 hours. Further studies demonstrated that these treatments signifi cantly improved both glucose tolerance over 30-150 min (P<0.01) and insulin sensitivity by insulin tolerance test. In conclusion: (i) we have identifi ed novel small molecule agonists for the NR4A3 orphan nuclear receptor; (ii) these agonists augment glucose transport and GLUT4 translocation in cultured muscle cells in a NR4A3-dependent manner, and administration to intact db/db mice improves systemic glucose tolerance and insulin sensitivity; (iii) these fi ndings provide proof-of-principle for NR4A3 as a novel therapeutic target, and for the development of agonists to treat insulin resistance in patients with Metabolic Syndrome and Type 2 Diabetes.

Supported by: NIH (DK038746), (DK083562); U.S. Dept. of Veterans Affairs (P60DK079626)

1788-PRole of Cholesterol Synthesis in Brain Glia in Diabetes Can Affect Function Independent of Changes in Glucose MetabolismHEATHER FERRIS, C. RONALD KAHN, Boston, MA

As people with diabetes live longer, healthier lives there has been an increasing recognition that there is an increase in the incidence of cognitive decline and Alzheimer’s in this population. Our previous studies have demonstrated that in mouse models of both type 1 and type 2 diabetes there is a decline in cholesterol synthesis in the brain, and that reduction can lead to impaired synapse and synaptosome formation. Diabetes is a complex disorder with both changes in glucose and lipid metabolism, and both glia and neurons are capable of cholesterol synthesis and altered glucose metabolism. Therefore, to understand the relative role of cholesterol metabolism in regulation of brain function independent of glycemia, we knocked out SREBP2, the key transcriptional regulator of cholesterol synthesis, selectively in glial cells by breeding SREBP2-fl oxed mice to mice carrying a glial specifi c GFAP-Cre. The resultant SREBP2GFAP

mice were born at normal Mendelian ratios with normal fertility, however, the brains of SREBP2GFAP-/- mice were 15% smaller than those of controls. They also had 15% less cholesterol per mg of brain tissue, without change in triglyceride content. Despite the knockout of SREBP2 in glia, there was no difference in mRNA levels in the whole brain of several enzymes in the cholesterol synthetic pathway that are regulated by SREBP2, and there was actually an increase in total brain SREBP2 protein, suggesting that neurons attempt to compensate for the decreased production of cholesterol in glia by overproducing SREBP2. Nonetheless, SREBP2GFAP-/- mice perform abnormally on a balance beam test, and take longer to cross the beam and have more foot slips than controls, both measures of abnormal neuronal control and/or peripheral neuropathy. These data indicate that changes in cholesterol production in brain glia that occur in animal models of diabetes may directly impact neurological function independent of the effects of glycemia on brain function.

Supported by: NIH (1K08DK097293-01)

1789-PInhibition of NF-kB Activity in the Liver by High Density Lipopro-teins Affords Protection Against Diet-Induced Insulin ResistanceKRISTINE C.Y. MCGRATH, XIAOHONG LI, ALISON K. HEATHER, Sydney, Australia

Obesity-induced infl ammation is most likely to occur in adipose tissue, liver and muscle. Studies have shown that infl ammation of the liver alone is enough to drive insulin resistance. Insulin resistance is associated with low high density lipoprotein (HDL) levels. HDL have anti-infl ammatory properties so it is compelling to hypothesize that low levels of HDL perpetuate infl ammatory responses in the liver that drive insulin resistance and that HDL treatment will suppress liver infl ammation and suppress insulin resistance. The aim of our study was to determine whether reconstituted HDL (rHDL) treatment can decrease liver infl ammation and improve insulin sensitivity in a mouse model of insulin resistance.

To induce insulin resistance, C57BL/6 mice were fed a high-fat diet (HFD) for 16 weeks. A subset of HFD mice were randomised to receive lipid-free AI injection 24 hours, 2- or 4-weeks prior to sacrifi ce. In vivo and in vitro approaches were used to determine the effects of apoA-I rHDL. Treatment for 2- or 4- weeks with lipid free apo A-I (8mg/kg) resulted in: (i) improved insulin sensitivity that was associated with decreased systemic and hepatic infl ammation; (ii) suppression of hepatic mRNA expression for the rate-limiting enzymes in gluconeogenesis and lipogenesis (PEPCK, G6Pase and SREBP1c) and; (iii) suppression of hepatic NF-κB activation. At the cellular level, cultured human hepatocytes exposed to apoA-I rHDL (16µM) or PBS (control) for 16 hrs before activation with TNFα (1ng/ml; 5 hrs) resulted in suppression of the NF-κB classical signalling pathway that correlated with suppression of NF-κB target gene expression.

In conclusion, our study show that HDL reverses insulin resistance in the HFD C57BL/6 mouse model, an effect mediated by suppression of the key infl ammatory mediator, NF-κB. These fi ndings may open new therapeutic options for a HDL-based treatment of insulin resistance.

1790-PKnockdown of Protein Phosphatase 1 Regulatory Subunit Nega-tively Affects Insulin ActionXIANGMIN ZHANG, DANJUN MA, MICHAEL CARUSO, MONIQUE LEWIS, OGNIAN IKONOMOV, ASSIA SHISHEVA, ZHENGPING YI, Detroit, MI

Extensive research has been carried out to study the role of kinases in insulin action. However, a mechanism for serine/threonine phosphatase action in insulin signaling is largely unknown. Protein Phosphatase 1 Regulatory Subunit 12A (PPP1R12A) of serine/threonine Protein Phosphatase 1 (PP1) binds to the delta isoform of PP1 catalytic subunit (PP1cdelta) to modulate the dephosphorylation activity of this holoenzyme towards its substrates. To determine the role PPP1R12A plays in insulin signal transduction, we constructed lentiviruses expressing shRNAs targeting PPP1R12A, which showed effi cient knock-down of PPP1R12A protein level in L6 rat skeletal muscle cells. Insulin stimulated IRS-1/p85 association transmits insulin signal downstream. To assess the effect of PPP1R12A knock-down on IRS-1/p85 association, L6 myotubes were transduced with lentiviruses encoding non-targeting scramble control shRNA or shRNA targeting PPP1R12A, respectively. 3 days after infection, cells were serum-starved and treated with/without 100nm insulin for 15min. IRS-1 was immunoprecipitated and IRS-1/p85 association was determined by the proteomics approach developed in our laboratory. The results indicated that either basal or insulin stimulated IRS-1/p85α association and IRS-1/p85β association were signifi cantly reduced to approximately half after PPP1R12A knock-down. Furthermore, to determine the effect of PPP1R12A knock-down on glucose uptake, we transduced L6 myoblasts stably overexpressing GLUT4 (L6 GLUT4myc) with shRNA lentiviruses. The results indicated that after PPP1R12A knock-down, glucose uptake was signifi cantly decreased to about half compared to scramble controls under both basal and insulin-stimulated conditions. In summary, PPP1R12A protein knock-down negatively affects insulin action, and led to reduced IRS-1/p85 association and impaired glucose uptake in L6 cells, suggesting that PPP1R12A may play a positive role in insulin signaling and glucose uptake.


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1791-PIdentifi cation of Regulatory Nodes in Glut4 Traffi ckingPAUL DUFFIELD BREWER, IRINA ROMENSKAIA, ESTIFANOS N. HABTEMICHAEL, ADELLE COSTER, CYNTHIA CORLEY MASTICK, Reno, NV, New Haven, CT, Sydney, Australia

In differentiated adipocytes, insulin increases cell surface Glut4 10-25-fold. In contrast, insulin has only a modest (1.5-fold) effect on surface Glut4 in undifferentiated preadipocytes (fi broblasts). To identify steps that account for the highly-insulin responsive traffi cking of Glut4, the effect of protein expression/knockdown on the kinetics of HA-Glut4/GFP traffi cking were measured in fi broblasts and adipocytes. These data were compared to effects on the kinetics of traffi cking of two endosomal proteins: the transferrin receptor (TfR) and LRP1. The TfR and Glut4 traffi c through kinetically distinct pathways in both fi broblasts and adipocytes, with the TfR cycling signifi cantly faster than Glut4 in both cell types. Interestingly, LRP1 traffi cs with kinetics similar to the TfR in fi broblasts, but with kinetics similar to Glut4 in adipocytes. This is consistent with the high degree of overlap between Glut4 and LRP1 that has been observed in adipocytes. Six steps account for the specialized traffi cking of Glut4: 1) endocytosis, 2) sorting/recycling from endosomes back to the cell surface (constitutive pathway), 3) rescue from degradation, 4) sequestration into GSVs (regulated pathway), 5) release from retention in GSVs, and 6) fusion of GSVs to the plasma membrane. The effects of treatments/proteins on each step were mapped by fi tting the data to minimal mathematical models. Using this approach, the sites of action of AS160, Rab10, Rab14, Sortilin, CDP138, Munc18c, and Syntaxin 4 were identifi ed.

1792-PFatty Acids Mediate AMPK Expression in Skeletal Muscle by a Mechanism Involving Protein Kinase CKIMBERLY A. COUGHLAN, THOMAS W. BALON, RUDY J. VALENTINE, NEIL B. RUDERMAN, ASISH K. SAHA, Boston, MA

Chronic exposure to fatty acids (FA) is known to lead to accumulation of DAG and ceramides and the development of insulin resistance in muscle. DAG is involved in activation of novel PKC isoforms, which have also been implicated to be involved in the development of insulin resistance. The purpose of this study was to investigate the effects of FA on PKC activation in myotubes and the timing of the development of insulin resistance. C2C12 myotubes were incubated with varying concentrations of palmitic or oleic acids (100 - 700µM) for a range of time periods (15min - 24h). Cell lysates were separated into membrane and cytosolic fractions to evaluate membrane association of various PKC isoforms by Western blot as an indicator of activation. Additionally, rat EDL muscle was incubated with 550µM palmitate for 1h and homogenates were fractionated in the same

way. The C2C12 cells showed a marked increase in membrane associated PKCε following 24h with 700µM palmitate. Interestingly, addition of 10mU insulin for the fi nal 10min prevented this increase. Similarly, in rat EDL, PKCε was increased in the membrane fraction following 1h with 550µM palmitate, but 10min with insulin prevented this increase. In contrast, palmitate decreased AMPK activation (Thr172 phosphorylation) at 24h in these cells, while it was increased in the EDL muscles after 1h. This disparity is likely due to the differences in time of incubation and concentrations of FA. After 2h, both palmitate and oleate increased AMPK activation in C2C12 cells. 400µM oleate decreased PKCε activation at 15min, but increased it at 30 and 2h. 400µM palmitate also decreased PKCε activation at 15min, as well as after 30min and 2h. These results suggest that short-term incubation with FA mimics the postprandial state, while long-term incubation (24h) mimics chronic overfeeding, leading to the development of insulin resistance.

1793-PUsing Mesenchymal Progenitor Cells to Dissect the Impact of Ge-netic Insulin Resistance on Cellular MetabolismBHARTI BALHARA, ALISON BURKART, YOUN-KYOUNG LEE, KRIS KRIAUCIU-NAS, SALVATORE IOVINO, CHAD COWAN, C. RONALD KAHN, MARY-ELIZABETH PATTI, Boston, MA

Inherited syndromes of insulin resistance, although rare, have provided insight into mechanisms of genetically determined insulin resistance. One example is Donohue syndrome (leprechaunism), due to mutations in the insulin receptor (INSR) gene. To identify molecular defects contributing to metabolic dysregulation in this syndrome, we generated mesenchymal progenitor cells (MPC) from induced pluripotent stem (iPS) cells, derived from control and patient fi broblasts.

Insulin signaling was assessed in MPC derived from a 6 week old female with Donohue syndrome (Minn1) and a healthy control (newborn male). MPC were stimulated with insulin (0-100nM) for 10 minutes, and protein and RNA extracted. Minn1 MPC showed a signifi cant decrease in mRNA expression of INSR (68.5%; p<0.05, n=3) and an 18% reduction in INSR protein (p<0.01, n=3) but similar IGF1R mRNA and protein levels. Minn1 MPC are responsive to insulin (100 nM), as indicated by western blot demonstrating phosphorylation of IRS-1 and AKT (1.5 & 1.4 fold respectively as compared to basal, n=4), patterns that did not differ in amplitude from control MPC. However, Minn1 MPC showed an 11% reduction in ERK phosphorylation (p=0.05, n=3) in response to insulin. Metabolic gene expression by qRT-PCR revealed downregulation of HK1 (↓42%; p=0.02) and similar trend for GLUT4 (↓55%; p=0.08). In contrast, genes regulating mitochondrial function, including PGC1α and Tfam showed an upward trend. Bioenergetic analysis (Seahorse XF24) indicated that Minn1 MPC had higher basal OCR (↑63%; p=0.05) and increased ECAR (↑67%; p=0.03).

Together our data show that Minn1 MPC maintain downstream INSR signaling, suggesting that IGF1 receptor signaling may compensate in part for INSR mutations. However, alterations in receptor expression or pathway-specifi c defects in insulin signaling, even in undifferentiated cells, can disrupt the regulation of cellular metabolism, potentially via transcriptional mechanisms.

Supported by: HSCI; PES

1794-PIsoform-Selective Inhibition of Facilitative Glucose Transporters: Elucidation of the Molecular Mechanism of HIV Protease Inhibitor BindingRICHARD C. HRESKO, THOMAS KRAFT, ANATOLY TZEKOV, LAURA SILBERMANN, PAUL W. HRUZ, St. Louis, MO

Pharmacologic inhibitors of the HIV Protease (PI) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter GLUT4. Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, the ability of chimeric transporters containing transmembrane (TM) and cytosolic (Cyt) domains from GLUT1 and GLUT4 were generated and expressed in HEK293 cells. Structural integrity was confi rmed via measurement of ATB-BMPA labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose uptake. Indinavir inhibited ATB-BMPA binding to the cytosolic surface of GLUT4 in a concentration-dependent manner with a Ki of 7.4 µM. The fl ag-tagged photoactivatable peptide zHFFBpa-Ahx-Ahx-

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INSULIN ACTION—SIGNAL TRANSDUCTION, INSULIN, AND OTHER HORMONES

DYDDDDK (WUCC-2) similarly inhibited ATB-BMPA binding to GLUT4 with an IC50 of 1.1 ± 0.1 µM but had no effect on GLUT1. Indinavir and WUCC-2 blocked ATB-BMPA binding to a chimeric protein containing GLUT4 TM and GLUT1 Cyt domains whereas labeling of the converse chimeric transporter was unaffected by these compounds. To facilitate confi rmation of involved TM segments, GLUT4 was photolabeled with WUCC-2 and purifi ed via affi nity chromatography. Simulated docking of PIs to modeled GLUTs was also performed. Taken together, these data demonstrate that the PI-binding domain is located within the transmembrane domains of GLUT4 and provide insight into the structural contributions to isoform-selective transporter inactivation.

Supported by: NIH (DK064572); Children's Discovery Institute

1795-PInhibition of Ceramide Danger Signal Through the Activation of Se-rum and Glucocorticoid Inducible Kinase (SGK)-1DONATELLA PASTORE, FRANCESCA PACIFICI, FRANCESCA FERRELLI, BARBARA CAPUANI, ANDREA COPPOLA, SARA CARATELLI, ROBERTO ARRIGA, MARIA PAOLA CAPUTO, MARIA ELENA RINALDI, ALFONSO BELLIA, MASSIMO FED-ERICI, PAOLO SBRACCIA, GIUSEPPE SCONOCCHIA, GIULIA DONADEL, DAVIDE LAURO, Rome, Italy

Several studies have shown that ceramide production triggers cell apoptosis. We hypothesized that SGK-1, a Serine Threonine protein kinase, can also regulate endogenous ceramide dependent apoptosis, since SGK-1 activates cell survival pathways. To this aim, SGK-1 wild type (SGK-1 wt) or its dominant negative gene, (SGK-1dn) were stable transfected into different cell lines. Transfected cells were incubated with different stimuli to increase endogenous cell ceramides production like UV, TNF alpha and also with synthetic ceramides like C2-Ceramide. Apoptosis was confi rmed by DNA fragmentation, FACS analysis and caspases-3 activation. We found that over expression of SGK-1 wt in different cell lines reduced the susceptibility to the apoptotic process in response to different stimuli. Moreover, C2-Ceramide triggered cell apoptosis and signifi cantly increased SGK-1 protein expression and activity while inhibited AKT-1 activation. Upon activation with the indicated stimuli, HEK-293 SGK-1 wt had a statistically signifi cant reduction of apoptotic levels than that observed in HEK-293 SGK-1 dn. To study the specifi c anti-apoptotic role of SGK-1, we used SGK-1 inhibitor, GSK 650394, that enhanced TNF-alpha-dependent HEK-293 SGK-1 wt and SGK-1 dn cells apoptosis. To investigate which was the apoptotic pathways involved in these processes, we analyzed if the activation of caspases and in particular of caspases-3-was affected using Z-VAD-FMK or Z-DEVD, general caspases and caspases-3 inhibitors, respectively. To study which the molecular pathways were involved in regulating C2-Ceramide induced apoptosis after the induction of SGK-1 over expression, we utilized specifi c metabolic inhibitors of p38MAPK, cAMP/PKA and PI3K. We found that the inhibition of the indicated kinases induced a preferential reduction of SGK-1 expression in cell over-expressing SGK-1 wt.

In conclusion SGK-1 may represent a target molecule to defence cell against sphingolipid damage.

Supported by: Fondazione Roma


Guided Audio Tour: Insulin Signaling and Action (Posters: 1796-P to 1802-P), see page 15.

& 1796-PChronic Exenatide Infusion Ameliorates Insulin Signaling in Skel-etal Muscle and Liver of Partially Pancreatectomized BaboonsTERESA VANESSA FIORENTINO, FRANCESCA CASIRAGHI, ANA M. PAEZ, SUB-HASH KAMATH, ALBERTO DAVALLI, GREGORY ABRAHAMIAN, GIUSEPPE DAN-IELE, FRANCESCO ANDREOZZI, ANTHONY COMMUZZIE, EDWARD DICK, RALPH A. DEFRONZO, GLENN HALFF, FRANCO FOLLI, San Antonio, TX, Milan, Italy

The GLP-1 analogue exenatide (Exe) is known to improve glycemic control in T2DM enhancing insulin secretion. We aimed to evaluate the effect of Exe on insulin sensitivity and signaling in non-human primates (baboons) underwent partial pancreatectomy. To this end 24 baboons, after partial pancreatectomy, were randomized to either Exe (0.014 ug/kg.h, n=12) or Saline (Sal, n=12) iv infusion for 14 weeks. Liver and skeletal muscle (SKLM) samples were obtained at the end of the 2nd hyperglycemic clamp (i.e. under maximal insulin stimulation), and insulin signaling cascade components

were analyzed by Western Blot. We observed a signifi cant 41% increase of M/I after Exe treatment (p=0.01) and a 19% decrease in Sal treated baboons (p=0.68). Interestingly, the improvement in whole body insulin sensitivity was associated to an amelioration of insulin signaling in liver and SKLM in Exe treated animals. A signifi cant 25% increase in p-Akt ser473 levels was observed in SKLM of baboons treated with Exe in comparison to Sal (p=0.03). This was associated to an increase of IR expression by 13% (p=0.26) and a 23% decrease of the insulin signaling inhibitor PTEN expression (p=0.07) and the stress kinase JNK phosphorylation (p=0.25). In addition, in liver of EXE treated baboons we observed a signifi cant 45% decrease in p-ERK levels (p=0.03), reduced levels of p-JNK and PHLPP [14% (p=0.3) and 10% decrease (p=0.1), respectively] which are known inhibitors of insulin signaling, as well as a 14% increase of p-Akt ser473 levels (p=0.1). These data demonstrate that Exe, in addition to improve β cell function, increases insulin sensitivity, possibly by improving insulin signaling in key target tissues such as liver and SKLM in non-human primates.

Supported by: NIH (R01080148)

& 1797-PInterleukin-10 Attenuates Aging-Associated Insulin Resistance in MiceSEZIN DAGDEVIREN, EUNJUNG LEE, XIAODI HU, YOSHIHIRO AZUMA, NICHO-LAS TSITSILIANOS, ANDREW V. TSITSILIANOS, DUY A. TRAN, GEORGE H. TSOU-GRANIS, DAE YOUNG JUNG, KI WON LEE, JASON K. KIM, Worcester, MA, Seoul, Republic of Korea

Aging is associated with obesity, infl ammation, and insulin resistance. We have previously shown that anti-infl ammatory cytokine IL-10 prevents lipid-mediated insulin resistance. To determine the role of IL-10 in aging, we examined male wild-type (WT) and transgenic mice with muscle-specifi c overexpression of IL-10 (MCK-IL10) at 18 months of age (n=4~5/group). WT and MCK-IL10 mice showed similar body weights and whole body fat mass (Fig. 1). A hyperinsulinemic-euglycemic clamp was performed to measure insulin sensitivity in awake mice. During clamps, WT mice showed low glucose infusion rates (87±14 µmol/kg/min) to maintain euglycemia, suggesting insulin resistance, and these rates tended to be higher in MCK-IL10 mice (129±14 µmol/kg/min; P=0.1). Whole body glucose turnover was signifi cantly higher in MCK-IL10 mice compared to WT mice, indicating improved systemic insulin sensitivity in these mice (Fig. 2; P=0.009). Insulin-stimulated glucose uptake in skeletal muscle was increased by almost 2-fold in MCK-IL10 mice (Fig. 3; P=0.04). Insulin sensitivity was selectively improved in muscle because glucose uptake in white adipose tissue was not different between the groups (Fig. 4). These results indicate that muscle-selective expression of IL-10 attenuates aging-associated insulin resistance in skeletal muscle, and our fi ndings suggest a potential role of anti-infl ammatory therapy in aging-mediated metabolic abnormalities.

Supported by: NIH

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& 1798-PA Novel Insulin Signaling Cascade that Selectively Drives Hepatic Lipogenesis, Despite Persistently Active PTENXIANGDONG WU, KEVIN JON WILLIAMS, Philadelphia, PA

In type 2 diabetes (T2DM), obesity, and related syndromes, insulin still stimulates hepatic lipogenesis and hence fatty liver. Surprisingly, in these same syndromes, insulin fails to stimulate endothelial lipogenesis. Lipo-genesis in endothelium is benefi cial, because it mediates normal palmi toyla-tion of eNOS.

We recently reported that injection of insulin into T2DM db/db mice failed to inactivate hepatic PTEN, a crucial action of NOX4. Insulin-stimulated db/db livers produced an unusual monophosphorylated form of AKT at Thr308 (pT308-AKT) with only weak phosphorylation at Ser473. Importantly, pT308-AKT activated downstream lipogenic pathways, but failed to phosphorylate FOXO1 or regulate glucose handling. All of these features were recapitulated in cultured hepatocytes by knocking down or inhibiting NOX4.

Here, we examined the KKAy mouse, a hyperphagic T2DM model with an intact leptin-leptin receptor axis. Insulin injection likewise stimulated hepatic production of pT308-AKT, which activated downstream lipogenic targets but bypassed FOXO1. In endothelium-rich lung from the same animals, however, insulin failed to trigger normal phosphorylation of AKT at either T308 or S473 - or to activate downstream lipogenic targets.

We hypothesized a role for the class II PI3K, PI3K-C2γ. It is unique to liver; it responds to insulin; and it generates PtdIns3P and PtdIns(3,4)P2, which are poor substrates for destruction by persistent PTEN. Knock-down of PI3K-C2γ in NOX4-defi cient hepatocytes blocked insulin-stimulated formation of pT308-AKT and activation of downstream lipogenic targets. The liver cells became like endothelium.

Our work identifi es a novel cascade from the insulin receptor, to PI3K-C2γ, to its unusual PtdIns products, to the Thr308 site on AKT, to selectively drive lipogenesis in T2DM liver, but not in endothelium. The cascade may explain fatty liver and dyslipoproteinemia, yet also poor endothelial production of NO and hence hypertension.

Supported by: Temple University

& 1799-PPTEN Upregulation May Explain Development of Insulin Resistance and New Onset Diabetes With High Dose StatinsYOCHAI BIRNBAUM, YUMEI YE, SHUKUAN LING, MANJYOT NANHWAN, JOSE R. PEREZ-POLO, MANDEEP BAJAJ, Houston, TX, Galveston, TX

Statin therapy may increase the incidence of new onset diabetes in patients. Studies in animal models have suggested that prolonged (>3d) statin therapy upregulates Phosphatase and Tensin Homologue on Chromosome 10 (PTEN) expression. This has been reported to offset the myocardial protection effects of statins against ischemia-reperfusion injury. PTEN levels are also elevated in skeletal muscles of animals with type 1 and type 2 diabetes, as well as the myocardium of diabetic patients. Increasing intracellular cAMP levels with subsequent activation of protein kinase A (PKA) has been reported to decrease PTEN expression. We assessed whether prolonged treatment with high dose statins induces insulin resistance or diabetes and upregulates PTEN in the skeletal muscle of rats receiving Western Diet and whether concomitant treatment with cilostazol (CIL, a phosphodiesterase-3 inhibitor that increases cAMP levels) will attenuate these effects. Rats received normal diet or Western diet without (control) or with rosuvastatin (ROS, 10 mg/kg/d), CIL (10 mg/kg/d), or ROS+CIL for 30 days. Fasting glucose, HbA1c and insulin were measured as well as PTEN, Ser-473 P-Akt and total Akt levels in skeletal muscle. Western diet alone caused an increase (p<0.05) in glucose, HbA1c and insulin. These levels were also signifi cantly higher (p<0.05) in the ROS group. Western diet alone increased PTEN expression and decreased P-Akt levels (p<0.05). Levels of PTEN were signifi cantly higher and P-Akt lower (p<0.05) in the ROS versus the control group (western diet). CIL normalized fasting glucose, HbA1c and insulin levels and attenuated the changes in PTEN and P-Akt concentrations in the ROS+CIL group (P<0.05 vs. controls). We conclude that long-term high dose statins can induce diabetes by upregulating PTEN that attenuates Akt activation. CIL attenuates these changes. Further studies are needed to assess the effects of increasing cAMP levels by CIL to prevent induction of diabetes by statins.

& 1800-PAS160 Phosphorylation in Human Skeletal Muscle Biopsies Ob-tained During Hyperinsulinemic Euglycemic Clamp StudiesZAHER MSALLATY, XIANGMIN ZHANG, MICHAEL CARUSO, DANJUN MA, MONIQUE K. LEWIS, WISSAM ANTEER, RODNEY BERRY, NOUR DABOUL, MI-CHAEL P. DIAMOND, ABDUL B. ABOU-SAMRA, GREGORY D. CARTEE, JEFFREY F. HOROWITZ, BERHANE SEYOUM, ZHENGPING YI, Detroit, MI, Ann Arbor, MI

Insulin resistance in skeletal muscle contributes to the development of type 2 diabetes. Phosphorylation of the 160 kDa Akt substrate (AS160, or TBC1D4) has been shown to be critical in regulating insulin action in skeletal muscle. A few AS160 phosphorylation sites showed reduced response to insulin in muscle from type 2 diabetics (T2D) compared to lean healthy controls (LC), while AS160 protein abundance was unchanged. Nonetheless, a comprehensive analysis on AS160 phosphorylation in LC, obese non-diabetic controls (OC), and T2D is lacking. We have applied the targeted proteomics approach for label-free protein phosphorylation quantifi cation developed in our laboratory to investigate AS160 phosphorylation in humans. Muscle biopsies were performed before and after hyperinsulinemic euglycemic clamp from 3 LC, 3 OC, and 3 T2D. Muscle protein lysates from the basal and 2h insulin infused conditions were subjected to AS160 immunoprecipitation and proteomics analysis. Twenty fi ve distinct AS160 phosphorylation sites were identifi ed, and among them, 7 (S108, S330, S694, S695, S698, T701, and S789) were not listed in 4 large phosphorylation site databases, and thus appeared to be novel. S108 and S330 are located within two phosphotyrosine interaction domains (PTB/PID) of AS160 (aa 53-209 and 312-468, respectively), and their phosphorylation might play a role in the interaction between AS160 and tyrosine phosphorylated signaling proteins. Another novel site S789 is close to the calmodulin-binding domain of AS160 and might regulate contraction-stimulated glucose transport. Data analysis is on-going to obtain quantitative information of identifi ed phosphorylation sites in OC and T2D. Furthermore, proteomics data acquisition on additional participants is being conducted. This study provides the largest in vivo phosphorylation map of AS160 in human muscle and provides novel targets to better understand the pathogenesis of type 2 diabetes.


& 1801-PAutophagy in Skeletal Muscle in Obesity and Type 2 DiabetesRIKKE K. HENRIKSEN, JONAS M. KRISTENSEN, STINE J. PETERSSON, BIRGITTE F. VIND, KURT HØJLUND, Odense, Denmark

Insulin resistance in obesity and type 2 diabetes (T2D) is associated with mitochondrial dysfunction and increased proteolysis in skeletal muscle. Autophagy is a catabolic process that maintains cellular homeostasis by degradation of proteins and organelles such as e.g. defective mitochondria. The role of autophagy in skeletal muscle insulin resistance remains to be determined. Here, we examined autophagy in skeletal muscle of patients with T2D and lean and obese controls.

Skeletal muscle biopsies were obtained from patients with T2D (n=10) and glucose tolerant lean (n=12) and obese (n=9) individuals before and after a 4-h euglycaemic-hyperinsulinemic clamp using physiological hyperinsulinemia. The expression of a selected set of genes involved in autophagy was examined by RT-qPCR, and abundance of proteins involved in autophagy as well as the LC3-I/LC3-II ratio, were determined by western blotting.

Muscle transcript levels of ULK1, ATG5, ATG7, ATG12, BECN1, BNIP3, BNIP3L, GABARAPL1, FOXO3A, MAP1LC3, SQSTM1, and PIK3C3 were similar in the lean, obese and T2D groups, and there were no differences in the protein abundance of ATG7, LC3, and BNIP3, and FOXO3A between the groups. Insulin decreased the mRNA expression of FOXO3A in lean controls (p=0.002) and patients with T2D (p=0.008) with a tendency (p=0.07) in obese controls. Moreover, insulin increased the LC3-I/LC3-II ratio by 2.5 fold in lean (p=0.0009), 1.8 fold in obese (p=0.03) controls, and with a tendency of 1.4 fold in patients with T2D (p=0.09), corresponding to inhibition of autophagy. However, the effect of insulin on the LC3-I/LC3-II ratio was decreased in patients with T2D when compared to lean controls (p=0.02).

Our results show that conditions such as obesity and T2D are not associated with abnormalities in autophagic markers in human skeletal muscle in the basal state. Furthermore, we show that physiological insulin concentrations regulate autophagy by decreasing the lipidation of LC3-I to LC3-II, an effect that appears to be impaired in patients with T2D.

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& 1802-PTen Nights of Exposure to Moderate Hypoxia Improves Insulin Sen-sitivity in Obese MenVIRGILE LECOULTRE, COURTNEY M. PETERSON, PHILIP J. EBENEZER, ELIZABETH A. FROST, JEFFREY D. COVINGTON, JEAN-MARC SCHWARZ, ERIC RAVUSSIN, Baton Rouge, LA, Vallejo, CA

It has been proposed that enlarged adipocytes become hypoxic in obesity, which triggers low-grade chronic infl ammation, thereby impairing insulin signaling and contributing to insulin resistance. On the other hand, exercise-induced hypoxia appears to promote glucose uptake and mitochondrial biogenesis in skeletal muscle and thus improve insulin sensitivity. Therefore, we hypothesized that exposure to moderate hypoxia will improve whole-body insulin sensitivity in obese humans, overriding any deleterious infl ammatory effects in adipose tissue. Eight non-diabetic male obese subjects (28 ± 3 years old, BMI 32.7 ± 3.5 kg/m2) were assessed before and after sleeping 10 nights (100 hours) in normobaric hypoxia (15 ± 0.5% O2, ca. 2400 meters elevation). At baseline and after 10 nights, insulin sensitivity was measured by a two-step hyperinsulinemic euglycemic clamp, and skeletal muscle and adipose tissue biopsies were obtained. After ten nights of mild hypoxia, fasting glucose dropped by 3.0 ± 4.2 mg/dL (p = 0.04). Glucose disposal rate (GDR) at low dose insulin infusion (20 mU/min/m2) tended to improve from 2.6 ± 1.4 to 3.2 ± 2.1 mg/min/kg (p = 0.09), whereas insulin-mediated suppression of endogenous glucose production (hepatic insulin sensitivity) increased by 8% (p = 0.007). Similarly, GDR at a high insulin infusion rate (80 mU/min/m2) increased from 8.3 ± 5.2 to 9.2 ± 4.6 mg/min/kg (p = 0.03). In an in vitro model, primary human skeletal muscle myotubes exposed to similar hypoxic conditions had improved basal- and insulin-stimulated glucose uptake. Unexpectedly, Akt phosphorylation (S478) did not increase. These data indicate for the fi rst time that short-term nightly exposure to moderate hypoxia improves whole-body and liver insulin sensitivity, through mechanisms probably unrelated to Akt phosphorylation. This may open a new therapeutic avenue to treat pre-diabetes.

Supported by: NIDDK (2P30DK072476)

1803-PSkeletal Muscle and Adipose Tissue Glycoprotein 130 Expression Is Associated With Insulin Resistance in HumansMONIKA KARCZEWSKA-KUPCZEWSKA, AGNIESZKA NIKOLAJUK, AGNIESZKA ADAMSKA, NATALIA MATULEWICZ, MAGDALENA STEFANOWICZ, IRINA KOW-ALSKA, MAREK STRACZKOWSKI, Bialystok, Poland, Olsztyn, Poland

Glycoprotein 130 (gp130) cytokines, like interleukin 6 (IL-6), act through plasma membrane receptors consisting of 2 glycoproteins: a cytokine binding subunit (like IL-6R) and gp130, responsible for signal transduction. IL-6 may regulate insulin sensitivity in a dual manner, as both insulin-sensitizing and -desensitizing actions have been reported. Other gp130 cytokines may have a benefi cial metabolic effects. The aim of the present study was to assess skeletal muscle and adipose tissue IL-6R and gp130 expression in relation to insulin sensitivity and obesity in male subjects. We examined 86 young (age 23.22±2.41 years), apparently healthy male subjects with normal glucose tolerance, 45 lean (BMI below 25 kg/m2) and 41 with overweight or obesity (BMI between 25 and 40 kg/m2). Euglycemic hyperinsulinemic clamp, indirect calorimetry and biopsies of vastus lateralis muscle and subcutaneous adipose tissue were performed. Tissue mRNA expression of IL-6R, gp130, suppressor of cytokine signaling 3 (SOCS3), AMP-activated protein kinase (AMPK) and nuclear factor κ B (NFκB) was analyzed with Real Time PCR. Obese subjects had higher adipose tissue gp130 expression (p=0.008). No difference in tissue IL-6R was found. Both muscle and adipose tissue gp130 expression was inversely related to insulin sensitivity (r=-0.32, p=0.004 and r=-0.28, p=0.035, respectively). Only adipose tissue gp130 was related to its serum soluble form (r=0.34, p=0.01). Muscle gp130 was related to lipid oxidation during the clamp (r=0.24, p=0.035) and muscle AMPK (r=0.48, p=0.042). Adipose tissue gp130 was associated with adipose tissue SOCS3 (r=0.60, p=0.007) and NFκB expression (r=0.49, p=0.035). Our data show that both muscle and adipose tissue gp130 expression is inversely related to insulin action. This association may be linked to lipid oxidation in muscle and infl ammation in adipose tissue.

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

1804-PANG II Receptor Blocker Azilsartan Ameliorates ANG II-Induced Insulin Resistance of Glucose Transport and Impaired Akt/AS160 Signaling in Rat Skeletal MuscleERIK J. HENRIKSEN, FERNANDO R. SANTOS, PAYAM HOOSHMAND, PARIA HOOSHMAND, IRINA MUGERFELD, ANNAYYA R. AROOR, GUIDO LASTRA, JAMES R. SOWERS, VINCENT G. DEMARCO, Tucson, AZ, Columbia, MO

Hypertension and insulin resistance induced by the renin-angiotensin system (RAS) can be ameliorated with angiotensin receptor (type 1) blockers (ARB). Azilsartan (AZL) is a newly introduced imidazole-based ARB antihypertensive medication, and its effectiveness in facilitating metabolic improvements in conditions of angiotensin II (Ang II)-associated insulin resistance is currently unknown. Therefore, the purpose of the present study was to assess the effects of chronic AZL treatment on glucose transport activity and key insulin signaling elements in ANG II-induced insulin-resistant skeletal muscle. Male Sprague-Dawley rats were treated for 8 weeks by subcutaneous osmotic mini-pump with or without ANG II (200 ng/kg/min) combined with either vehicle or a known blood pressure-lowering dose of AZL (1 mg/kg/day). ANG II induced signifi cant insulin resistance of glucose transport activity in soleus strips, and AZL co-treatment increased insulin-stimulated glucose transport activity by more than 50% (p<0.05). In red gastrocnemius muscle, AZL treatment of ANG II-infused animals was associated with signifi cant enhancements of critical insulin signaling factors, including phosphorylation of Akt (both Ser473 (80%) and Thr308 (23%)) and AS160 Thr642 (42%). Phosphorylation of AMPKα (Thr172) was also increased (66%) by AZL. Importantly, Thr389 phosphorylation of p70 S6K, a serine/threonine kinase known to negatively regulate insulin signaling, was decreased (51%) with AZL treatment. These data provide evidence that the novel ARB AZL can improve insulin action on glucose transport activity and key insulin signaling elements in ANG II-induced insulin-resistant skeletal muscle, possibly associated with enhanced AMPK activation and suppressed p70 S6K function. These results provide further support for targeting the RAS to improve both hemodynamic and metabolic regulation in conditions of insulin resistance.

Supported by: Takeda Pharmaceuticals

1805-PNitric Oxide (NO) Reverses the Impairment in Insulin Transport Seen With Insulin Resistance by Inhibition of PTP1B ActivityHONG WANG, AILEEN X. WANG, KEVIN AYLOR, EUGENE BARRETT, Charlottes-ville, VA

We previously found that inhibiting insulin signaling by wortmannin or PD98059 or inhibition of Src by PP1 or treatment with TNFα signifi cantly impaired endothelial cell (EC) insulin uptake. Insulin signaling is tightly controlled via a balanced regulation by protein tyrosine kinases and protein tyrosine phosphatase 1B (PTP1B), respectively. We examined whether NO can reverse the impairment in insulin trans-endothelial transport (TET) caused by inhibiting insulin action and tested the hypothesis that NO acts by enhancing protein S-nitrosylation (PSN). We used both cultured bovine aortic ECs and aortic ECs freshly harvested from rats and measured either 50 nM fl uoroisothiocyanate (FITC)-insulin uptake (the fi rst step of insulin TET) or insulin TET using 200 pM ([125I] TyrA14) with ECs grown in Transwell plates. We found that 0.3 µM sodium nitroprusside (SNP), a NO donor, strikingly enhanced both FITC-insulin uptake and [125I] TyrA14 insulin TET in ECs and reversed the impaired FITC-insulin uptake in the ECs pre-treated with wortmannin, PD98059, PP1 or TNFα. Neither SNP, insulin nor both changed intracellular cGMP levels and adding 8-bromo-cGMP did not affect FITC-insulin uptake. However, inhibition of PSN by siRNA knockdown of Txnip (a strong endogenous inhibitor of de-nitrosylases) almost completely eliminated SNP-enhanced FITC-insulin uptake. Furthermore, treatment with either insulin or SNP or both signifi cantly increased general PSN and inhibited PTP1B activity. Additionally, the increased S-nitrosothiol induced by SNP and/or insulin strikingly co-localized with PTP1B visualized by confocal imaging. Finally, SNP treatment signifi cantly increased insulin-stimulated Akt phosphorylation at Ser473. We conclude that NO can directly promote insulin transport via inhibiting PTP1B activity by enhancing its PSN and also suggest this as a potential site for diminishing pro-infl ammatory cytokine-induced insulin resistance.

Supported by: NIH

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1806-PInsulin-Stimulated Phosphorylation of Grb10 (Growth Factor Recep-tor-Bound Protein 10) by Mechanistic Target of Rapamycin (mTORC1) Mediates its Negative Feedback on Phosphatidylinositol 3-Kinase (PI3K)/Akt Signaling in Human Skeletal MuscleSERGIO BURGOS, STÉPHANIE CHEVALIER, JOSE A. MORAIS, MARIE LAMA-RCHE, ERROL B. MARLISS, Montreal, QC, Canada

Grb10 is a negative regulator of insulin signaling implicated in mTORC1-mediated negative feedback to PI3K/Akt signaling. We determined the function of Grb10 and its regulation by insulin in human skeletal muscle. We studied 8 healthy men (21.4±1.3 yr, 21.5±0.3 kg/m2) during a two-stage (2×3hr) pancreatic clamp, with octreotide suppression and basal glucagon and hGH replacement. Glycemia was clamped at ~5.5 mmol/L and branched-chain amino acids at ~350 µmol/L. Insulin was infused to maintain glycemia during the basal state and raised to peripheral postprandial levels during hyperinsulinemic state. Glucose turnover was measured by 3-3H-glucose infusion. Vastus lateralis muscle biopsies were taken immediately before then 30 and 120 min after the start of the hyperinsulinemic stage. Primary human skeletal muscle cells were isolated and differentiated into myotubes. Hyperinsulinemia was reached by 30 min (589.2±37.0 pmol/L) and maintained throughout (150-180 min, 551.2±23.2 pmol/L). Glucose disposal increased from 2.73±0.08 mg/kg/min at the basal state to 9.54±0.62 mg/kg/min at 120 min (P<0.00001) of hyperinsulinemia. Akt phosphorylation at Ser473 was maximal within 30 min and did not change at 120 min hyperinsulinemia. Ser476 phosphorylation of Grb10 (n=4) did not change by 30 min (2.36±0.91 vs. 2.14±0.68; NS), but nearly doubled by 120 min (4.16±1.14; P=0.067). In human myotubes, knockdown of Grb10 increased basal and insulin-stimulated Akt phosphorylation. Acute inhibition of mTORC1 by rapamycin eliminated basal and insulin-stimulated Grb10 phosphorylation, while enhancing insulin-stimulated phosphorylation of Akt. These results establish a physiological role for Grb10 as an mTORC1 target that mediates its inhibition of PI3K/Akt signaling in human skeletal muscle.

Supported by: CIHR

1807-PElevation of Circulating Free Fatty Acids Abolishes Down-Regula-tion of Skeletal Muscle Adiponectin Receptor 1 (AdipoR1) Expres-sion Caused by Insulin InfusionMAREK STRACZKOWSKI, MONIKA KARCZEWSKA-KUPCZEWSKA, AGNIESZKA ADAMSKA, MAGDALENA STEFANOWICZ, NATALIA MATULEWICZ, IRINA KOW-ALSKA, AGNIESZKA NIKOLAJUK, Bialystok, Poland, Olsztyn, Poland

Adiponectin is an adipocytokine with insulin-sensitizing and anti-infl ammatory properties. In skeletal muscle it increases fatty acid oxidation and insulin-stimulated glucose uptake through binding with adiponectin receptor 1 (AdipoR1). The aim of our study was to assess the effect of insulin and Intralipid/heparin infusions on muscle AdipoR1 expression in humans. Twenty healthy male subjects (age 25.2±3.2 years; BMI, 26.5±4.6 kg/m2) with normal glucose tolerance were studied. Six-hours euglycemic hyperinsulinemic clamp was performed 2 times: with and without concurrent Intralipid/heparin infusion. Indirect calorimetry was performed at baseline and every 2 hours of the clamp. Biopsy of vastus lateralis muscle was performed before and after each clamp. Muscle AdipoR1 and insulin receptor substrate 1 (IRS1) mRNA expression was analyzed with Real Time PCR. Intralipid/heparin infusion resulted in a decrease in insulin sensitivity by approx. 40% (p<0.0001). Serum adiponectin concentration decreased similarly during both clamp (approx. -25%, both p<0.001). Muscle AdipoR1 was positively related to serum adiponectin (r=0.46, p=0.046), insulin sensitivity (r=0.50, p=0.023), muscle IRS1 (r=0.55, p=0.014), but negatively to respiratory quotient in insulin-stimulated conditions (r=-0.45, p=0.046). Insulin infusion decreased muscle AdipoR1 expression by approx. 30% (p=0.006). This effect was almost completely abolished by concurrent Intralipid/heparin infusion (-7%, p=0.44; the difference between 2 experiments, p=0.027). Our data indicate that elevation of circulating free fatty acids abolishes down-regulation of muscle AdipoR1 expression caused by insulin, independently of the changes in serum adiponectin. This may be a potential compensatory mechanism for free fatty acid-induced insulin resistance.

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

1808-PDigenic Short Stature and Severe Insulin Resistance Associated With Heterozygous Mutations of IGF1R and INSR Genes: A Physi-ologic Role of IGF1R-INSR Hybrids in Longitudinal Growth?CARLO COLOMBO, LAURA PROIETTI PANNUNZI, VALERIA GRASSO, CRISTINA ROFANI, ORNELLA MASSA, MARIA GRAZIA DEIANA, CECILIA MOTTA, DOMENI-CO ACCILI, VINCENZO TOSCANO, FABRIZIO BARBETTI, Rome, Italy, New York, NY

Mature insulin and IGF1 receptors (INSR, IGF1R) are constituted by an alfa and a beta subunit. INSR and IGF1R intracellular beta subunits share about 80% of aminoacid sequence; extracellular alfa subunits are more diverse. INSR and IGF1R can form hybrids that bind IGF1 with higher affi nity than insulin.

Heterozygous, dominant-negative INSR mutations give rise to severe insulin resistance type A (SIR-A), characterized by hyperinsulinemia and hirsutism in adolescent females, while mutations of IGF1R can cause short stature. In two sisters of 21 and 16 years of age with SIR-A (post-load insulinemia 2100 and 1100 microU/ml, and severe hirsutism) and short stature (133 and 138 cm, respectively) we identifi ed the heterozygous INSR p.Gly1146Arg mutation, that was paternally inherited (father’s height=152 cm, poast-load insulin=692 microU/ml). This mutation was already described in a Japanese girl of normal stature with SIR-A. Screening of IGF1R gene revealed that both sisters bear the p.Val422Asp (PolyPhen2: damaging, score of 0.994) heterozygous mutation, inherited from their mother, 149 cm tall. A younger sister,12 y old, with normal INSR/IGF1R genotype, is 151 cm tall.

GFP-FOXO1 transfected in fi broblast of normal sister localized mainly in the cytoplasm and became more nuclear upon FCS starvation for 16 h. In contrast, almost no effect was observed after FCS starvation of fi broblast of the two affected sisters, with GFP-FOXO1 remaining mostly nuclear, even after incubation with high dose insulin+IGF1. RNA micro-array and real-time PCR analysis revealed that IGF2, along with other genes, was underexpressed in fi broblasts of the affected sisters as compared to the younger sister and the father. INSR and IGF1R expression was normal.

We speculate that short stature of these two girls it is likely due to reduced phosphrylation of INSR/IGF1R hybrids. Experiments are under way to verify our hypothesis.

1809-PProteomic Analysis of Insulin-Stimulated Insulin Receptor Sub-strate-1 Protein Interaction Partners in Human Skeletal Muscle and Type 2 DiabetesMICHAEL CARUSO, XIANGMIN ZHANG, DANJUN MA, MONIQUE LEWIS, WIS-SAM ANTEER, ZAHER MSALLATY, BERHANE SEYOUM, JEFFERY HOROWITZ, MICHAEL DIAMOND, ZHENGPING YI, Detroit, MI, Ann Arbor, MI

Insulin resistance and type 2 diabetes (T2D) are metabolic disorders characterized by diminished cellular uptake and utilization of glucose, particularly within skeletal muscle. Insulin receptor substrate-1 (IRS-1) is a keystone mediator necessary for proper downstream insulin signal transduction. Perturbations in protein-protein interaction involving IRS-1 may lead to the development of insulin resistance and T2D. Co-immunoprecipitation was coupled with label-free HPLC-ESI-Orbitrap tandem mass spectrometry (MS/MS) to identify and quantify endogenous protein interaction partners of IRS-1. Subjects were grouped according to BMI (lean, n=6; obese, n=6) or as T2D (n=5); muscle biopsies were analyzed under fasting (basal) conditions as well as at the end of 2-hour hyperinsulinemic euglycemic clamps. A large number of IRS-1 protein partners were identifi ed, including known as well as novel interaction partners, in the 3 groups. A putative increase in protein-protein interaction (n=6, p<0.05) was identifi ed between IRS-1 and p85α/β under insulin stimulation for lean and obese subjects, but was not signifi cantly increased in T2D subjects (confi rmed by immunoblot analysis). These data suggest the potential for several endogenous novel IRS-1 protein interaction partners in humans during fasting and insulin-stimulated conditions.


1810-PDysfunctional PKA Signaling in the Diabetic HeartKENNETH HUMPHRIES, LEE BOCKUS, Oklahoma City, OK

In the heart, cAMP-dependent protein kinase (PKA) is the primary effector of the adrenergic pathway, concertedly increasing contractility and metabolism. The diabetic heart is known to have contractile and metabolic dysfunction. In addition, there is documented chronic adrenergic stimulation as well as a highly oxidative environment resulting from excess production of free radicals. In rodent models of type 1 diabetes we have made the novel fi nding that both cardiac PKA content and total activity are

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reduced. This is manifested in differential PKA phosphorylation of cardiac proteins in the diabetic heart versus control mice. Given the central role in cardiac physiology, this decrease in PKA may be causally related to the cardiac dysfunction that arises from diabetes. We hypothesize that the chronic adrenergic stimulation and PKA activation that occurs with diabetes results in decreased functional PKA content by ROS-mediated degradation. To test this hypothesis, mechanistic studies have been undertaken in H9c2 cardiomyoblasts to determine how chronic stimulation and the cellular redox environment affects PKA content and signaling. We have found that chronic activation of PKA leads to an increased rate of turnover that is exacerbated by oxidizing conditions. In contrast, antioxidants signifi cantly attenuated the rate of PKA degradation. This increased rate of PKA turnover, evidenced by a decrease in total PKA content and activity, is also evident in control mice chronically stimulated with the adrenergic agonist isoproterenol. Continuing studies are determining how the in vivo redox environment affects this process. Results from this work will provide therapeutic insight in treating diabetic heart failure.

Supported by: P20RR024215

1811-PPioglitazone Increases Expression of Hepatic Insulin Degrading En-zyme in Diet-Induced Obese C57BL/6 MiceXIUQING WEI, ZHANGOU GAO, JIANPING YE, Baton Rouge, LA

It is generally accepted that a decrease in insulin production by pancreatic beta-cells is responsible for the reduced fasting plasma insulin in type 2 diabetic patients treated by pioglitazone, an insulin sensitizing medicine. It is not known if insulin clearance plays a role in the therapeutic activity of pioglitazone. In this study, we addressed this issue by examining insulin degrading enzyme (IDE) in the liver of type 2 diabetic mice treated by pioglitazone. C57BL/6 mice were fed a high fat diet for six month to induce type 2 diabetes. Pioglitazone (10 mg/kg BW) was administrated through dietary supplement for last 2 months in the 6 month feeding. The pioglitazone treatment reduced fasting insulin by 50% (2.3±0.76 nM vs 5.5±1.43 nM, p=0.001, n=10) and blood glucose by 20% (123.67±5.71 mg/dl vs 158.44±6.60 mg/dl, p=0.005). The treatment increased body weight modestly (54.8±1.15 gram vs 51.54±1.76 gram, p=0.07). IDE activity was determined in the liver for protein and mRNA by western blot and real-time RT-PCR. The treatment induced IDE protein by 1.6 fold (p=0.016) and mRNA by 0.6 fold (p=0.011) in the liver of treated mice. In Hepa 1C1C7 cells, a mouse hepatoma cell line, pioglitazone (5 µM) increased IDE protein at 2-4 hours of treatment. In summary, pioglitazone directly increased the hepatic IDE expression in liver of type 2 diabetic mice and in mouse hepatoma cells. These effects are associated with reduced circulating insulin in T2D mice. Given that IDE controls the rate of insulin clearance in the liver, this study suggests that an increase in IED activity may involve in the control of hyperinsulinemia by pioglitazone.

Supported by: NIH

1812-PShort-Term Caloric Restriction Differently Affects mRNA Expres-sion of Insulin Receptor Signaling Cascade in Subcutaneous Adi-pose Tissue of Obese Subjects With and Without Type 2 Diabetes MellitusMILOS MRAZ, ZDENKA LACINOVA, PETRA KAVALKOVA, JANA DRAPALOVA, PAVEL TRACHTA, MONIKA URBANOVA, MARTIN MATOULEK, STEPAN SVACINA, MARTIN HALUZIK, Prague, Czech Republic

Insulin resistance, one of the key mechanisms in the development of type 2 diabetes mellitus (T2DM), is mainly caused by downstream defects in signal transduction from the insulin receptor. The aim of our study was to analyze mRNA expression of members of the insulin receptor signaling cascade in adipose tissue of obese patients with and without T2DM undergoing short-term caloric restriction.

mRNA expression of 44 genes involved in insulin receptor signaling was assessed in subcutaneous adipose tissue (SAT) of 15 subjects with obesity and T2DM (T2DM group), 14 obese non-diabetic patients (OB group) and 14 healthy lean control subjects (C group) at baseline and after 2 weeks of very-low-calorie diet (VLCD, energy intake 2500 kJ/day). Adipose tissue samples were obtained using aspiration biopsy.

At baseline, SAT of both T2DM and OB groups showed decreased mRNA expression of most of the genes involved in insulin signaling including insulin receptor and insulin receptor substrates 1 and 2, GLUT-4 (glucose transporter 4), phosphatidylinositol triphosphate and Akt pathway and MAPK (mitogen-activated proteinkinase) pathway. Two weeks of VLCD reduced body weight in both cohorts (BMI 48.0±2.41 vs. 50.4±2.62, p<0.05 for T2DM and 48.8±2.7

vs. 50.7±2.53, p<0.05 for OB) and improved glycemia in T2DM group (7.6±0.7 vs. 10.6±1.1 mmol/l, p<0.05). In SAT of OB group VLCD increased mRNA expression of several key signal transducers including insulin receptor and mTOR (mammalian target of rapamycin), while no such effect could be seen in T2DM subjects.

We conclude that obesity and T2DM are accompanied by decreased mRNA expression of insulin signaling cascade members in subcutaneous adipose tissue. Short-term caloric restriction attenuates this decrease only in obese subjects without T2DM.

Supported by: RVO-VFN64165/2012; IGA13299-4; SVV264503

1813-PInsulin Glargine Does Not Increase Breast Cancer Growth in an Ani-mal ModelEMILY JANE GALLAGHER, AVIVA TOBIN-HESS, JEFFREY BLANK, NICHOLAS BUFFIN, ASHLEY POLIN, NORBERT TENNAGELS, ULRICH WERNER, DEREK LE-ROITH, New York, NY, Frankfurt, Germany

Individuals with type 2 diabetes are at greater risk of developing breast cancer. Of the contributing factors, endogenous hyperinsulinemia appears to play an important role. Some epidemiological studies suggest that exogenous insulin therapy may increase the risk of developing cancer in those with diabetes, while other studies report no increased risk. We previously found that rhIGF-1 and the insulin analog, AspB10 increase mammary tumor growth in a mouse model of insulin resistance and endogenous hyperinsulinemia. In this study, we aimed to determine whether insulin glargine and increased orthotopic mammary tumor growth in a mouse with insulin resistance and endogenous hyperinsulinemia (MKR mouse). 50,000 MVT1 (c-myc/vegf overexpressing) and 250,000 Met1 (polyoma virus middle T antigen expressing) cells were injected orthotopically into 8-10 week old MKR mice. Mice were divided into groups with equal tumor volume and treated with vehicle, rhIGF1 (1mg/kg) or glargine (12.5 U/kg) twice daily for 2 weeks. rhIGF1 treated mice had signifi cantly larger MVT1 (229.1±19.8mm3) and Met1 tumor volumes (246.9±18.8mm3) than the vehicle treated group (MVT1 121.99±9.6 mm3, p<0.05, Met1 162.4±14.1mm3, p<0.05). Tumor volumes in mice treated with insulin glargine did not differ from the vehicle treated groups and were signifi cantly lower than rhIGF1 treated groups. Ex-vivo analysis of protein lysates from MVT1 and Met1 tumors revealed that rhIGF1 treatment led to activation of the IGF1 receptor (IGF-1R), hybrid IGF-1R/insulin receptors (IR) and the Akt signaling pathway, with sustained activation of the receptors and Akt an hour after rhIGF1 injection. Treatment with glargine led to Akt phosphorylation that was reduced an hour after injection in both Met1 and MVT1 tumors. Our studies show that chronic administration of insulin glargine in an animal model of endogenous hyperinsulinemia did not increase the growth of orthotopic mammary tumors, an important fi nding for the management of patients with diabetes.

Supported by: Sanofi

1814-PExenatide, a GLP-1 Receptor Agonist, Activates Glucose Transport in L6 Muscle Cell Line by an AMPK-Dependent MechanismFRANCESCO ANDREOZZI, CECILIA NIGRO, GREGORY ALEXANDER RACITI, CLAU-DIA MIELE, FRANCO FOLLI, San Antonio, TX, Naples, Italy

Exenatide (EXE) is a GLP-1 analogue which acts on pancreatic beta cells potentiating glucose-induced insulin secretion in type 2 diabetes treatment. We explore the effects of EXE on L6 skeletal muscle myotubes as well as the existence of a cross-talk between insulin- and EXE- signalling pathways. 10-7 M EXE induced glucose uptake up to 48 h, being maximal after 20’ (2-deoxyglucose nmol/min/mg prot: basal 1.14±0.09; EXE 20’ 2.32±0.18; EXE 2h 2.12±0.27; EXE 4h 1.92±0.14; EXE 24h 2.22±0.15; EXE 48h 2.09±0.37; p<0.01) to an extent similar to insulin 100nM 30’ (2.84±0.17;p<0.001). Interestingly, EXE differently from insulin, was not able to induce IR beta subunit and IRS1 tyrosine phosphorylation, activation of AKT and GSK3beta defi ned by the phosphorylation at Ser473 and Ser21/9 respectively, as well as ERK1/2 and JNK1/2 phosphorylation. Thus, we hypothesized that an alternative pathway, such as AMPK, could be involved in EXE effect on glucose uptake. EXE acutely increased AMPKalpha phosphorylation at Thr172 by 2.5-fold compared to control (p<0.01). By contrast, insulin neither activated AMPK nor increased EXE effect on AMPK activation when cells were stimulated with both insulin and EXE.To clarify the role of AMPK in the effects of EXE on glucose uptake in L6 myotubes, cells were incubated either with LY294002 (LY), a PI 3-kinase inhibitor, or with tubercidin (Tub), an AMPK inhibitor.LY inhibited insulin but not EXE-induced glucose uptake (basal 1.23±0.12; Insulin 100nM 30’ 2.94±0.15; Insulin 100nM 30’ + LY 1.26±0.08; EXE 20’ 2.42±0.16; EXE 20’ +LY 2.13±0.17 nmol/min/mg prot, p<0.001), whilst Tub pre-treatment

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almost abolished EXE effect on glucose uptake (EXE 20’ 2.42±0.16; EXE 20’ + Tub 1.45±0.07; p<0.01) but decreased insulin-induced glucose uptake only by 20% (Insulin 100nM 30’ 2.94±0.15; Insulin 100nM 30’ + Tub 2.36±0.18, nmol/min/mg prot, p<0.05) In conclusion, Exenatide stimulates glucose uptake by activating AMPK, in L6 myotubes.

1815-PInhibitory Mechanism of TNF-α-Induced Serine Phosphorylation of IRS-1 at 636/639 Mediated by AMP-Activated Protein Kinase in 3T3-L1 AdipocytesKUMI SATOH, TOMOHITO SHIBATA, AKIRA TAKAGURI, KAZUO ICHIHARA, Otaru, Japan

Adenosine monophosphate-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and monitors cellular energy charge, acting as a “metabolic master switch” for regulating adenosine triphosphate concentrations in the face of stresses that reduce cellular energy levels. Although the activation of AMPK stimulated is known to contribute to the acceleration of insulin signaling, the mechanism by which AMPK regulates insulin signaling remains unclear.

Serine phosphorylation of insulin receptor substrate (IRS)-1 negatively regulates insulin signaling. Here we investigated the role of AMPK in serine phosphorylation of IRS-1 at 636/639, which is induced by tumor necrosis factor (TNF)-α in 3T3L1 adipocytes. We demonstrated that the AMPK activator 5-aminoimidazole-4-carboxamide-1-d-ribofuranoside (AICAR) signifi cantly inhibited the TNF-α-induced serine phosphorylation of IRS-1 at 636/639 by suppression of extracellular signal regulated kinase (ERK) phosphorylation but not c-Jun-NH2-terminal kinase (JNK) phosphorylation. In addition, AICAR stimulation resulted in enhanced interaction between ERK and MAP kinase phosphatase-4 (DUSP9/MKP-4) without affecting DUSP9/MPK4 mRNA synthesis. Moreover, intraperitoneal administration (0.25 g/kg) of AICAR to db/db mice improved blood glucose levels and inhibited the phosphorylation of ERK in adipose tissue. In conclusion, we propose a new mechanism in which AICAR suppresses TNF-α-induced serine phosphorylation of IRS-1 at 636/639 by enhancing the interaction between ERK and DUSP9/MKP-4. Taken together, these fi ndings provide evidence that AMPK plays a crucial role in improving of Type 2 diabetes.

1816-PDietary Carbohydrates are Not Necessary for HFD Induction of In-sulin Resistance but a Small Amount of it can Push the HFD-Induced Insulin Resistance to a Maximal LevelWENHONG CAO, Kannapolis, NC

It is clear that dietary compositions play a critical role in the development of insulin resistance. Both high fat diet (HFD) and high carbohydrate diet have been shown to be associated with insulin resistance. However, some key questions have not been answered and that is why all kinds of diets are out there now with mixed results. In this study, we addressed two key questions. First, are dietary carbohydrates (carbs) necessary for the HFD-induced insulin resistance? Second, how much dietary carb is too much or suffi cient to promote the HFD-induced insulin resistance? Our results show that after a 5-week HFD (58%) with no carbs, mice developed severe insulin resistance. Addition of 5% carbs to the HFD dramatically elevated insulin resistance level and 10% carbs in the HFD induced a maximal level of insulin resistance. The HFD-induced ectopic fat accumulation and lipogenesis in liver and skeletal muscle occurred in the absence of dietary carbs, but was dramatically enhanced by addition of carbs into the HFD. Similarly, the HFD-induced oxidative stress in liver and skeletal muscle occurred without dietary carbs, and addition of carbs into the HFD elevated oxidative stress dramatically. Finally, HFD containing no/little carbs increased expression of key gluconeogenic genes (PEPCK and glucose-6-phosphatase) in liver most dramatically compared to other HFD groups with 5-25.5% carbs. In cultured hepatocytes, our results show that fat is necessary for development of insulin resistance induced by chronic exposure to a pathological level of insulin. Together, our results conclude that: a) fat is necessary for development of insulin resistance induced by diet or chronic exposure to a pathological level of insulin; and b) dietary carbs are not necessary for HFD to induce insulin resistance but a small amount of carbs (10%) in HFD can induce a maximal level of insulin resistance.

Supported by: NIH

1817-PMicrorna-103 Mediated Pro-Infl ammatory Cytokine-Induced Inhibi-tion of Vascular Endothelial Insulin UptakeHONG WANG, KEVIN AYLOR, EUGENE BARRETT, Charlottesville, VA

Insulin’s trans-endothelial transport (TET) is a rate-limiting step for insulin action in muscle, and insulin delivery to muscle interstitium is delayed in insulin-resistant subjects. We previously reported that vascular endothelial cell (EC) insulin uptake (the fi rst step of insulin TET) required caveolin-1 for the formation of transporting caveolae. Knockdown or overexpression of caveolin-1 in ECs strikingly decreased or increased EC insulin uptake, respectively. TNFα or IL6 treatment signifi cantly inhibited caveolin-1 mRNA and protein expression and profoundly inhibited EC insulin uptake. However, the mechanism by which these pro-infl ammatory cytokines inhibit caveolin-1 expression is unknown. Emerging evidence suggests that microRNAs regulate many cellular processes including caveolin-1 expression. Here we examined whether TNFα or IL6 treatment affected microRNA-103 expression as upregulation of this microRNA has previously been reported to inhibit caveolin-1 expression by adipocytes. Bovine aorta ECs were cultured in basal medium with either 20ng/ml TNFα or IL6 or vehicle for 24 hr before enriched microRNA was extracted. cDNAs were reversely transcribed with TaqMan microRNA Reverse Transcription Kit and Real-time quantifi cation to measure microRNA-103 expression was performed with the TaqMan microRNA assay kit. U6 small nucleolar RNA was used as the housekeeping small reference gene. Data analysis was performed by the 2-ΔΔCt method. Compared to the vehicle control, IL6 treatment caused a two-fold upregulation of microRNA-103 (1 ± 0.14 vs. 2.03 ± 0.36, p=0.034); TNFα treatment caused even greater upregulation of microRNA-103 (1 ± 0.14 vs. 3.75 ± 0.24, p<0.001). We conclude that pro-infl ammatory cytokines TNFα or IL6 can regulate micro-RNA 103 expression in ECs. This may be one mechanism by which these cytokines modulate EC insulin uptake.

Supported by: NIH

1818-PHPLC-ESI-MS/MS Analysis of Insulin-Stimulated Akt2 Protein In-teraction Partners in L6 MyotubesMICHAEL CARUSO, DANJUN MA, XIANGMIN ZHANG, REBECCA TAGETT, SORIN DRAGHICI, ZHENGPING YI, Detroit, MI

Insulin resistance and Type 2 diabetes are characterized by an aberrant response in the insulin signaling network. The isoform-specifi c serine/threonine kinase Akt2 has been implicated to play a key role in insulin-stimulated skeletal muscle glucose uptake. Activation of Akt2 is regulated via phosphorylation -- notably Thr309 and Ser478 -- whereas dephosphorylation has been shown to decrease Akt2 activity. Insulin-stimulated L6 myotubes were used to examine Akt2 protein interaction partners utilizing co-immunoprecipitation coupled with HPLC-ESI-MS/MS analysis. The pull-down assay displayed specifi city for the Akt2 isoform; Akt1 and Akt3 were not detected. Nearly 100 proteins indicated a signifi cant difference (n=7; p<0.05) between basal and insulin-stimulated conditions. Of the potential insulin-responsive Akt2 interaction partners, several notable kinases and phosphatases were detected: protein kinase A (PKA); mitogen-activated protein kinase (MAPK) 1 and 3; 5’-AMP-activated protein kinase alpha 1 (AMPK); serine/threonine protein phosphatase alpha (PP1A); phosphatidylinositide phosphatase (SAC1); all of which decreased following insulin stimulation. The insulin-stimulated decrease in Akt2 kinase and phosphatase interaction partners may have the potential to mediate site-specifi c Akt2 phosphorylation affecting insulin signaling transduction, as well as alternative signaling pathways through Akt2 substrate interaction. These data suggest that several novel proteins may associate with endogenous Akt2 under basal, as well as insulin-stimulated conditions, and may provide further insight into the insulin signaling network.


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1819-PErythropoietin Improves Palmitate-Induced Hepatic Insulin Re-sistance Through PI3K/Akt Signalling and Infl ammation in HepG2 CellsHONG ZHANG, YAN BI, DALONG ZHU, Nanjing, China

Several studies have shown that insulin resistance in hemodialysis patients was ameliorated by erythropoietin (Epo) treatment, insulin resistance was also improved in diet-induced obese mice while Epo was over-expressed in muscle tissue, and mice lacking EpoR in non-haematopoietic tissue became obese and insulin-resistant with disruption of Epo signaling. However, the potential molecular mechanisms were still unresolved.

Hepatic insulin resistance was induced by addition of 0.25 mmol/l palmitate (Sigma) for 24 h to HepG2 cells. 5 or 10 U/ml Epo was administered beginning 1 h before incubation with palmitate.

The results showed that insulin-stimulated glycogen content and glucose disposal by Epo treatment were signifi cantly enhanced in insulin-resistant HepG2 cells. Epo improved insulin-induced increases in PI3K, AKT, GSK-3β and FOXO1 activity. Furthermore, PI3K inhibitors markedly blocked Epo-mediated increase in AKT, GSK-3β and FOXO1 phosphorylation. Meanwhile, the increase in p-IRS-1(Ser) and p-IRS-2(Ser731) induced by palmitate was signifi cantly suppressed Epo treatment. In addition, levels of TNF-α, IL-1β, NF-κB p65 and JNK1 activity were obviously suppressed by 5 or 10 U/ml Epo treatment, as detected by western blot or real time PCR.

This is the fi rst report of the effect of Epo on hepatic glucose metabolism and indicates that Epo improved hepatic insulin resistance through increasing PI3K activity, promoting the phosphorylation of AKT, FOXO1 and GSK-3β in palmitate-induced HepG2 cells. Furthermore, these effects are correlated with inhibition of Epo on infl ammation, since reduced Ser phosphorylation of IRS-1 and IRS-2 activates insulin signalling and also crosstalks with JNK1 and NF-κB.

Supported by: NSFC (81070636), (8120059)

1820-PTo Study the Effects of Sulindac on Glucose Metabolism in Rats of Insulin Resistance Induced by High Fat DietX.Y. DING, Y.D. PENG, Shanghai, China

Objective: By blocking the activity of IKK in vivo, to observe the infl uence of sulindac on insulin sensitivity in obese rats of insulin resistance induced by high fat diet and investigate the effects of interfering in infl ammation on the expression of insulin receptor substrate1(IRS-1) and its Ser/Tyr phos-phory la tion.

Methods: Forty-eight male SD rats were divided into CHOW and HFD group. 20 week after feeding, oral glucose tolerance test and insulin releasing test were performed for estimating insulin sensitivity. Continued to take high fat diet, 18 rats of insulin resistance were subdivided into three groups randomly(n=6/group): high fat diet, plus sulindac(an inhibitor of IKK) or rosiglitazone on the other two groups for periods of 6 weeks. The insulin sensitivity in different groups were estimated by hyperinsulinemic-euglycemic clamp technique. The expression of IKK as well as the IRS-1 protein and its Ser/Tyr phosphorylation in insulin target tissues were detected by western blot. Co-immunoprecipitation was performed to investigate whether IRS-1 or insulin receptor β subunit (IR-β) could serve as a direct substrate for IKK. Results: Hyperinsulinemic-euglycemic clamp study revealed a signifi cantly impaired insulin action after high-fat feeding. Glucose disposal rate was signifi cantly higher in both rosiglitazone and sulindac treatment group than that of control group. There was an signifi cant decrease in expression of the IKKα/β (ser 180/181) protein and the IRS-1 Ser phosphorylation in liver and skeletal muscle of rats treated with sulindac, while the expression of IRS-1 Tyr phosphorylation and IκBα were increased. The physical association between IRS-1 and the IKK complex as well as IκBα and IRβ was confi rmed using co-immunoprecipitation..

Conclusion: Treatment with sulindac could in part improve insulin resistance of obese rats probably through regulating the expression of IRS-1 Ser/Tyr phosphorylation. IKK and IRS-1 could directly interact in intact cells.

Supported by: NSFC (81070682)

1821-PDiurnal Pattern of Hepatic Insulin Sensitivity in Healthy vs. Type 1 Diabetic SubjectsFRANCESCA PICCININI, CHIARA DALLA MAN, LING HINSHAW, RITA BASU, YOGISH C. KUDVA, CLAUDIO COBELLI, ANANDA BASU, Padova, Italy, Rochester, MN

We recently demonstrated that the diurnal pattern of whole-body insulin action is different in healthy subjects and people with type 1 diabetes. Here we sought to determine the presence of a diurnal pattern of insulin action on the liver (hepatic insulin sensitivity, SI

L) in both these groups.For this purpose, 19 healthy and 19 C-peptide negative subjects with type

1 diabetes spent 88 hours in the Mayo clinical research unit and ingested three identical triple tracer mixed meals each containing 50 grams CHO at 0700 (B), 1300 (L), and 1900 (D) in randomized Latin Square order. Postprandial glucose turnover was determined with the tracer-to-tracee clamp technique and SI

L was estimated with a model of endogenous glucose production. Two ways ANOVA demonstrated that SI

L was different in healthy and type 1 diabetes (p<0.001), and as well as at B, L and D (p=0.01), with the same trend for both healthy and type 1 diabetes. Post-hoc comparison showed that SI

L was signifi cantly lower in healthy than in type 1 diabetes (p<0.0001), and lower at B than L or D, both in healthy (p<0.005) and in type 1 diabetes subjects (p<0.03) (Figure 1).

These results reveal the presence of a similar diurnal pattern in postprandial hepatic insulin sensitivity, both in healthy and type 1 diabetes subjects. Further studies are needed to determine possible neuro-humoral causes for and regulation of this pattern and the greater hepatic insulin sensitivity in type 1 diabetes.

Supported by: NIDDK (085516), (DP3094331); FIRB

1822-PGALNT2 Expression Is Reduced in Patients With Type 2 Diabetes, Possible Role of HyperglycemiaANTONELLA MARUCCI, LAZZARO DI MAURO, CLAUDIA MENZAGHI, SABRINA PRUDENTE, DAVIDE MANGIACOTTI, GRAZIA FINI, GIUSEPPE LOTTI, VINCENZO TRISCHITTA, ROSA DI PAOLA, San Giovanni Rotondo, Italy, Rome, Italy

Insulin resistance (IR) plays a major role in the pathogenesis of type 2 diabetes (T2D). Unraveling the molecular mechanisms underlying IR would improve setting up preventive and treatment strategies of T2D. Down-regulation of GALNT2, an UDPN-acetyl-alpha-D-galactosamine polypeptideN-acetylgalactosaminyltransferase-2, impairs insulin signaling and action in cultured human liver cells (1625P, ADA 2012). In addition, GALNT2 is down-regulated in liver of Goto-Kakizaki rats with IR and diabetes. We investigated the role of GALNT2 expression in human hyperglycemia, by measuring GALNT2 mRNA levels in peripheral whole blood cells (PWBC) of 130 (84 non-obese, 46 obese) non-diabetic individuals and 98 patients with T2D. We also studied the effect of in vitro high glucose concentration on GALNT2 expression in human cultured U937 cells.

Samples were obtained from volunteers at “Casa Sollievo della Sofferenza” Institute (S. Giovanni. Rotondo, Italy). Diabetes was defi ned as fasting glucose levels > 126 mg/dl. GALNT2 mRNA levels were measured by real-time PCR and calculated by the comparative ΔCT method by using GAPDH as endogenous reference.

GALNT2 mRNA levels were reduced in diabetic as compared to both non-obese and obese non-diabetic individuals (31% and 26% reduction, respectively; p<0.001 for both). In addition, GALNT2 expression was

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INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

negatively associated with HbA1c in T2D patients (r = -0.291, p = 0.005), thus pointing to a direct relationship between GALNT2 expression and glycemic levels.

In vitro, GALNT2 mRNA levels were reduced by 38 % (p = 0.02) in U937 cells exposed to 25 mmol/l glucose as compared to cells exposed to 5.5 mmol/l glucose + 19.5 mmol/l mannitol.

In conclusion, our data indicate that GALNT2 is down-regulated in T2D and suggest that this association is secondary to hyperglycemia. Together with previous in vitro and animal data, our fi nding support the hypothesis, that GALNT2 plays a major role in the pathogenesis of hyperglycemia-induced IR.

Supported by: Italian Ministry of Health

1823-PGLP-1-Receptor Agonist Increases Insulin Action in the Human Liver Cell Line HepG2JUTTA HAAS, JÖRG KOTZKA, BIRGIT KNEBEL, DIRK MÜLLER-WIELAND, Ham-burg, Germany, Düsseldorf, Germany

GLP-1 as an incretin regulates not only islets cell biology, but appears to be a hormone with ubiquitous actions affecting cells of different organs, like brain, heart, and adipose tissue. As it is still unclear whether GLP-1 has a direct effect on liver, the aim of the study was to investigate effects of the GLP-1-receptor agonist liraglutide on cellular metabolism of the human liver cell line HepG2 with a genetic and proteomic approach. Cells were maintained 72h in culture medium with 11 or 22 mM glucose and for further 72h with or without 10-7 M liraglutide. Before harvesting, cells were then left untreated or stimulated with 10-7 M insulin for 60 min. Oil red staining showed that lipid content was signifi cantly raised in cells maintained in 22 mM glucose. Total fat content of these cells was increased >4 fold and a signifi cant shift from saturated to unsaturated fatty acids was observed. The incubation with liraglutide did not affect the glucose effect on lipid contend directly. Nevertheless liraglutide incubation potentiated the observations by strongly improving insulin responsiveness. To get a hind of cellular pathways involved, we investigated key actors and transcription factors of gluconeogenesis/ glycolysis, cholesterol synthesis and fatty acid synthesis by RT PCR. Liraglutide alone increased ABCA1, malic enzyme, HSL, SREBP-1, and PFK mRNA levels by ~50% and decreased SCAP and LDLR gene mRNA levels by ~30-50%. The mRNA levels of c-fos, SREBP-2, HMG-CoAR, FAS, Glut-2, G6PD, PEPCK, LXR, FXR, HNF1α or ABCG5 were not directly affected by liraglutide. However, there was a statistical signifi cant synergistic effect of liraglutide on insulin response increasing expression levels of most targets by at least 2-3 fold. These data show, that GLP-1 can infl uence gene expression in human liver cells directly, but the major effects appear to be due to enhancement of insulin action. This might also have clinical implications for combination therapies of incretin based pharmaceuticals with insulin.

1824-PGLP-1 Receptor as a Biomarker for Incretin Function in Type 2 Dia-betesSINDHU RAJAN, JOHN B. ANCSIN, ELIZABETH MATHEW, JYOTHI JANARDAN-AN, LOUIS PHILIPSON, Chicago, IL

GLP-1 (Glucagon-Like Peptide-1) is a gut derived incretin hormone secreted in response to food intake and it plays a crucial role in glucose homeostasis. The success of incretin-based therapies depends on the abundance of plasma membrane GLP-1 receptors (GLP-1R) and effi cient GLP-1R signaling. Several studies have reported diminished GLP-1 responsiveness in hyperglycemia. Our recent study in pancreatic beta cells showed that impaired forward traffi cking of GLP-1R to the plasma membrane is glucose dependent, correlated to SUMO (Small Ubiquitin Like Modifi er protein) modifi cation of the receptor that is regulated by Protein Kinase A (PKA) activity. Besides pancreatic beta cells, many cell types including peripheral blood cells express GLP-1 receptor. We found that both hyperglycemia and chronic exposure to GLP-1R agonists cause reduced receptor density at the cell membrane of peripheral blood monocytes. In particular, human monocytic THP-1 cells and mouse macrophage cells, cultured in high glucose conditions showed diminished GLP-1R density at the plasma membrane. Moreover, prolonged stimulation of GLP-1R by a GLP-1 analog, Exendin-4 also resulted in severe reduction of cell surface GLP-1R. This observation is particularly signifi cant since long-acting agonists of GLP-1R are now available. We have therefore, developed a blood-based assay that can evaluate cell surface GLP-1 receptors in peripheral blood cells as a diagnostic tool to assist determination of patient suitability, drug dosage and responsiveness to incretin based therapies.

INTEGRATED PHYSIOLOGY—INSULIN SECRETION IN VIVO

Guided Audio Tour: Gastric Bypass, Incretins, and Insulin Secretion (Post-ers: 1825-P to 1830-P), see page 17.

& 1825-PGreater Improvement in β-Cell Function after Gastric Bypass Is In-dependent of Weight LossROXANNE DUTIA, KATRINA BRAKONIECKI, GARY WANG, SHARON MOGUL, KEESANDRA AGENOR, JAMES MCGINTY, SCOTT J. BELSLEY, DANIEL J. ROSEN, BLANDINE LAFERRÈRE, New York, NY

Evidence suggests that Roux-en-Y gastric bypass (RYGBP) superiorly remits type 2 diabetes (T2DM), compared to either diet or gastric banding (GB)--this effect is rapid and may be independent of weight loss. To investigate if RYGBP improves β-cell function independently of weight loss, obese subjects with T2DM were studied before and A) 4 wk post-RYGBP (n=23), or B) after equivalent weight loss via either GB or diet (GB/D, n=23, avg wt loss duration 10 wk). Before and after intervention, blood samples were collected during: 1) a 3h 50g oral glucose tolerance test (OGTT), and 2) an isoglycemic IV glucose clamp (Iso-IVGC; peripheral glucose administration matching OGTT plasma glucose), and the following were calculated: Insulin secretion rate (ISR), derived from C-peptide deconvolution; β-cell glucose sensitivity (BCGS)=slope of ISR vs. plasma glucose; Disposition index (DI)=BCGS/HOMA-IR. At baseline, subjects in the RYGBP and GB/D groups were matched for T2DM duration and control, ISR, BCGS, HOMA-IR, weight and BMI, and after intervention matched for weight, BMI (39.3±0.8 vs. 38.7±0.9 kg/m2, p=.60) and weight loss (9.9±0.6 vs. 9.3±0.4%, p=.42; 11.2±0.7 vs. 10.4±0.5 kg, p=.32). After equivalent weight loss, there was a comparable reduction in fasting glucose and HOMA-IR, but a greater improvement in glucose tolerance (AUC p<.05 and 2h glucose p<.001) after RYGBP. BCGS and DI during the OGTT improved signifi cantly in both groups, however, they were ~60% higher in RYGBP vs. GB/D (BCGS: 1.53±0.14 vs. 0.97±0.16, p=.01; DI: 0.43±.07 vs. 0.27±.08, p=.01); this greater increase in BCGS and DI in the RYGBP group was not observed during the IV glucose challenge (Iso-IVGC). This study shows that in patients with T2DM, some of the improvements in β-cell function after RYGBP, including BCGS and DI, are independent of weight loss. Furthermore, this greater improvement only occurred after oral, but not IV glucose stimulation, suggesting an incretin or other gut-mediated effect after RYGBP.

Supported by: R01-DK67561; PO1-DK58398; UL1TR000040; DK-26687, DK-63068-05

& 1826-PDifferential Effects of 36 Hours of Fasting on Metabolism in Low Birth Weight SubjectsSINE W. JOERGENSEN, CHARLOTTE BRØNS, LES J. BLUCK, KRISTINE FÆRCH, DAVID DUNGER, ALLAN A. VAAG, København N., Denmark, Cambridge, United Kingdom, Gentofte, Denmark

Low birth weight (LBW) is associated with an increased risk of type 2 diabetes when exposed to a westernized lifestyle, but may be benefi cial during sparse living conditions. Therefore, young healthy individuals born with LBW may display an improved metabolic risk profi le when subjected to short time fasting compared with controls born with a normal birth weight (NBW).

Eighteen NBW and 21 LBW young healthy males matched for age and BMI were included. LBW was defi ned as a birth weight ≤ 10th percentile and NBW between the 50th and the 75th percentile of the birth cohort. All participants were exposed to 36 hr of fasting, followed by a frequently sampled intravenously glucose tolerance test (IVGTT) to be used for minimal modeling. Frequent sampling of glucose and insulin were performed throughout the study, and a [6,6-D2]-2H2 glucose tracer was used to assess endogenous glucose production after 36 hr of fasting. Glucose tracer and insulin were included in the IVGTT to assess glucose mediated glucose uptake (Sg) and insulin sensitivity (Si). We performed indirect calorimetry after 12, 31 and 34 hr of fasting.

During 36 hr of fasting, s-insulin decreased signifi cantly more in LBW compared with NBW individuals (p<0.001), whereas energy expenditure decreased in LBW (p=0.11) and increased in NBW (p=0.02) during fasting.

After fasting, LBW expressed lower endogenous glucose production (p=0.046) and lower Sg (p=0.054) than NBW individuals, but no difference were found regarding insulin sensitivity between the two groups. After the IVGTT, excretion of glucose in the urine was lower than expected (p=0.027) in the LBW but not in the NBW individuals.

INSULIN ACTION—ADIPOCYTE BIOLOGY 1745-P …...A455 For author disclosure information, see page...

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