Restored Insulin Sensitivity Research Articles

Suppression of GATA-3 increases adipogenesis, reduces inflammation and improves insulin sensitivity in 3T3L-1 preadipocytes

Impaired adipogenesis plays an important role in the development of obesity-associated insulin resistance and type 2 diabetes. Adipose tissue inflammation is a crucial mediator of this process. GATA-3 plays important roles in adipogenesis and inflammation. The aim of this study is to investigate the impact of GATA-3 suppression on improving adipogenesis, lowering inflammation and reversing insulin resistance. GATA-3 levels were measured in subcutaneous (SC) and omental (OM) adipose tissues obtained from insulin sensitive (IS) and insulin resistant (IR) obese individuals during weight reduction surgeries. The effect of GATA-3 suppression on adipogenesis, expression of inflammatory cytokines and insulin resistance biomarkers was performed in 3T3L-1 mouse preadipocytes via transfection with GATA-3-specific DNAzyme. GATA-3 expression was higher in OM compared to SC adipose tissues and in stromal vascular fraction-derived differentiating preadipocytes from IR obese individuals compared to their IS counterparts. Suppression of GATA-3 expression in 3T3L-1 mouse preadipocytes with GATA-3 specific inhibitor reversed 4-hydroxynonenal-induced impaired adipogenesis and triggered changes in the expression of insulin signaling-related genes. GATA-3 inhibition also modulated the expression of IL-6 and IL-10 and lowered the expression of insulin resistance biomarkers (PAI-1 and resistin) and insulin resistance phosphoproteins (p-BAD, p-PTEN and p-GSK3β). Inhibiting GATA-3 improves adipocytes differentiation, modulates the secretion of inflammatory cytokines and improves insulin sensitivity in insulin resistant cells. Suppression of GATA-3 could be a promising tool to improve adipogenesis, restore insulin sensitivity and lower obesity-associated inflammation in insulin resistant individuals.

Serum Ceramide Reduction by Blueberry Anthocyanin-Rich Extract Alleviates Insulin Resistance in Hyperlipidemia Mice

Blueberry anthocyanin-rich extract (BAE) was supplemented to high-fat diet (HFD)-fed mice to investigate sphingolipid metabolism modulating factors involved in the attenuated hyperinsulinemia and hyperlipidemia. A BAE-containing diet effectively controlled food intake and liver weight and significantly attenuated insulin resistance triggered by a HFD. Higher BAE (200 mg/kg of body weight) administration performed more efficiently in the improvement of hepatic steatosis and adipocyte hypertrophy, together with distinct suppressions in serum triacylglycerol and cholesterol in total and species. Serum lipid compositions revealed 200 mg/kg of BAE supplementation remarkably suppressed ceramide accumulation. Consistently, genes encoding enzymes associated with sphingomyelin conversion and ceramide de novo synthesis were modulated toward a healthy direction for restrained sphingolipid accumulation. Further, the inhibited mRNA expressions of protein phosphatase 2A and protein kinase Cζ involved in blocking Akt phosphorylation connected the controlled ceramides with the restored insulin sensitivity.

Rutaecarpine exhibits anti-diabetic potential in high fat diet–multiple low dose streptozotocin induced type 2 diabetic mice and in vitro by modulating hepatic glucose homeostasis

Rutaecarpine, an indolopyridoquinazoline alkaloid, attracted attentions because of possessing various biological activities. The objective of this study was to investigate the effect of rutaecarpine on glucose and lipid metabolism in high fat diet–multiple low dose streptozotocin induced type 2 diabetic (HFD-db) mice and to understand the mechanism of action. HFD-db mice showed impaired glucose metabolism and lipid profile. Oral administration of rutaecarpine reduced the blood glucose levels, decreased blood hemoglobin A1c (HbA1c) levels, improved glucose tolerance and restored insulin sensitivity in HFD-db mice. Rutaecarpine also decreased body weight gain, water intake and visceral fat gain in HFD-db mice. Total cholesterol, triglycerides, very low density lipoprotein and low density lipoprotein were reduced and high density lipoprotein level was augmented in rutaecarpine treated HFD-db mice. Rutaecarpine also reduced the elevated levels of serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, urea and creatinine in HFD-db mice. Rutaecarpine significantly promoted the rate of glucose consumption, glucose uptake and glycolysis in C2C12 myotubes. Western blotting results showed that rutaecarpine augmented p-GSK-3β and p-AMPK expression, and suppressed G6Pase expression in HepG2 cells. These results suggest that rutaecarpine might be having therapeutic importance to fight against type 2 diabetes mellitus associated with dyslipidemia.

1752-P: Discovery of Targets for Insulin Resistance by Microfluidic Organ-on-Chip Models

Metabolism of glucose and lipids is essential for nutrient balance and metabolic homeostasis, and is dynamically regulated by adipose tissue and liver. Dysfunction in regulation of these molecules as well as the secreted factors results in metabolic syndrome and insulin resistance (IR), which is a precursor to type 2 diabetes. Studies have shown that as the main site of adaptive thermogenesis, brown adipose tissue (BAT) improves hepatic insulin sensitivity, whereas white adipose tissue (WAT) can induce hepatic IR. However, how these organs communicate to regulate insulin sensitivity is not completely understood due to the lack of representative experimental models. Animals models are useful but suffer from confounding effects from other organs; traditional in vitro models have limited physiological representation. We have developed an organ-on-chip platform to study the interactions between BAT/WAT and liver under different metabolic states. The adipose-liver organ-chip consists of two interconnected microfluidic devices - brown/white adipocytes were cultured in one device and hepatocytes (insulin sensitive and IR) in the other, both under perfusion. We show that (a) adipocytes exhibited higher intracellular lipid and fatty acid secretion and (b) the hepatocytes secreted higher albumin compared to traditional co-cultures. We found that brown adipocytes secretions restored insulin sensitivity, and improved glucose/lipid metabolism in IR hepatocytes. In contrast, white adipocytes worsened insulin sensitivity and glucose/lipid metabolism. IR hepatocytes lowered Akt-phosphorylation and glucose uptake while upregulated adipocyte fatty acids secretion. Through RNA-seq, we have identified adipose genes responsible for improving hepatic IR. We have discovered potential new targets for improving hepatic insulin sensitivity. The novel organ-chip platform represented the cells physiologically and will be useful in the discovery and validation of new therapeutic targets for T2D. Disclosure N. Tanataweethum: None. A. Trang: None. L. Annepureddy: None. J. Mehta: None. R.N. Cohen: None. A. Bhushan: None. Funding DRTC (P30DK020595)

Anthocyanin Improves Glucose Homeostasis in Obese Mice via Regulation of Intestinal Microbiota and Barrier Function

Abstract Objectives Anthocyanin (ACN) with low bioavailability possess anti-diabetic effect, the mechanism of the action remained unclear. This study aimed to investigate whether the anti-diabetic effects of anthocyanin were related to regulation of gut microbiota and epithelial barrier function. Methods Male C57Bl/6N mice were randomly assigned into 3 groups and fed chow, high fat/high sugar diet (HFHS, 45 kcal% fat, 17 kcal% sucrose) or HFHS + 0.4% anthocyanin for 8 weeks. Furthermore, a separate cohort of HFHS-fed C57Bl/6N mice were oral gavage with fecal microbiota from HFHS, HFHS + ACN and heat-killed fecal microbial from HFHS + ACN-fed donors (HK-ACN) for 4 weeks, The composition of gut microbiota, systemic inflammation, glucose homeostasis and intestinal barrier function were determined. Results The area under the curve (AUC) of the oral glucose tolerance test (GTT) and insulin tolerance test (ITT) in HFHS-fed mice were 61% and 79% higher than those in Chow-fed mice (P < 0.05). ACN supplementation improved HFHS-induced glucose intolerance in the GTT (P < 0.05) and restore insulin sensitivity in the ITT (P < 0.05). ACN supplementation also attenuated HFHS-induced systemic inflammation, intestinal permeability and metabolic endotoxemia (P < 0.05). In addition, ACN administration increased expression of epithelial barrier function genes ZO-1, Occludin and decreased inducible NO-synthase (iNOS) protein levels in intestine of HFHS-fed mice. Gut microbiota analysis showed that ACN greatly altered gut microbiota of HFHS-fed mice. Transplantation of the gut microbiota from ACN-fed mice, but not HFHS-fed or HK-ACN-fed mice, significantly improved intestinal epithelial barrier function, systemic inflammation and glucose homeostasis. Conclusions The present study demonstrated that ACN improves HFHS-induced impairment of glucose metabolism by regulating the gut microbiota and epithelial barrier functions. Funding Sources No funding sources to report.

Discovery of Highly Selective Inhibitors of Calmodulin-Dependent Kinases That Restore Insulin Sensitivity in the Diet-Induced Obesity in Vivo Mouse Model

Polymorphisms in the region of the calmodulin-dependent kinase isoform D (CaMK1D) gene are associated with increased incidence of diabetes, with the most common polymorphism resulting in increased recognition by transcription factors and increased protein expression. While reducing CaMK1D expression has a potentially beneficial effect on glucose processing in human hepatocytes, there are no known selective inhibitors of CaMK1 kinases that can be used to validate or translate these findings. Here we describe the development of a series of potent, selective, and drug-like CaMK1 inhibitors that are able to provide significant free target cover in mouse models and are therefore useful as in vivo tool compounds. Our results show that a lead compound from this series improves insulin sensitivity and glucose control in the diet-induced obesity mouse model after both acute and chronic administration, providing the first in vivo validation of CaMK1D as a target for diabetes therapeutics.

SAT-653 Exploring the Role of Brown Adipokines on Hepatic Insulin Resistance Using a Microfluidic Organ-On-Chip

The development of insulin resistance (IR) in liver is a key of pathophysiologic response in type 2 diabetes. Although insulin resistance impairs its ability to suppress hepatic glucose production, insulin regulation of lipogenesis is maintained (1). Currently available insulin sensitizers are effective at lowering glucose levels, but have significant adverse effect on weight gain due to triglyceride accumulation, which highlights a need to develop new therapeutic treatment options for type 2 diabetes. Brown adipose tissue (BAT) has been studied as a new target for anti-obesity and type 2 diabetes as BAT stimulation increases energy expenditure, reduces adiposity, and improves insulin sensitivity (2). However, the underlying mechanisms are not completely understood. To identify the role of BAT adipokines on hepatic insulin resistance, we developed an insulin resistant liver organ-on-chip model and then perfused primary mouse brown adipocyte conditioned media through the hepatocytes. Our results demonstrate that IR hepatocytes treated with brown adipocyte - conditioned media restores insulin sensitivity and improves glucose metabolism. This was verified by significantly increased expression of Phospho-Akt (Ser473) and glucose production gene markers (G6pc and PEPCK), lowered glucose production, increased glucose uptake, and increased glycogen synthesis in treated hepatocytes over IR group (p < 0.05). Our results also indicate that brown adipocyte - conditioned media treatment has the potential to suppress lipogenesis in hepatic insulin resistance. This was confirmed by significantly reduced expression of a lipogenesis gene marker (SREPB1) and fatty acid uptake in treated hepatocytes over IR group (p < 0.05). Current efforts are focused towards identifying the BAT adipokine via mass spectrometry. We conclude that BAT-derived endocrine factors could be a potential target for new drug discovery for obesity and type 2 diabetes treatment. Reference: (1) Langlet et al. Cell. 2017 Nov;171(4):824-835. (2) Subhadraw et al. Am J Physiol Endocrinol Metlab. 2015 Jun;308(12):E1043-E1055. Nothing to Disclose: NT, CL, AT, FZ, KK, JM, RC, AB

2-Methoxyestradiol inhibits high fat diet-induced obesity in rats through modulation of adipose tissue macrophage infiltration and immunophenotype

Recently, experimental studies demonstrated that 2-methoxyestradiol (2ME2) ameliorates high fat diet (HFD)-induced obesity and restores insulin sensitivity. However, the mechanisms underlying these effects are unveiled yet. The current study was undertaken to test the hypothesis that 2ME2 exerts its effects by modulating adipose tissue macrophages (ATMs) accumulation, polarization and immunophenotypes. The experiment was carried out in males Wistar rats (n = 28) for 13 consecutive weeks. In HFD-fed group; body weight, glucose intolerance, serum insulin, HOMA-IR, lipid profile and adipose tissue (AT) weight were significantly higher compared to normal standard diet (NSD)- fed rats. However, treatment of HFD-fed rats with 2ME2 (200 μg/kg/day; i.p. from the beginning of the 9th week) resulted in significant enhancements in all these parameters as compared to HFD-fed rats. Treatment with 2ME2 was associated with a significant reduction in macrophage infiltration in the AT, shifting macrophage polarization towards M2 phenotype as indicated by significant decrease in the expression of pro-inflammatory M1 macrophages markers (IL-6, IL-1β, CD11c and iNOS) and concurrent significant increase in the M2 anti-inflammatory macrophage markers (Arginase 1 and IL-10). 2ME2 ameliorates HFD-induced obesity and glucose intolerance through inhibition of ATM infiltration in AT and shifting macrophage polarization from pro-inflammatory M1 to M2 anti-inflammatory phenotypes.

Validation of PAS Kinase, a Regulator of Hepatic Fatty Acid and Triglyceride Synthesis, as a Therapeutic Target for Nonalcoholic Steatohepatitis.

Hyperactivation of sterol regulatory element binding protein 1c (SREBP‐1c), which transcriptionally induces expression of enzymes responsible for de novo lipogenesis and triglyceride (TG) formation, is implicated in nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH) pathogenesis. Posttranslational SREBP‐1c maturation and activation is stimulated by the protein per–arnt–sim kinase (PASK). PASK‐knockout mice are phenotypically normal on a conventional diet but exhibit decreased hypertriglyceridemia, insulin resistance, and hepatic steatosis on a high‐fat diet. We investigated the effects of pharmacologic PASK inhibition using BioE‐1115, a selective and potent oral PASK inhibitor, in Zucker fatty (fa)/fa) rats, a genetic model of obesity, dyslipidemia, and insulin resistance, and in a dietary murine model of NAFLD/NASH. Female Zucker (fa/fa) rats and lean littermate (fa/+) controls received BioE‐1115 (3‐100 mg/kg/day) and/or omega‐3 fatty acids, and blood glucose, hemoglobin A1c, glucose tolerance, insulin, and serum TG were measured. C57BL/6J mice fed a high‐fat/high‐fructose diet (HF‐HFrD) were treated with BioE‐1115 (100 mg/kg/day) or vehicle. Body weight and fasting glucose were measured regularly; serum TG, body and organ weights, and liver TG and histology were assessed at sacrifice. Messenger RNA (mRNA) abundance of SREBP‐1c target genes was measured in both models. In Zucker rats, BioE‐1115 treatment produced significant dose‐dependent reductions in blood glucose, insulin, and TG (all greater than omega‐3 fatty acids) and dose dependently restored insulin sensitivity assessed by glucose tolerance testing. In HF‐HFrD mice, BioE‐1115 reduced body weight, liver weight, fasting blood glucose, serum TGs, hepatic TG, hepatic fibrosis, hepatocyte vacuolization, and bile duct hyperplasia. BioE‐1115 reduced SREBP‐1c target mRNA transcripts in both models. Conclusion: PASK inhibition mitigates many adverse metabolic consequences associated with an HF‐HFrD and reduces hepatic fat content and fibrosis. This suggests that inhibition of PASK is an attractive therapeutic strategy for NAFLD/NASH treatment.

Interleukin-4 Restores Insulin Sensitivity in Insulin-Resistant Osteoblasts by Increasing the Expression of Insulin Receptor Substrate 1.

Obesity and latent inflammation can give rise to insulin resistance and type 2 diabetes. Here we established an insulin resistance model of osteoblasts to explore the restoration effect of anti-inflammatory interleukin-4 (IL-4) on insulin sensitivity and its mechanism. We found that IL-4 inhibited cell proliferation in a concentration- and time-dependent manner. Insulation resistance significantly reduced the phosphorylation levels of the insulin receptor substrate 1 (IRS1; Tyr612), Akt (Ser473), and AS160 (Ser318) proteins. The addition of IL-4 to the insulin resistance model led to a dose-dependent stimulation of the phosphorylation of IRS1, Akt, and AS160. IL-4 fully restored the activation of the insulin cascade in insulin-resistant cells at the concentration of 50ng/ml. Additionally, IL-4 promoted the expression of IRS1 in a time-dependent manner. We conjecture that IL-4 restores insulin sensitivity in osteoblasts by upregulating the expression of IRS1. It was also found that IL-4 promoted the expression of osteoprotegerin depending on the time of exposure. This effect may play an important role in the regulation of the energy metabolism in the whole body.

Chaperone-Based Therapeutic Target Innovation: Heat Shock Protein 70 (HSP70) for Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is still a global health problem. Current T2DM treatments are limited to curing the symptoms and have not been able to restore insulin sensitivity in insulin-sensitive tissues that have become resistant. In the past decade, some studies have shown the significant role of a chaperone family, heat shock protein 70 (HSP70), in insulin resistance pathogenesis that leads to T2DM. HSP70 is a cytoprotective molecular chaperone that functions in protein folding and degradation. In general, studies have shown that decreased concentration of HSP70 is able to induce inflammation process through JNK activation, inhibit fatty acid oxidation by mitochondria through mitophagy decrease and mitochondrial biogenesis, as well as activate SREBP-1c, one of the lipogenic gene transcription factors in ER stress. The overall molecular pathways are potentially leading to insulin resistance and T2DM. Increased expression of HSP70 in brain tissues is able to improve insulin sensitivity and glycemic control specifically. HSP70 modulation-targeting strategies (including long-term physical exercise, hot tub therapy (HTT), and administration of alfalfa-derived HSP70 (aHSP70)) in subjects with insulin resistance are proven to have therapeutic and preventive potency that are promising in T2DM management.

Serum Level of Galectin -3 and Its Relation to Insulin Resistance in Patients with Androgenetic Alopecia

Androgenetic alopecia (AGA) is the most common cause of hair loss in both genders, characterized by follicular miniaturization in a patterned hair loss occurring due to systemic androgen and genetic factors. Galectin-3 is a macrophage-derived instigator of insulin resistance and impaired glucose tolerance, which are two phenotypes associated with obesity-induced type 2 diabetes. Their findings highlight Gal3 as a candidate new therapeutic target to restore insulin sensitivity . to evaluate serum Galectin -3, fasting insulin and fasting blood glucose levels in patients with androgenetic alopecia and to assess its relation to insulin resistance in androgenetic alopecia patients. This case control study was conducted on 60 patients suffering from Androgentic alopecia (Group A) and 20 apparently healthy individuals of matched age and sex as a control group (Group B). Each patient were subjected to full history taking, complete clinical examination and Estimation of serum levels of Galectin-3 and fasting insulin using enzyme linked-immunosorbent assay (ELISA) technique, and fasting blood glucose level in both androgenetic alopecia patients and control group. The mean age of AGA patients was 39.9 years. Male patients represented 66.7%, GAL3 showed excellent AUC (AUC=0.978). At cut off value of 7.9, sensitivity was 93.3%, specificity was 93.3%, HOMA IR showed excellent AUC (AUC=0.967). At cut off value of 1.85, sensitivity was 93.3%, specificity was 93.3%. Conclusion: higher HOMA IR, GAL3 were considered as independent predictors for AGA development. Higher HOMA IR, GAL3 were considered as independent predictors for more severe AGA in both males a females.

Loss of TAZ Boosts PPARγ to Cope with Insulin Resistance

In this issue of Cell Metabolism, El Ouarrat etal. identify the Hippo signaling terminal effector TAZ as an endogenous negative regulator of PPARγ, a master transcriptional regulator of lipid metabolism and insulin sensitivity. Selective destruction of TAZ in adipocytes lowers inflammation and restores insulin sensitivity and glucose homeostasis.

EEF1A2 exacerbated insulin resistance in male skeletal muscle via PKCβ and ER stress.

Recent studies raise the possibility that eukaryotic translation elongation factor 1 alpha (eEF1A) may play a role in metabolism. One isoform, eEF1A2, is specifically expressed in skeletal muscle, heart and brain. It regulates translation elongation and signal transduction. Nonetheless, eEF1A2's function in skeletal muscle glucose metabolism remains unclear. In the present study, suppression subtractive hybridisation showed a decrease in Eef1a2 transcripts in the skeletal muscle of diabetic Mongolian gerbils. This was confirmed at mRNA and protein levels in hyperglycaemic gerbils, and in db/db and high-fat diet-fed mice. Further, this downregulation was independent of Eef1a2 promoter methylation. Interestingly, adeno-associated virus-mediated eEF1A2 overexpression in skeletal muscle aggravated fasting hyperglycaemia, hyperinsulinaemia and glucose intolerance in male diabetic gerbils but not in female gerbil models. The overexpression of eEF1A2 in skeletal muscle also resulted in promoted serum glucose levels and insulin resistance in male db/db mice. Up- and downregulation of eEF1A2 by lentiviral vector transfection confirmed its inhibitory effect on insulin-stimulated glucose uptake and signalling transduction in C2C12 myotubes with palmitate (PA)-induced insulin resistance. Furthermore, eEF1A2 bound PKCβ and increased its activation in the cytoplasm, whereas suppression of PKCβ by an inhibitor attenuated eEF1A2-mediated impairment of insulin sensitivity in insulin-resistant myotubes. Endoplasmic reticulum (ER) stress was elevated by eEF1A2, whereas suppression of ER stress or JNK partially restored insulin sensitivity in PA-treated myotubes. Additionally, eEF1A2 inhibited lipogenesis and lipid utilisation in insulin-resistant skeletal muscle. Collectively, we demonstrated that eEF1A2 exacerbates insulin resistance in male murine skeletal muscle via PKCβ and ER stress.

9 - Therapeutic Targeting of Skeletal Muscle Nix in Early-Onset Insulin Resistance

Therapeutic targeting of skeletal muscle Nix in early-onset insulin resistance lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that Nix, a lipotoxicity-responsive gene, accumulates in response to diacylglycerols induced by high-fat and sucrose (HFS) feeding and exacerbated by exposure to gestational diabetes (GDM) during fetal development. Here we identify a novel phosphorylation residue, activated by cilomilast treatment that can prevent Nix-induced mitochondrial dysfunction in muscle cells. In a series of gain- and loss-of-function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium-dependent activation of DRP-1 and mitophagy. In addition, Nix-induced mitophagy leads to myotube insulin resistance through activation of mTOR-S6K inhibition of IRS-1. Through detailed phospho-peptide mapping of Nix, we identified a novel phosphorylation residue within the transmembrane domain, modulated by PKA activating agents, such as adrenergic agonist clenbuterol and the phosphodiesterase-4 inhibitor cilomilast. Treatment of myotubes with these agents serves to prevent Nix-induced mitochondrial dysfunction and restore insulin sensitivity. Furthermore, Nix knock-down or clenbuterol/cilomilast treatment rescued palmitate-induced phosphorylation of Ser1101 on the insulin receptor substrate-1 (IRS-1) and prevented insulin resistance. These findings provide insight into the role of Nix-induced mitophagy and muscle insulin resistance during an overfed state. Finally, our data supports the hypothesis that Nix regulates mitochondrial metabolism and insulin signalling in myotubes and suggests a mechanism by which pharmacological activation of PKA may circumvent the mitochondrial dysfunction characteristic of insulin resistance.

Oridonin prevents insulin resistance-mediated cognitive disorder through PTEN/Akt pathway and autophagy in minimal hepatic encephalopathy.

Minimal hepatic encephalopathy (MHE) was characterized for cognitive dysfunction. Insulin resistance (IR) has been identified to be correlated with the pathogenesis of MHE. Oridonin (Ori) is an active terpenoid, which has been reported to rescue synaptic loss and restore insulin sensitivity. In this study, we found that intraperitoneal injection of Ori rescued IR, reduced the autophagosome formation and synaptic loss and improved cognitive dysfunction in MHE rats. Moreover, in insulin‐resistant PC12 cells and N2a cells, we found that Ori blocked IR‐induced synaptic deficits via the down‐regulation of PTEN, the phosphorylation of Akt and the inhibition of autophagy. Taken together, these results suggested that Ori displays therapeutic efficacy towards memory deficits via improvement of IR in MHE and represents a novel bioactive therapeutic agent for treating MHE.

Iron overload inhibits late stage autophagic flux leading to insulin resistance.

Iron overload, a common clinical occurrence, is implicated in the metabolic syndrome although the contributing pathophysiological mechanisms are not fully defined. We show that prolonged iron overload results in an autophagy defect associated with accumulation of dysfunctional autolysosomes and loss of free lysosomes in skeletal muscle. These autophagy defects contribute to impaired insulin-stimulated glucose uptake and insulin signaling. Mechanistically, we show that iron overload leads to a decrease in Akt-mediated repression of tuberous sclerosis complex (TSC2) and Rheb-mediated mTORC1 activation on autolysosomes, thereby inhibiting autophagic-lysosome regeneration. Constitutive activation of mTORC1 or iron withdrawal replenishes lysosomal pools via increased mTORC1-UVRAG signaling, which restores insulin sensitivity. Induction of iron overload via intravenous iron-dextran delivery in mice also results in insulin resistance accompanied by abnormal autophagosome accumulation, lysosomal loss, and decreased mTORC1-UVRAG signaling in muscle. Collectively, our results show that chronic iron overload leads to a profound autophagy defect through mTORC1-UVRAG inhibition and provides new mechanistic insight into metabolic syndrome-associated insulin resistance.

Arachidonic acid-rich ARASCO oil has anti-inflammatory and antidiabetic actions against streptozotocin + high fat diet induced diabetes mellitus in Wistar rats.

The aim of this study was to investigate the effects of arachidonic acid (AA)-rich ARASCO oil on high-fat diet (HFD) + streptozotocin (STZ)-induced diabetes mellitus in male Wistar rats and its possible mechanisms of action. Male Wistar rats with HFD + STZ-induced diabetes were employed in the present study. ARASCO oil was administered orally for the first 7 d consecutively, followed by once weekly throughout the study (14 wk). At various time points, blood glucose and body weight and oral glucose tolerance tests were measured. At the end of the study, animals were sacrificed to collect plasma and various organs and stored at -80°C. Plasma insulin, tumor necrosis factor-α, interleukin-6, and lipoxin A4 were measured. Expression of the following genes was determined: nuclear factor-κΒ (NF-κB), cyclooxygenase-2 (COX-2), 12-lipoxygenase (12-LOX) in pancreas and lipocalin 2 (LPCLN2) in adipose tissue. Various antioxidants were measured in the plasma and other tissues. Area under the curve and insulin sensitivity index were assessed by computing homeostatic model of assessment for insulin resistance, quantitative insulin check index, Matsuda, and Belfiore indices. ARASCO oil treatment decreased hyperglycemia, restored insulin sensitivity, suppressed inflammation, enhanced plasma lipoxin A4 levels, and reversed altered antioxidant status to near normal in animals with HFD + STZ-induced diabetes. These results suggest that ARASCO, a rich source of AA, can prevent HFD + STZ-induced diabetes in Wistar rats owing to its anti-inflammatory action. It remains to be seen whether ARASCO oil is useful in preventing or postponing the development of type 2 diabetes mellitus in humans.

270-LB: Amelioration of Hepatic Insulin Resistance by Brown Adipocyte Secretion

Insulin resistance (IR) is a hallmark of T2D. In the IR state, insulin fails to inhibit hepatic glucose production yet promotes lipogenesis. Although thiazolidinediones can partially reinstate insulin sensitivity, there are concerns about long-term usage. Recent studies have shown positive effects of paracrine signaling from BAT transplant on metabolic homeostasis. Transplant of embryonic BAT into diabetic mice can improve glucose tolerance. However, the mechanisms are unclear. In this study, we show that brown adipocyte secretions can partially resolve hepatic IR. We cultured primary hepatocytes in a microfluidic organ-on-chip device. IR was induced by cytokine exposure and the cells were treated with brown adipocyte secretions among other controls. The group treated with brown adipocytes had restored insulin sensitivity. This was verified by an increased Akt phosphorylation (Figure 1), lowered glucose production, increased glucose uptake, and increased glycogen synthesis. We finally confirmed that genes important for glucose production (G6pc, PEPCK) and lipogenesis (SREBP1) were downregulated in the treated cells. We will present results from ongoing mechanistic studies. Our data imply that brown adipocyte secretions contain factors that regulate hepatic insulin sensitivity and could be potential targets for drug development. Disclosure A. Bhushan: None. N. Tanataweethum: None. C. Lee: None. F. Zhong: None. A. Trang: None. A. Budiman: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK095984NIH, P30DK020595)

Cefdinir Microsphere Modulated Microflora and Liver Immunological Response to Diet Induced Diabetes in Mice.

Gut microbiota is currently targeted for various diseases especially metabolic disorders such as diabetes. Our strategy is to alter gut microflora via specific antibiotic to reduce load of inflammation in the liver that increases as a result of high carbohydrate diet. Th1, Th17 and Treg are important immune cell types which decide the type of inflammatory response. Liver is tolerogenic in nature with low Th17/Treg ratio. In diabetics, this ratio decreases even more, and can cause liver trauma. The present study tries to find relationship between gut flora and immune cells such as Th1/Th17/Treg and their role in liver metabolism using diet induced diabetic mice model. Upon alteration of flora using Cefdinir in different forms, one could help lower the level of Treg cells thus increasing the ratio. Gut flora is strongly associated with the immunity in the liver. Targeted alteration of gut flora helps us to restore insulin sensitivity. Colon targeted Cefdinir gives more promising results, opens colonic bacteria as target for improving gut, liver inflammation and insulin sensitivity.