Reverse Insulin Resistance Research Articles

Supplementation of endogenous Ahr ligands reverses insulin resistance and associated inflammation in an insulin-dependent diabetic mouse model

Aryl-hydrocarbon receptor (Ahr) plays an important role in the regulation of intestinal homeostasis.Diabetes is characterized by vascular complications and intestinal dysfunction. We aimed at understanding the relationship between intestinal defense impairment and inflammation in diabetes and effects of Ahr ligands on diabetes-induced insulin resistance, endovascular inflammation, and intercellular adhesion molecule (ICAM) and flavin mono-oxygenase (FMO3) expression. Effects of Ahr ligands, such as tryptophan (Trp) and indole-3-carbinol (I3C) on intestinal barrier and inflammation of Ins2Akita mice were examined. Myeloid differentiation primary response 88 (MYD88) is the adaptor for inflammatory signaling pathways. Ins2Akita-MyD88-/- mice were used to study the role of MyD88. Ins2Akita mice demonstrated decreased Ahr and regenerating islet-derived 3-β (Reg3β) expression, and increased Klebsiella pneumoniae translocation. Ins2Akita mice demonstrated increased inducible nitric oxide synthase (iNOS) expression of intestine; ICAM, iNOS, interleukin 1 beta (IL-1β), and FMO3 expression of liver; and ICAM, iNOS, and FMO3 expression in aorta. Trp and I3C decreased diabetes-induced translocation and increased Ahr and Reg3β expression of intestine. Ahr ligands reduced diabetes-induced ICAM and FMO3 expression in liver and aorta; IL-6, tumor necrosis factor alpha (TNF-α), and iNOS expression in Kupffer cells; plasma IL-6 and TNF-α levels; dipeptidyl peptidase (DPP4) activity; and insulin insensitivity. Ins2Akita-MyD88-/- mice demonstrated decreased expression of p-NF-κB of liver and ICAM of aorta compared with Ins2Akita mice. Altogether, our data suggest that diabetes induces ICAM and FMO3 expression through the decrease in intestinal defense and MyD88. Ahr ligands reverse diabetes-induced intestinal defense impairment, insulin insensitivity, FMO3/ICAM expression, and systemic inflammation.

SCR-1693 inhibits tau phosphorylation and improves insulin resistance associated cognitive deficits

Except for few symptoms-improved drugs for Alzheimer's disease (AD), no disease-modified drug has been developed, especially for AD in type 2 diabetes mellitus (T2DM). SCR-1693, a disease-mortified candidate for AD, which is now in Phase I clinical study in China, improves Aβ25–35-impaired cognitive function in rodent's models. Here we report the effect of SCR-1693 on regulation of tau phosphorylation and insulin resistance associated cognition, and illustrate its underlying mechanism. We found that in intracerebroventricular injection of streptozotcin (STZ) rats, oral administration of SCR-1693 dose-dependently improved the learning and memory in Morris water maze test, decreased tau hyperphosphorylation, astrogliosis and postsynaptic protein loss in hippocampus. In Neura-2a cells with stable transfection of full-length human tau (Neura-2a-tau), treatment of SCR-1693 concentration-dependently enhanced the activation of protein phosphatase (PP1) and protein phosphatase 2A (PP2A), decreased cellular tau phosphorylation, and increased insulin-induced cellular signaling to reverse insulin resistance. Pre-treatment with the inhibitor of PP1 and PP2A inhibited the effect of SCR-1693 on both of tau phosphorylation and insulin signaling in Neura-2a-tau cells. All data suggest that an increase of activity of tau phosphatase was involved in the mechanism of SCR-1693 on the regulation of tau phosphorylation and insulin signaling, and SCR-1693 is considerable candidate for insulin resistance associated sporadic AD.

Intranasal insulin administration may be highly effective in improving cognitive function in mice with cognitive dysfunction by reversing brain insulin resistance.

It is well known in clinical practice that Alzheimer's disease (AD) is closely associated with brain insulin resistance, and the cerebral insulin pathway has been proven to play a critical role in the pathogenesis of AD. However, finding the most efficient way to improve brain insulin resistance remains challenging. Peripheral administration of insulin does not have the desired therapeutic effect and may induce adverse reactions, such as hyperinsulinemia, but intranasal administration may be an efficient way. In the present study, we established a brain insulin resistance model through an intraventricular injection of streptozotocin, accompanied by cognitive impairment. Following intranasal insulin treatment, the learning and memory functions of mice were significantly restored, the neurogenesis in the hippocampus was improved, the level of insulin in the brain increased, and the activation of the IRS-1-PI3K-Akt-GSK3β insulin signal pathway, but not the Ras-Raf-MEK-MAPK pathway, was markedly increased. The olfactory bulb-subventricular zone-subgranular zone (OB-SVZ-SGZ) axis might be the mechanism through which intranasal insulin regulates cognition in brain-insulin-resistant mice. Thus, intranasal insulin administration may be a highly efficient way to improve cognitive function by increasing cerebral insulin levels and reversing insulin resistance.

An Acetylation Switch of the NLRP3 Inflammasome Regulates Aging-Associated Chronic Inflammation and Insulin Resistance

It is well documented that the rate of aging can be slowed, but it remains unclear to which extent aging-associated conditions can be reversed. How the interface of immunity and metabolism impinges upon the diabetes pandemic is largely unknown. Here, we show that NLRP3, a pattern recognition receptor, is modified by acetylation in macrophages and is deacetylated by SIRT2, an NAD+-dependent deacetylase and a metabolic sensor. We have developed a cell-based system that models aging-associated inflammation, a defined co-culture system that simulates the effects of inflammatory milieu on insulin resistance in metabolic tissues during aging, and aging mouse models; and demonstrate that SIRT2 and NLRP3 deacetylation prevent, and can be targeted to reverse, aging-associated inflammation and insulin resistance. These results establish the dysregulation of the acetylation switch of the NLRP3 inflammasome as an origin of aging-associated chronic inflammation and highlight the reversibility of aging-associated chronic inflammation and insulin resistance.

Conditions for Better uses of Some Cameroonian Plants Potentially Anti-Diabetic and Reversing Insulin Resistance

Background: Hyperglycemia-induced oxidative and inflammatory harm are the major causes of chronic and fatal complications of diabetes. In many developing countries the products of socio-cultural medicine are more used by low income populations to fight against diseases particularly diabetes. The economic crises, the slump of agricultural product’s prizes and the significant increase of the population, are at the origin of the strong dependence on African traditional medicine. Objective: The objectives were to identify factors that influenced the better uses of potential bioactive plants published by Cameroonians, particularly used for diabetes management in order to select those that can improve insulin sensitivity and can be principally used to avoid diabetic complications. Methods: To achieve this objective, the review was carried out in online databases including Google, Google Scholar and Pubmed, between 2018 and 2019. For the ethnopharmacological standardization of recipes, we proposed in this work the doses calculated by deduction from the doses used to treat in vivo alloxan or streptozotocin induced diabetic rats. The presence of one or several antihyperglycemic compounds in recorded plants and the hypoglycemic effects of their extract reinforced the herbal use of these species. Results: All the admitted plants exhibited antidiabetic properties. Twenty-eight point fifty-seven percent (28, 57%) of them were confirmed antihyperglycemic and improved insulin sensitivity. Permanent stress is the important factor influencing the better management of diabetes by these plants. 1.5. Conclusion: The results of this study can be the scientific basis for antidiabetic drugs discovery that can prevent insulin resistant and consequently complications of diabetes type 2.

Polyphenol-rich green tea extract induces thermogenesis in mice by a mechanism dependent on adiponectin signaling

Adiponectin is downregulated in obesity negatively impacting the thermogenesis and impairing white fat browning. Despite the notable effects of green tea (GT) extract in the enhancement of thermogenesis, if its effects are being mediated by adiponectin has been scarcely explored. For this purpose, we investigated the role of adiponectin in the thermogenic actions of GT extract by using an adiponectin-knockout mice model. Male wild-type (WT) and knockout (AdipoKO) C57Bl/6 mice (3 months) were divided into 6 groups: mice fed a standard diet+gavage with water (SD WT, and SD AdipoKO), high-fat diet (HFD)+gavage with water (HFD WT, and HFD AdipoKO), and HFD + gavage with 500 mg/kg of body weight (BW) of GT extract (HFD + GT WT, and HFD + GT AdipoKO). After 20 weeks of experimentation, mice were euthanized and adipose tissue was properly removed. Our findings indicate that treatment with GT extract reversed complications of obesity in WT mice by decreasing final BW gain, adiposity index, adipocyte size and insulin resistance (IR). However, the action of the GT extract was not effective in reversing those markers in the AdipoKO mice, although GT acts independently in the reversal of IR. GT-treatment induced enhancement in energy expenditure (EE), BAT thermogenesis, and promoted browning phenotype in the subcutaneous WAT (scWAT) of WT mice. On the other hand, the thermogenic program was markedly impaired in BAT and scWAT of AdipoKO mice. Our outcomes unveiled adiponectin as a key direct signal for GT extract inducing adaptive thermogenesis and browning in scWAT.

Lycopene prevents the progression of lipotoxicity-induced nonalcoholic steatohepatitis by decreasing oxidative stress in mice

Excessive fatty acid uptake-induced oxidative stress causes liver injury and the consecutive recruitment of inflammatory immune cells, thereby promoting the progression of simple steatosis to nonalcoholic steatohepatitis (NASH). Lycopene, the most effective singlet oxygen scavenger of the antioxidant carotenoids, has anti-inflammatory activity. Here, we investigated the preventive and therapeutic effects of lycopene in a lipotoxic model of NASH: mice fed a high-cholesterol and high-fat diet. Lycopene alleviated excessive hepatic lipid accumulation and enhanced lipolysis, decreased the proportion of M1-type macrophages/Kupffer cells, and activated stellate cells to improve hepatic inflammation and fibrosis, and subsequently reduced the recruitment of CD4+ and CD8+ T cells in the liver. Importantly, lycopene reversed insulin resistance, as well as hepatic inflammation and fibrosis, in pre-existing NASH. In parallel, lycopene decreased LPS-/IFN-γ-/TNFα-induced M1 marker mRNA levels in peritoneal macrophages, as well as TGF-β1-induced expression of fibrogenic genes in a stellate cell line, in a dose-dependent manner. These results were associated with decreased oxidative stress in cells, which might be mediated by the expression of NADPH oxidase subunits. In summary, lycopene prevented and reversed lipotoxicity-induced inflammation and fibrosis in NASH mice by reducing oxidative stress. Therefore, it might be a novel and promising treatment for NASH.

Phellinus linteus polysaccharide extract improves insulin resistance by regulating gut microbiota composition.

The hypoglycemic effect of Phellinus linteus polysaccharide extract (PLPE) has been documented in several previous studies, but the functional interactions among PLPE, gut microbiota, and the hypoglycemic effect remain unclear. We examined the regulatory effect of PLPE on gut microbiota, and the molecular mechanism underlying improvement of insulin resistance, using a type 2 diabetic rat model. Here, 24 male Sprague-Dawley rats were randomly divided into four groups that were subjected to intervention of saline (normal and model control group), metformin (120 mg/kg.bw), and PLPE (600 mg/kg.bw) by oral administration. After 8 weeks of treatment, PLPE increased levels of short-chain fatty acids (SCFAs) by enhancing abundance of SCFA-producing bacteria. SCFAs maintained intestinal barrier function and reduced lipopolysaccharides content in blood, thereby helping to reduce systemic inflammation and reverse insulin resistance. Our findings suggest that PLPE (in which polysaccharides are the major component) has potential application as a prebiotic for regulating gut microbiota composition in diabetic patients.

Cytotoxicity Analysis of Morphologically Different Sol-Gel-Synthesized MgO Nanoparticles and Their In Vitro Insulin Resistance Reversal Ability in Adipose cells.

Insulin resistance is one of the major factors that leads to type 2 diabetes. Although insulin therapies have been shown to overcome insulin resistance, overweight and hypoglycemia are still observed in most cases. The disadvantages of insulin therapies have driven the interest in developing novel curative agents with enhanced insulin resistance reversibility. Magnesium deficiency has also been recognized as a common problem which leads to insulin resistance in both type 1 and 2 diabetes. Oxide nanoparticles demonstrate highly tunable physicochemical properties that can be exploited by engineers to develop unique oxide nanoparticles for tailored applications. Magnesium supplements for diabetic cells have been reported to increase the insulin resistance reversibility. Hence, it is hypothesized that magnesium oxide (MgO) nanoparticles could be molecularly engineered to offer enhanced therapeutic efficacy in reversing insulin resistance. In the present work, morphologically different MgO nanoparticles were synthesized and evaluated for biophysical characteristics, biocompatibility, cytotoxicity, and insulin resistance reversibility. MTT assay revealed that hexagonally shaped MgO nanoparticles are less toxic to 3T3-L1 adipose cells (diabetic) compared with spherically and rod-shaped MgO nanoparticles. MTT assays using VERO cells (normal, non-diabetic) showed that 400 μg/ml of hexagonal MgO nanoparticles were less toxic to both diabetic and non-diabetic cells. DNS glucose assay and western blot showed that hexagonally shaped MgO nanoparticles had reversed 29.5% of insulin resistance whilst fluorescence microscopy studies indicated that the insulin resistance reversal is due to the activation of intracellular enzymes. The probable mechanism for MgO nanoparticles to induce cytotoxic effect and insulin resistance reversal is discussed.

Molecular effects of Moringa leaf extract on insulin resistance and reproductive function in hyperinsulinemic male rats.

Many studies have reported that insulin resistance impairs the antioxidant defense system and causes male infertility. Moringa oleifera is a medicinal plant that has been employed for the medicament of many disorders. It controls the levels of glucose and manages male sexual disorders. However, its extracts can reverse insulin resistance-linked metabolic alterations remains unknown. Therefore, the current study investigated the potential of the aqueous leaves extract from Moringa oleifera to reverse insulin resistance and testicular disorders in rats. Rats were fed either a chow (as a control group) or a high fructose diet (HFD, to persuade a state of insulin resistance), in addition to a group of rats fed HFD and treated with Moringa (300mg/kg) for 4weeks. Moringa reversed hepatic insulin insensitivity and this was linked to up-regulation of genes involved in insulin receptors and glucose uptake in the liver. These results were associated with amended the insulin level in serum and standardization of insulin sensitivity. In addition, it improved the serum testosterone level and the gene expression of the testicular steridogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD). Taken together, our findings demonstrate that Moringa reversed HFD diet-induced insulin resistance and improved the testicular function.

Controlled-release mitochondrial protonophore (CRMP) reverses dyslipidemia and hepatic steatosis in dysmetabolic nonhuman primates.

Nonalcoholic fatty liver disease (NAFLD) is estimated to affect up to one-third of the general population, and new therapies are urgently required. Our laboratory previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides. Although we previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, hepatic inflammation, and fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess its safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of longer-term CRMP treatment on hepatic mitochondrial oxidation and on the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in high-fat, fructose-fed cynomolgus macaques (n = 6) and spontaneously obese dysmetabolic rhesus macaques (n = 12). Using positional isotopomer nuclear magnetic resonance tracer analysis (PINTA), we demonstrated that acute CRMP treatment (single dose, 5 mg/kg) increased rates of hepatic mitochondrial fat oxidation by 40%. Six weeks of CRMP treatment reduced hepatic triglycerides in both nonhuman primate models independently of changes in body weight, food intake, body temperature, or adverse reactions. CRMP treatment was also associated with a 20 to 30% reduction in fasting plasma triglycerides and low-density lipoprotein (LDL)-cholesterol in dysmetabolic nonhuman primates. Oral administration of CRMP reduced endogenous glucose production by 18%, attributable to a 20% reduction in hepatic acetyl-coenzyme A (CoA) content [as assessed by whole-body β-hydroxybutyrate (β-OHB) turnover] and pyruvate carboxylase flux. Collectively, these studies provide proof-of-concept data to support the development of liver-targeted mitochondrial uncouplers for the treatment of metabolic syndrome in humans.

A Complex Relationship: Dietary Folate, Arsenic Metabolism, and Insulin Resistance in Mice.

A Complex Relationship: Dietary Folate, Arsenic Metabolism, and Insulin Resistance in Mice.

Pirfenidone prevents and reverses hepatic insulin resistance and steatohepatitis by polarizing M2 macrophages

Nonalcoholic steatohepatitis (NASH) is associated with lipotoxic liver injury, leading to insulin resistance, inflammation, and fibrosis. Despite its increased global incidence, very few promising treatments for NASH are available. Pirfenidone is an antifibrotic agent used to treat pulmonary fibrosis; it suppresses the pulmonary influx of T cells and macrophages. Here, we investigated the effect of pirfenidone in a mouse model of lipotoxicity-induced NASH via a high-cholesterol and high-fat diet. After 12 weeks of feeding, pirfenidone administration attenuated excessive hepatic lipid accumulation and peroxidation by reducing the expression of genes related to lipogenesis and fatty acid synthesis and enhancing the expression of those related to fatty acid oxidation. Flow cytometry indicated that pirfenidone reduced the number of total hepatic macrophages, particularly CD11c+CD206–(M1)-type macrophages, increased the number of CD11c–CD206+(M2)-type macrophages, and subsequently reduced T-cell numbers, which helped improve insulin resistance and steatohepatitis. Moreover, pirfenidone downregulated the lipopolysaccharide (LPS)-induced mRNA expression of M1 marker genes and upregulated IL-4-induced M2 marker genes in a dose-dependent manner in RAW264.7 macrophages. Importantly, pirfenidone reversed insulin resistance, hepatic inflammation, and fibrosis in mice with pre-existing NASH. These findings suggest that pirfenidone is a potential candidate for the treatment of NASH.

Effect of 1-Deoxynojirimycin Isolated from Mulberry Leaves on Glucose Metabolism and Gut Microbiota in a Streptozotocin-Induced Diabetic Mouse Model.

1-Deoxynojirimycin (DNJ) exerts hypoglycemic effects. However, the traditional method for DNJ extraction is inefficient, and the hypoglycemic mechanism of DNJ remains unclear. In this study, the mixed fermentation by Lactobacillus fermentum and Saccharomyces cerevisiae was used to enhance DNJ extraction efficiency. It was found that this strategy was more efficient than the traditional method as the yield improved from the original 3.24 mg/g to 5.97 mg/g. The purified DNJ significantly decreased serum glucose (P < 0.01) and insulin levels (P < 0.05), improved serum lipid levels (P < 0.05), and reversed insulin resistance (P < 0.05) in diabetic mice. These changes were caused by up-regulating the protein expression of insulin receptor and glycolysis enzymes (GK, PK, and PFK) (P < 0.05) and down-regulating the protein expression of insulin receptor substrate-1 and gluconeogenesis enzymes (PCB, PEPCK, FBPase, and G-6-Pase) (P < 0.05), thus alleviating glucose tolerance. Additionally, DNJ treatment relieved gut dysbiosis in diabetic mice by promoting the growth of Lactobacillus, Lachnospiraceae NK4A136 group, Oscillibacter, norank Lachnospiraceae, Alistipes, and Bifidobacterium (P < 0.05) and suppressing the growth of Ruminococcaceae UCG-014, Weissella, Ruminococcus, Prevotellaceae Ga6A1 group, Anaerostipes, Klebsiella, Prevotellaceae UCG-001, and Bacteroidales S24-7 group (P < 0.05).

Pterostilbene reverses palmitic acid mediated insulin resistance in HepG2 cells by reducing oxidative stress and triglyceride accumulation

Insulin resistance (IR) is known to precede onset of type 2 diabetes and increased oxidative stress appears to be a deleterious factor leading to IR. In this study, we evaluated ability of pterostilbene (PTS), a methoxylated analogue of resveratrol and a known antioxidant, to reverse palmitic acid (PA)-mediated IR in HepG2 cells. PTS prevented reactive oxygen species (ROS) formation and subsequent oxidative lipid damage by reducing the expression of NADPH oxidase 3 (NOX3) in PA treated HepG2 cells. Hepatic glucose production was used as a measure of IR and PTS reversed PA-mediated increase in hepatic glucose production by reducing expression of genes coding for gluconeogenic enzymes namely glucose-6-phosphatase (G6Pase), phosphoenolpyruvate carboxykinase (PEPCK), and pyruvate carboxylase (PC); and their transcription factors cAMP response element binding protein (CREB) and fork head class Box O (FOXO1) along with its coactivator peroxisome proliferator-activated receptor gamma co-activator-1 α (PGC1α). PTS reversed PA-mediated activation of c-Jun N-terminal kinase (JNK), which in turn altered insulin signalling pathway by phosphorylating IRS-1 at Ser 307, leading to inhibition of phosphorylation of Akt and GSK-3β. PTS also reduced PA-mediated lipid accumulation by reducing expression of transcription factors SREBP1c and PPARα. SREBP1c activates genes involved in fatty acid and triglyceride synthesis while PPARα activates CPT1, a rate limiting enzyme for controlling entry and oxidation of fatty acids into mitochondria. PTS, however, did not influence PA uptake confirmed by using BODIPY-labelled fluorescent C16 fatty acid analogue. Thus, our data provides a possible mechanistic explanation for reversal of PA-mediated IR in HepG2 cells.

19-OR: Controlled-Release Mitochondrial Protonophore (CRMP) Reverses Hypertriglyceridemia and Hepatic Steatosis in Dysmetabolic Nonhuman Primates

NAFLD is estimated to affect up to one third of the general population and is a major predisposing factor in the pathogenesis of hepatic insulin resistance, NASH, and T2D. While weight loss is highly effective at reversing NAFLD, insulin resistance and T2D, it is difficult to sustain and new therapies are required. Our lab has recently developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-targeted and promotes oxidation of hepatic triglycerides by promoting a subtle sustained increase in hepatic mitochondrial inefficiency. While we have previously demonstrated that CRMP safely reverses hypertriglyceridemia, fatty liver, and hepatic inflammation/fibrosis in diet-induced rodent models of obesity, there remains a critical need to assess the safety and efficacy in a model highly relevant to humans. Here, we evaluated the impact of CRMP treatment on hepatic mitochondrial oxidation and the reversal of hypertriglyceridemia, NAFLD, and insulin resistance in a spontaneous nonhuman primate model of NAFLD and the metabolic syndrome. Using Positional Isotopomer NMR Tracer Analysis (PINTA), we demonstrate that acute CRMP treatment (single dose of 5 mg/kg) significantly increases rates of hepatic mitochondrial fat oxidation by 40% (P&amp;lt;0.05). Importantly, CRMP treatment (5 mg/kg/day x 6 weeks) reduced hepatic and plasma triglycerides by 20% (both P=0.05) independently of changes in body weight, food intake, body temperature or adverse reactions. Furthermore, administration of CRMP significantly reduced endogenous glucose production by 18% (P&amp;lt;0.05), which could be attributed to a ~20% reduction in hepatic acetyl-CoA content (as assessed by whole-body βOHB turnover) and pyruvate carboxylase flux. Conclusion: These studies provide important proof-of-concept data to support the development of novel liver-targeted mitochondrial uncouplers for the treatment of NAFLD/NASH and T2D in humans. Disclosure L. Goedeke: None. V. Montalvo Romeral: None. G. Butrico: None. M. Kahn: None. S. Dufour: None. X. Zhang: None. G. Cline: None. K. Petersen: Advisory Panel; Spouse/Partner; Aegerion Pharmaceuticals, AstraZeneca, AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc., Novo Nordisk A/S. Consultant; Spouse/Partner; Gilead Sciences, Inc. Consultant; Self; Merck &amp; Co., Inc. Consultant; Spouse/Partner; Nimbus Discoverey. Consultant; Self; Novo Nordisk A/S. K. Chng: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding National Institutes of Health

991-P: Potential of a Novel Long-Acting Glucagon Analog, HM15136, for the Treatment of Obesity

Obesity is considered as a main risk factor for metabolic syndrome. Although there are several anti-obesity drugs, their main mode of action (MoA) is appetite suppression. Thus, body weight loss (BWL) efficacy is limited with benefits fading when the drugs stopped. Glucagon (GCG) has been used to treat hypoglycemia. Recent studies indicated a stimulatory effect of GCG on energy expenditure, suggesting its utilization as an anti-obese drug. Since BWL is a key for insulin resistance (IR) reversal and blood glucose (BG) normalization, we hypothesized that sustained GCG treatment may reduce hyperglycemic risk via IR improvement following efficient BWL. To investigate this hypothesis, we developed a novel long-acting GCG analog, HM15136, and evaluated the effect of HM15136 on BW and BG in vivo. In obese animals, HM15136 demonstrated dose-dependent BWL (-21 ∼ -53% vs. Veh. for DIO mice, -10 ∼ -64% vs. Veh. for DIO rats). Of note, despite an initial increase, BG level rapidly normalized. As to the responsible MoA, a reduced blood lipid and HOMA-IR were observed. In vitro studies demonstrated the increased FGF-21 and adiponectin expression by HM15136 in 3T3-L1 adipocytes. These results suggest that HM15136 could improve insulin sensitivity via multiple mechanisms, leading to normal BG maintenance under chronic GCG receptor stimulation. To further investigate the therapeutic potential of HM15136, BWL efficacy was compared to liraglutide: Greater BWL was observed with HM15136 (-34% and -21% vs. Veh. for HM15136 and liraglutide, respectively). Indirect calorimetry demonstrated enhanced energy expenditure by HM15136. Most interestingly, efficient BWL achieved by HM15136 was maintained during 4 weeks drug-holiday, compared to liraglutide. In conclusion, our results demonstrate the potential for prolonged GCG receptor stimulation with agent like HM15136 as a novel therapeutic option for obesity treatment. Human studies are needed to confirm these findings and to evaluate the potential safety of HM15136. Disclosure J. Kim: None. J. Kim: None. S. Lee: None. J. Lee: None. S. Lee: Employee; Self; Hanmi Pharm. Co., Ltd. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

165-OR: The Mechanisms by Which Adiponectin Reverses Insulin Resistance in High-Fat Diet Fed Mice

Adiponectin has emerged as an antidiabetic adipokine, and a potential therapy to treat type 2 diabetes. However, the mechanism by which adiponectin improves insulin sensitivity remains unclear. To address this question, we examined the effects of a 2-week continuous infusion of globular adiponectin (gAcrp30) or saline (CON) on glucose and lipid metabolism in a high fat diet fed (HFD) mouse model. Whole-body and tissue-specific insulin action was assessed by a hyperinsulinemic-euglycemic clamp (HEC). gAcrp30-treated mice displayed reduced fasting plasma glucose and insulin concentrations (both P&amp;lt;0.05 vs. CON) and increased glucose infusion rate (14 vs. 22 mg/(kg-min), P&amp;lt;0.001) during the HEC reflecting increased whole-body insulin-sensitivity. Increased insulin sensitivity could be attributed to reduced endogenous glucose production (16.7 vs.14.2 mg/(kg-min), P&amp;lt;0.05), increased glucose uptake in muscle (191 vs. 285 nmol/(kg-min), p=0.006) and adipose tissues (BAT: 112 vs. 217, WAT: 11 vs. 22 nmol/(kg-min), both P&amp;lt;0.05) during the HEC. These improvements in liver and muscle insulin sensitivity were associated with ∼50% reductions in liver and muscle triglyceride (TAG) and ∼40% diacylglycerol (DAG) (both P&amp;lt;0.01 vs. CON) content but no changes in hepatic ceramide content. Reduced tissue TAG/DAG content occurred independent of changes in WAT lipolysis, hepatic AMPK activation, ACC phosphorylation or mitochondrial oxidation. Reduced tissue DAG content was associated with decreased PKCε activation in liver and PKCθ activation in muscle and increased insulin signaling in liver, muscle and white adipose tissue. Conclusion: Taken together these data support the hypothesis that adiponectin improves insulin sensitivity in liver and muscle of HFD fed mice by reducing ectopic lipid (DAG) content in these tissues leading to decreased nPKC activity and increased insulin signaling, and contrary to prior studies, is independent of changes in hepatic ceramide content. Disclosure X. Li: None. D. Zhang: None. R.J. Perry: Research Support; Self; AstraZeneca. D.F. Vatner: None. L. Goedeke: None. Y. Zhang: None. G.I. Shulman: Advisory Panel; Self; AstraZeneca, Janssen Research &amp; Development, Merck &amp; Co., Inc. Advisory Panel; Spouse/Partner; Merck &amp; Co., Inc. Board Member; Self; Novo Nordisk A/S. Consultant; Self; Aegerion Pharmaceuticals, IMetabolic BioPharma Corporation, Longitude Capital, Nimbus Discovery, Inc., Staten Biotechnology B.V. Funding American Heart Association (19PRE34380268); National Institutes of Health (R01DK116774)

108-LB: Sustained Glucagon Effect on Blood Glucose and Improvements of Insulin Resistance Mediated by a Novel Long-Acting Glucagon Analog, HM15136, in Animal Models

HM15136 is a novel long-acting glucagon (GCG) analog conjugated with human IgG FC fragment via a short PEG linker. Previously, HM15136 treatment led to body weight loss (BWL) in obese animal models while showing transient effect on blood glucose (BG). Insulin resistance (IR) can lead to imbalances in BG homeostasis in obesity, representing a key risk factor for T2DM. Considering the potential benefit of BWL in IR improvement, HM15136 could be a novel therapeutic option for obesity treatment. To assess this concept, the present study evaluated the effect of HM15136 on BG as well as BW, and investigated the mechanism leading to IR improvement in vitro and in vivo. In DIO rats, 4 weeks treatment of HM15136 led to remarkable BWL (-17 ∼ -42% vs. Veh). BG monitoring showed that an initial BG elevation was rapidly normalized and maintained normal thereafter, with no HbA1c increase (3.4 and 3.7% for Veh and HM15136). Similar results were also observed in DIO mice, showing no abnormal glycemic control by HM15136. Interestingly, BWL effect were correlated with free-fatty acid (FFA) and HOMA-IR reduction. In vitro studies in 3T3-L1 adipocytes further confirmed the deteriorating effect of FFA on insulin signaling. These results suggested that improvements in the lipotoxic milieu explains, at least in part, the IR improvement seen with HM15136. To further explore additional mechanism, inflammatory markers and adipokines affecting insulin signaling were evaluated. HM15136 not only inhibited F4/80 expression, but also increased FGF-21 and adiponectin expression in white adipose tissue and 3T3-L1 adipocytes. Our results demonstrated that BWL under by HM15136 therapy is accompanied by the reduction of 1) lipotoxicity, 2) inflammatory biomarkers, and the expression of 3) insulin-sensitizing adipokines, eventually leading to IR reversal and normal BG maintenance. Human studies are needed to confirm whether these findings will translate into humans. Disclosure J. Choi: None. S. Lee: None. J. Kim: None. J. Lee: None. Y. Kim: None. I. Choi: Employee; Self; Hanmi Pharm. Co., Ltd.

OR05-5 AZP-3404, a 9-Amino Acid Peptide Analog of Insulin-Like Growth Factor-Binding Protein 2, Reverses Insulin Resistance in Leptin-Deficient ob/ob Mice

Insulin-like growth factor-binding protein-2 (IGFBP-2), one of six highly-conserved binding proteins that modulate circulating IGF1, has significant regulatory actions on carbohydrate, lipid and bone metabolism that are independent of its IGF-1 binding role. The metabolic activity of IGFBP-2 has been localized to a short peptide sequence within its unique heparin-binding domain, HBD-1, that is able to reproduce the metabolic actions of the full IGFBP-2 on bone and fat. Through structure-activity studies of this sequence, an acylated 9-amino acid peptide, AZP-3404, with enhanced stability and pharmacokinetics, has been developed that retains the activity of IGFBP-2 on fat and bone metabolism. As IGFBP-2 treatment has also been demonstrated to improve glucose homeostasis in animal models of severe insulin resistance, including the leptin-deficient ob/ob mouse, the present study was undertaken to determine whether AZP-3404 is able to reverse insulin resistance and improve glucose homeostasis in the ob/ob mouse. Male B6.Cg-Lepob/J mice were implanted with HD-XG telemetric devices (Data Sciences International), which allows continuous, wireless monitoring of carotid blood glucose under awake, unrestrained conditions. Animals were randomly assigned to groups (n=8) receiving either saline or AZP-3404 at 1 or 3 mg/kg, b.i.d. for four weeks. The mean daily blood glucose level prior to study initiation was 507 ± 102 mg/dL. Treatment with AZP-3404 dose-dependently decreased the average daily glucose concentration, such that by day 27, glucose levels were reduced by 64 ± 29 and 100 ± 16 mg/dL in the 1 and 3 mg/kg AZP-3404-treated groups, respectively, while slightly increased by 14 ± 29 mg/dL in the vehicle-treated group. On days 7, 14, 21 and 28, following a 24-hour fast, the mice were given an intraperitoneal glucose tolerance test (IPGTT: 1g glucose/kg) to evaluate the impact of AZP-3404 on glucose disposal which is impaired in ob/ob mice. By day 28, the fasting glucose level was reduced by 8% and 20% in the 1 and 3 mg/kg AZP-3404-treated groups, respectively, as compared with fasting levels on day 7. The IPGTTs revealed a progressive, dose-dependent reduction in glucose excursion that reached a decrease of 40% after 4 weeks of treatment with 3mg/kg AZP-3404. In related studies, we have observed that AZP-3404, both alone and in an additive manner with insulin, stimulates glucose transport in mouse C2C12 differentiated myotubes through activation of AMP-activated protein kinase (AMPK). These findings suggest that AZP- 3404 stimulates glucose uptake by muscle tissue in ob/ob mice thereby improving glucose homeostasis. The results from these studies confirm that AZP-3404 retains the ability of IGFBP-2 to restore glucose homeostasis and support the development of AZP-3404 as a novel therapeutic approach for syndromes of severe insulin resistance.