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Insulin-induced hypoglycemia in diabetes is associated with impaired glucagon secretion. In this study, we tested whether stimulation of GPR119, a G-protein-coupled receptor expressed in pancreatic islet as well as enteroendocrine cells and previously shown to stimulate insulin and incretin secretion, might enhance glucagon secretion during hypoglycemia. In the study, GPR119 agonists were applied to isolated islets or perfused pancreata to assess insulin and glucagon secretion during hypoglycemic or hyperglycemic conditions. Insulin infusion hypoglycemic clamps were performed with or without GPR119 agonist pretreatment to assess glucagon counterregulation in healthy and streptozotocin (STZ)-induced diabetic rats, including those exposed to recurrent bouts of insulin-induced hypoglycemia that leads to suppression of hypoglycemia-induced glucagon release. Hypoglycemic clamp studies were also conducted in GPR119 knockout (KO) mice to evaluate whether the pharmacological stimulatory actions of GPR119 agonists on glucagon secretion during hypoglycemia were an on-target effect. The results revealed that GPR119 agonist-treated pancreata or cultured islets had increased glucagon secretion during low glucose perfusion. In vivo, GPR119 agonists also significantly increased glucagon secretion during hypoglycemia in healthy and STZ-diabetic rats, a response that was absent in GPR119 KO mice. In addition, impaired glucagon counterregulatory responses were restored by a GPR119 agonist in STZ-diabetic rats that were exposed to antecedent bouts of hypoglycemia. Thus, GPR119 agonists have the ability to pharmacologically augment glucagon secretion, specifically in response to hypoglycemia in diabetic rodents. Whether this effect might serve to diminish the occurrence and severity of iatrogenic hypoglycemia during intensive insulin therapy in patients with diabetes remains to be established. [ABSTRACT FROM AUTHOR]

Lipid lowering properties of glucagon have been reported. Blocking glucagon signaling leads to rise in plasma LDL levels. Here, we demonstrate the lipid lowering effects of acute dosing with Glp1r/Gcgr dual agonist (DualAG). All the experiments were performed in 25 week-old male diet-induced (60% kCal fat) obese mice. After 2 hrs of fasting, mice were injected subcutaneously with vehicle, liraglutide (25nmol/kg) and DualAG (25nmol/kg). De novo cholesterol and palmitate synthesis was measured by deuterium incorporation method using D2O. 13C18-oleate infusion was used for measuring fatty acid esterification. Simultaneous activation of Glp1r and Gcgr resulted in decrease in plasma triglyceride and cholesterol levels. DualAG enhanced hepatic LDLr protein levels, along with causing decrease in content of plasma ApoB48 and ApoB100. VLDL secretion, de novo palmitate synthesis and fatty acid esterification decreased with acute DualAG treatment. On the other hand, ketone levels were elevated with DualAG treatment, indicating increased fatty acid oxidation. Lipid relevant changes were absent in liraglutide treated group. In an acute treatment, DualAG demonstrated significant impact on lipid homeostasis, specifically on hepatic uptake, VLDL secretion and de novo synthesis. These effects collectively reveal that lipid lowering abilities of DualAG are primarily through glucagon signaling and are liver centric. [ABSTRACT FROM AUTHOR]

Objectives: Platensimycin (PTM) is a natural antibiotic produced by Streptomyces platensis that selectively inhibits bacterial and mammalian fatty acid synthase (FAS) without affecting synthesis of other lipids. Recently, we reported that oral administration of PTM in mouse models (db/db and db/+) with high de novo lipogenesis (DNL) tone inhibited DNL and enhanced glucose oxidation, which in turn led to net reduction of liver triglycerides (TG), reduced ambient glucose, and improved insulin sensitivity. The present study was conducted to explore translatability and the therapeutic potential of FAS inhibition for the treatment of diabetes in humans. Methods: We tested PTM in animal models with different DNL tones, i.e. intrinsic synthesis rates, which vary among species and are regulated by nutritional and disease states, and confirmed glucose-lowering efficacy of PTM in lean NHPs with quantitation of liver lipid by MRS imaging. To understand the direct effect of PTM on liver metabolism, we performed ex vivo liver perfusion study to compare FAS inhibitor and carnitine palmitoyltransferase 1 (CPT1) inhibitor. Results: The efficacy of PTM is generally reproduced in preclinical models with DNL tones comparable to humans, including lean and established diet-induced obese (eDIO) mice as well as non-human primates (NHPs). Similar effects of PTM on DNL reduction were observed in lean and type 2 diabetic rhesus and lean cynomolgus monkeys after acute and chronic treatment of PTM. Mechanistically, PTM lowers plasma glucose in part by enhancing hepatic glucose uptake and glycolysis. Teglicar, a CPT1 inhibitor, has similar effects on glucose uptake and glycolysis. In sharp contrast, Teglicar but not PTM significantly increased hepatic TG production, thus caused liver steatosis in eDIO mice. Conclusions: These findings demonstrate unique properties of PTM and provide proof-of-concept of FAS inhibition having potential utility for the treatment of diabetes and related metabolic disorders. [ABSTRACT FROM AUTHOR]

Objective: Bariatric surgery induces weight loss and improvement of insulin resistance; one aspect of both is an amelioration of hepatic steatosis. This study was undertaken to assess the changes in the hepatic lipidome after duodenal-jejunal bypass (DJB) surgery.Methods: A DJB surgical model was developed and characterized in diet-induced obese mice. In comparison with sham-operated mice, an unbiased lipidomic profiling of hepatic lipids was performed together with measurements of gene expression within key pathways of hepatic lipid metabolism.Results: In the liver of DJB mice, a dramatic reduction (by 77%) in hepatic triacylglycerols was observed. Global lipidomic profiling identified marked decreases of triacylglycerols comprised of medium length fatty acids and with low double bond content. Specific diacylglycerol species were also among the most dramatic decreases in hepatic lipids, whereas lysophosphatidic acids and phosphatidic acids were increased. Expression of fatty acid transporter and lipogenic genes was down-regulated.Conclusions: From in-depth analysis of hepatic lipid composition, specific lipid intermediates were identified that are preferentially changed following DJB surgery. These changes were most likely due to DJB-induced weight loss, and only further studies will be able to distinguish weight loss-dependent from weight loss-independent changes. [ABSTRACT FROM AUTHOR]

Biliopancreatic diversion (BPD) improves insulin sensitivity and decreases serum cholesterol out of proportion with weight loss. Mechanisms of these effects are unknown. One set of proposed contributors to metabolic improvements after bariatric surgeries is bile acids (BAs). We investigated the early and late effects of BPD on plasma BA levels, composition, and markers of BA synthesis in 15 patients with type 2 diabetes (T2D). We compared these to the early and late effects of Roux-en-Y gastric bypass (RYGB) in 22 patients with T2D and 16 with normal glucose tolerance. Seven weeks after BPD, insulin sensitivity had doubled and serum cholesterol had halved. At this time, BA synthesis markers and total plasma BAs, particularly unconjugated BAs, had markedly risen; this effect could not be entirely explained by low FGF19. In contrast, after RYGB, insulin sensitivity improved gradually with weight loss and cholesterol levels declined marginally; BA synthesis markers were decreased at an early time point (2 weeks) after surgery and returned to the normal range 1 year later. These findings indicate that BA synthesis contributes to the decreased serum cholesterol after BPD. Moreover, they suggest a potential role for altered enterohepatic circulation of BAs in improving insulin sensitivity and cholesterol metabolism after BPD. [ABSTRACT FROM AUTHOR]

Objective Identify a gene expression signature in white adipose tissue (WAT) that reports on WAT browning and is associated with a healthy phenotype. Methods RNA from several different adipose depots across three species were analyzed by whole transcriptome profiling, including 1) mouse subcutaneous white fat, brown fat, and white fat after in vivo treatment with FGF21; 2) human subcutaneous and omental fat from insulin-sensitive and insulin-resistant patients; and 3) rhesus monkey subcutaneous fat from healthy and dysmetabolic individuals. Results A 'browning' signature in mice was identified by cross-referencing the FGF21-induced signature in WAT with the brown adipose tissue (BAT) vs. WAT comparison. In addition, gene expression levels in WAT from insulin-sensitive/healthy vs. insulin-resistant/dysmetabolic humans and rhesus monkeys, respectively, correlated with the gene expression levels in mouse BAT vs. WAT. A subset of 49 genes were identified that were consistently regulated or differentially expressed in the mouse and human data sets that could be used to monitor browning of WAT across species. Conclusions Gene expression profiles of WATs from healthy insulin-sensitive individuals correlate with those of BAT and FGF21-induced browning of WAT. [ABSTRACT FROM AUTHOR]

Insulin resistance is the key feature of type 2 diabetes and is manifested as attenuated insulin receptor (IR) signaling in response to same levels of insulin binding. Several small molecule IR activators have been identified and reported to exhibit insulin sensitization properties. One of these molecules, TLK19781 (Cmpd1), was investigated to examine its IR sensitizing action in vivo. Our data demonstrate that Cmpd1, at doses that produced minimal efficacy in the absence of insulin, potentiated insulin action during an OGTT in non-diabetic mice and enhanced insulin-mediated glucose lowering in diabetic mice. Interestingly, different from insulin alone, Cmpd1 combined with insulin showed enhanced efficacy and duration of action without increased hypoglycemia. To explore the mechanism underlying the apparent glucose dependent efficacy, tissue insulin signaling was compared in healthy and diabetic mice. Cmpd1 enhanced insulin’s effects on IR phosphorylation in both healthy and diabetic mice. In contrast, the compound potentiated insulin’s effects on Akt phosphorylation in diabetic but not in non-diabetic mice. These differential effects on signaling corresponding to glucose levels could be part of the mechanism for reduced hypoglycemia risk. The in vivo efficacy of Cmpd1 is specific and dependent on IR expression. Results from these studies support the idea of targeting IR for insulin sensitization, which carries low hypoglycemia risk by standalone treatment and could improve the effectiveness of insulin therapies. [ABSTRACT FROM AUTHOR]

The liver is a crossroad for metabolism of lipid and carbohydrates, with acetyl-CoA serving as an important metabolic intermediate and a precursor for fatty acid and cholesterol biosynthesis pathways. A better understanding of the regulation of these pathways requires an experimental approach that provides both quantitative metabolic flux measurements and mechanistic insight. Under conditions of high carbohydrate availability, excess carbon is converted into free fatty acids and triglyceride for storage, but it is not clear how excessive carbohydrate availability affects cholesterol biosynthesis. To address this, C57BL/6J mice were fed either a low-fat, high-carbohydrate diet or a high-fat, carbohydrate- free diet. At the end of the dietary intervention, the two groups received 2H2O to trace de novo fatty acid and cholesterol synthesis, and livers were collected for gene expression analysis. Expression of lipid and glucose metabolism genes was determined using a customdesigned pathway focused PCR-based gene expression array. The expression analysis showed downregulation of cholesterol biosynthesis genes and upregulation of fatty acid synthesis genes in mice receiving the high-carbohydrate diet compared with the carbohydratefree diet. In support of these findings, 2H2O tracer data showed that fatty acid synthesis was increased 10-fold and cholesterol synthesis was reduced by 1.6-fold in mice fed the respective diets. In conclusion, by applying gene expression analysis and tracer methodology, we show that fatty acid and cholesterol synthesis are differentially regulated when the carbohydrate intake in mice is altered. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Genome-wide association studies (GWAS) are useful in studying the complex pathways underlying diseases such as atherosclerosis; however, additional testing is often necessary to identify the disease causal genes linked to GWAS loci. We used siRNA-mediated gene knockdown in primary human hepatocytes (PHuH) to identify potential GWAS causal genes affecting the hepatic secretion of apolipoprotein B (ApoB), ApoA1, and proprotein convertase subtilisin/kexin type 9. Materials and methods: Candidate causal genes within GWAS loci affecting human plasma levels of total cholesterol, LDL-cholesterol, HDL-cholesterol, and triglycerides were identified from the literature; 191 genes were selected from 74 loci. A functional siRNA screen was performed using PHuH. Results: Four genes: poly (ADP-ribose) polymerases member 10, haptoglobin, fucosyltransferase 1, and lysophosphatidic acid receptor 2 were identified and confirmed. Knocking down these genes reduced cell-associated and secreted ApoB levels. Conclusion: Modification of these four genes may affect plasma lipids through modulation of ApoB secretion. [Copyright &y& Elsevier]

Summary: The emergence of drug-resistant bacteria coupled with the limited discovery of novel chemical scaffolds and druggable targets inspires new approaches to antibiotic development. Here we describe a chemical genomics strategy based on 245 Staphylococcus aureus antisense RNA strains, each engineered for reduced expression of target genes essential for S. aureus growth. Attenuation of gene expression can sensitize cells to compounds that inhibit the activity of a gene product or associated process. Pools of strains grown competitively in the presence of bioactive compounds generate characteristic profiles of strain sensitivities reflecting compound mechanism of action. Here, we validate this approach with a structurally and mechanistically diverse set of reference antibiotics and, in the accompanying paper in this issue of Chemistry & Biology (), demonstrate its use in the discovery of new cell wall inhibitors. [Copyright &y& Elsevier]