Featured ArticlesVolume 4 | No. 12 | December 2015
|Bioenergetic cues shift FXR splicing towards FXRα2Farnesoid X receptor (FXR) consists of four distinct proteins and is a promising target for the treatment of non-alcoholic fatty liver disease (NAFLD). Correia and colleagues show that FXR variants regulate hepatic lipid metabolism in an isoform-dependent manner. This constitutes a novel mechanism by which alternative FXR splicing in the liver integrates systemic energetic demands with a gene program of enhanced lipid handling/utilization and ketogenesis that positively impacts hepatic steatosis and insulin sensitivity.|
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Objective: Farnesoid X receptor (FXR) plays a prominent role in hepatic lipid metabolism. The FXR gene encodes four proteins with structural differences suggestive of discrete biological functions about which little is known.
Methods: We expressed each FXR variant in primary hepatocytes and evaluated global gene expression, lipid profile, and metabolic fluxes. Gene delivery of FXR−/− variants to Fxr mouse liver was performed to evaluate their role in vivo. The effects of fasting and physical exercise on hepatic Fxr splicing were determined.
Results: We show that FXR splice isoforms regulate largely different gene sets and have specific effects on hepatic metabolism. FXRα2 (but not α1) activates a broad transcriptional program in hepatocytes conducive to lipolysis, fatty acid oxidation, and ketogenesis. Consequently, FXRα2 decreases cellular lipid accumulation and improves cellular insulin signaling to AKT. FXRα2 expression in Fxr−/− mouse liver activates a similar gene program and robustly decreases hepatic triglyceride levels. On the other hand, FXRα1 reduces hepatic triglyceride content to a lesser extent and does so through regulation of lipogenic gene expression. Bioenergetic cues, such as fasting and exercise, dynamically regulate Fxr splicing in mouse liver to increase Fxrα2 expression.
Conclusions: Our results show that the main FXR variants in human liver (α1 and α2) reduce hepatic lipid accumulation through distinct mechanisms and to different degrees. Taking this novel mechanism into account could greatly improve the pharmacological targeting and therapeutic efficacy of FXR agonists.[Hide abstract]
|High intensity interval training improves liver and adipose tissue insulin sensitivityMarcinko and colleagues show that high intensity interval training (HIIT) improves exercise capacity and whole-body glucose homeostasis by enhancing liver and adipose tissue insulin sensitivity. These effects are independent of reductions in adiposity/adipose tissue cell size. Improved insulin sensitivity is also independent of adipose tissue inflammation, macrophage infiltration/inflammation or reductions in liver lipid content, indicating dissociation between these parameters and insulin resistance. They demonstrate that HIIT exercise training improves insulin sensitivity independently of the AMPK-ACC signaling pathway.|
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Objective: Endurance exercise training reduces insulin resistance, adipose tissue inflammation and non-alcoholic fatty liver disease (NAFLD), an effect often associated with modest weight loss. Recent studies have indicated that high-intensity interval training (HIIT) lowers blood glucose in individuals with type 2 diabetes independently of weight loss; however, the organs affected and mechanisms mediating the glucose lowering effects are not known. Intense exercise increases phosphorylation and inhibition of acetyl-CoA carboxylase (ACC) by AMP-activated protein kinase (AMPK) in muscle, adipose tissue and liver. AMPK and ACC are key enzymes regulating fatty acid metabolism, liver fat content, adipose tissue inflammation and insulin sensitivity but the importance of this pathway in regulating insulin sensitivity with HIIT is unknown.
Methods: In the current study, the effects of 6 weeks of HIIT were examined using obese mice with serine–alanine knock-in mutations on the AMPK phosphorylation sites of ACC1 and ACC2 (AccDKI) or wild-type (WT) controls.
Results: HIIT lowered blood glucose and increased exercise capacity, food intake, basal activity levels, carbohydrate oxidation and liver and adipose tissue insulin sensitivity in HFD-fed WT and AccDKI mice. These changes occurred independently of weight loss or reductions in adiposity, inflammation and liver lipid content.
Conclusions: These data indicate that HIIT lowers blood glucose levels by improving adipose and liver insulin sensitivity independently of changes in adiposity, adipose tissue inflammation, liver lipid content or AMPK phosphorylation of ACC.[Hide abstract]
|A human beta cell line with drug inducible excision of immortalizing transgenesAccess to human beta cells is crucial to progress in understanding human-specific beta cell function. Benazra and colleagues have produced a novel human beta cell line derived from EndoC-βH2 cells that contains floxed immortalizing transgenes and an integrated Tamoxifen inducible form of CRE recombinase. This new line offers a simple and efficient procedure to mass produce non-proliferative human beta cells that can maintain a stable phenotype for 5 weeks in culture.|
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Objective: Access to immortalized human pancreatic beta cell lines that are phenotypically close to genuine adult beta cells, represent a major tool to better understand human beta cell physiology and develop new therapeutics for Diabetes. Here we derived a new conditionally immortalized human beta cell line, EndoC-βH3 in which immortalizing transgene can be efficiently removed by simple addition of tamoxifen.
Methods: We used lentiviral mediated gene transfer to stably integrate a tamoxifen inducible form of CRE (CRE-ERT2) into the recently developed conditionally immortalized EndoC βH2 line. The resulting EndoC-βH3 line was characterized before and after tamoxifen treatment for cell proliferation, insulin content and insulin secretion.
Results: We showed that EndoC-βH3 expressing CRE-ERT2 can be massively amplified in culture. We established an optimized tamoxifen treatment to efficiently excise the immortalizing transgenes resulting in proliferation arrest. In addition, insulin expression raised by 12 fold and insulin content increased by 23 fold reaching 2 µg of insulin per million cells. Such massive increase was accompanied by enhanced insulin secretion upon glucose stimulation. We further observed that tamoxifen treated cells maintained a stable function for 5 weeks in culture.
Conclusions: EndoC βH3 cell line represents a powerful tool that allows, using a simple and efficient procedure, the massive production of functional non-proliferative human beta cells. Such cells are close to genuine human beta cells and maintain a stable phenotype for 5 weeks in culture.[Hide abstract]
|Accumulation of 3-hydroxytetradecenoic acid: Cause or corollary of glucolipotoxic impairment of bioenergetics?One of the processes implicated in fatty acid-induced pancreatic beta cell dysfunction is impairment of bioenergetics. Doliba and colleagues show that islets cultured under glucolipotoxic conditions suffer a marked perturbation of energy metabolism. They propose defective bioenergetics as main cause of glucolipotoxicity.|
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Objective: Hyperglycemia and elevated blood lipids are the presumed precipitating causes of β-cell damage in T2DM as the result of a process termed “glucolipotoxicity”. Here, we tested whether glucolipotoxic pathophysiology is caused by defective bioenergetics using islets in culture.
Methods: Insulin secretion, respiration, ATP generation, fatty acid (FA) metabolite profiles and gene expression were determined in isolated islets treated under glucolipotoxic culture conditions.
Results: Over time, chronic exposure of mouse islets to FAs with glucose leads to bioenergetic failure and reduced insulin secretion upon stimulation with glucose or amino acids. Islets exposed to glucolipotoxic conditions displayed biphasic changes of the oxygen consumption rate (OCR): an initial increase in baseline and Vmax of OCR after 3 days, followed by decreased baseline and glucose stimulated OCR after 5 days. These changes were associated with lower islet ATP levels, impaired glucose-induced ATP generation, a trend for reduced mitochondrial DNA content and reduced expression of mitochondrial transcription factor A (Tfam). We discovered the accumulation of carnitine esters of hydroxylated long chain FAs, in particular 3-hydroxytetradecenoyl-carnitine.
Conclusions: As long chain 3-hydroxylated FA metabolites are known to uncouple heart and brain mitochondria [53–55] , we propose that under glucolipotoxic condition, unsaturated hydroxylated long-chain FAs accumulate, uncouple and ultimately inhibit β-cell respiration. This leads to the slow deterioration of mitochondrial function progressing to bioenergetics β-cell failure.[Hide abstract]
|α/β-Hydrolase domain-6 and saturated long chain monoacylglycerol regulate insulin secretionGlucose stimulated insulin secretion (GSIS) is a biphasic process with a rapid first phase followed by a sustained second phase. Zhao and colleagues provide evidence that α/β-hydrolase domain-6 (ABHD6) accessible monoacylglycerol (MAG), which is produced during nutrient metabolism, synergizes with other cellular signals arising from fuel and some non-fuel stimuli to potentiate insulin secretion and that MAG activates the KATP-independent pathway and second phase of secretion. The data highlight the importance of ABHD6 and MAG in regulating insulin secretion in response to all stimuli to control glucose homeostasis.|
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Objective: α/β-Hydrolase domain-6 (ABHD6) is a newly identified monoacylglycerol (MAG) lipase. We recently reported that it negatively regulates glucose stimulated insulin secretion (GSIS) in the β cells by hydrolyzing lipolysis-derived MAG that acts as a metabolic coupling factor and signaling molecule via exocytotic regulator Munc13-1. Whether ABHD6 and MAG play a role in response to all classes of insulin secretagogues, in particular various fuel and non-fuel stimuli, is unknown.
Methods: Insulin secretion in response to various classes of secretagogues, exogenous MAG and pharmacological agents was measured in islets of mice deficient in ABHD6 specifically in the β cell (BKO). Islet perifusion experiments and determinations of glucose and fatty acid metabolism, cytosolic Ca2+ and MAG species levels were carried out.
Results: Deletion of ABHD6 potentiated insulin secretion in response to the fuels glutamine plus leucine and α-ketoisocaproate and to the non-fuel stimuli glucagon-like peptide 1, carbamylcholine and elevated KCl. Fatty acids amplified GSIS in control and BKO mice to the same extent. Exogenous 1-MAG amplified insulin secretion in response to fuel and non-fuel stimuli. MAG hydrolysis activity was greatly reduced in BKO islets without changes in total diacylglycerol and triacylglycerol lipase activity. ABHD6 deletion induced insulin secretion independently from KATP channels and did not alter the glucose induced rise in intracellular Ca2+. Perifusion studies showed elevated insulin secretion during second phase of GSIS in BKO islets that was not due to altered cytosolic Ca2+ signaling or because of changes in glucose and fatty acid metabolism. Glucose increased islet saturated long chain 1-MAG species and ABHD6 deletion caused accumulation of these 1-MAG species at both low and elevated glucose.
Conclusion: ABHD6 regulates insulin secretion in response to fuel stimuli at large and some non-fuel stimuli by controlling long chain saturated 1-MAG levels that synergize with other signaling pathways for secretion.[Hide abstract]
|Hepatocyte TRAF3 promotes insulin resistance and type 2 diabetesChen and colleagues have identified hepatic TNF receptor-associated factor 3 (TRAF3) as a new metabolic regulator in obese mice. Liver TRAF3 levels are abnormally higher in obesity. Hepatocyte-specific deletion of TRAF3 attenuates insulin resistance, glucose intolerance, and hepatic steatosis in obese mice. Obesity-associated factors trigger and/or amplify a glucose-TRAF3 reinforcement loop in the liver, which is likely to drive type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) progression. Disrupting this glucose-TRAF3 positive feedback loop may serve as a new strategy to treat type 2 diabetes and NAFLD.|
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Objective: Metabolic inflammation is believed to promote insulin resistance and type 2 diabetes progression in obesity. TRAF3, a cytoplasmic signaling protein, has been known to mediate/modulate cytokine signaling in immune cells. The goal is to define the metabolic function of hepatic TRAF3 in the setting of obesity.
Methods: Hepatocyte-specific TRAF3 knockout mice were generated using the loxp/albumin-cre system. Liver TRAF3 was deleted in adult obese mice via Cre adenoviral infection. Both high fat diet-induced and genetic obesity were examined. TRAF3 levels and insulin signaling were measured by immunoblotting. Insulin sensitivity, hepatic glucose production, and glucose metabolism were examined by glucose, insulin, and pyruvate tolerance tests. Hepatic steatosis was examined by Oil red O staining of liver sections and measuring liver triacylglycerol levels.
Results: Liver TRAF3 levels were lower in the fasted states in normal mice, and were aberrantly higher in obese mice and in mice with streptozotocin-induced hyperglycemia. Glucose directly increased TRAF3 levels in primary hepatocytes. Hepatocyte-specific deletion of TRAF3 decreased hyperinsulinemia, insulin resistance, glucose intolerance, and hepatic steatosis in mice with either high fat diet-induced obesity or genetic obesity (ob/ob); conversely, in lean mice, adenovirus-mediated overexpression of TRAF3 in the liver induced hyperinsulinemia, insulin resistance, and glucose intolerance. Deletion of TRAF3 enhanced the ability of insulin to stimulate phosphorylation of Akt in hepatocytes, whereas overexpression of TRAF3 suppressed insulin signaling.
Conclusions: Glucose increases the levels of hepatic TRAF3. TRAF3 in turn promotes hyperglycemia through increasing hepatic glucose production, thus forming a glucose-TRAF3 reinforcement loop in the liver. This positive feedback loop may drive the progression of type 2 diabetes and nonalcoholic fatty liver disease in obesity.[Hide abstract]
|Hypothalamic CaMKKβ mediates glucagon anorectic effect and its diet-induced resistanceGlucagon counteracts hypoglycemia and opposes insulin actions by stimulating hepatic glucose synthesis and mobilization. Quiñones and colleagues show that glucagon causes a rapid and transient decrease in food intake through a mechanism involving the glucagon receptor, protein kinase A, Ca2+ -calmodulin-dependent protein kinase kinase β (CaMKKβ) and AMPK within the arcuate nucleus (ARC). This functional pathway is blunted in diet-induced obese rats, suggesting a high fat diet-induced resistance to the anorectic action of central glucagon. The data describe the molecular underpinnings by which glucagon controls feeding that may lead to a better understanding of anorexia and cachexia.|
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Objective: Glucagon receptor antagonists and humanized glucagon antibodies are currently studied as promising therapies for obesity and type II diabetes. Among its variety of actions, glucagon reduces food intake, but the molecular mechanisms mediating this effect as well as glucagon resistance are totally unknown.
Methods: Glucagon and adenoviral vectors were administered in specific hypothalamic nuclei of lean and diet-induced obese rats. The expression of neuropeptides controlling food intake was performed by in situ hybridization. The regulation of factors of the glucagon signaling pathway was assessed by western blot.
Results: The central injection of glucagon decreased feeding through a hypothalamic pathway involving protein kinase A (PKA)/Ca2+-calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMP-activated protein kinase (AMPK)-dependent mechanism. More specifically, the central injection of glucagon increases PKA activity and reduces protein levels of CaMKKβ and its downstream target phosphorylated AMPK in the hypothalamic arcuate nucleus (ARC). Consistently, central glucagon significantly decreased AgRP expression. Inhibition of PKA and genetic activation of AMPK in the ARC blocked glucagon-induced anorexia in lean rats. Genetic down-regulation of glucagon receptors in the ARC stimulates fasting-induced hyperphagia. Although glucagon was unable to decrease food intake in DIO rats, glucagon sensitivity was restored after inactivation of CaMKKβ, specifically in the ARC. Thus, glucagon decreases food intake acutely via PKA/CaMKKβ/AMPK dependent pathways in the ARC, and CaMKKβ mediates its obesity-induced hypothalamic resistance.
Conclusions: This work reveals the molecular underpinnings by which glucagon controls feeding that may lead to a better understanding of disease states linked to anorexia and cachexia.[Hide abstract]
|GLP-1 sensitivity despite insulin resistance in obese humansThe data of Heni and colleagues support a model in which GLP-1 is secreted after food intake, and subsequently reaches the brain where it regulates food-related activity of the orbitofrontal cortex, independently of insulin. While obese persons seem to be insulin-resistant in this brain area, they might remain sensitive to GLP-1. The orbitofrontal GLP-1 response might regulate reward and reduce hunger, which could help individuals to stop eating and prevent further food intake.|
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Objective: Glucagon-like peptide-1 (GLP-1) is released into the bloodstream after food intake. In addition to stimulating insulin release, it causes satiety and contributes to the termination of food intake. In this study, we investigated whether endogenous GLP-1 affects food-related brain activity and hunger.
Methods: Twenty-four volunteers (12 lean; 12 obese) underwent a 75 g oral glucose tolerance test that promotes GLP-1 secretion. Food cue-induced brain activity was assessed by functional magnetic resonance imaging and GLP-1 concentrations were measured before, 30, and 120 min after glucose intake.
Results: The significant increase in GLP-1 levels negatively correlated with a change in the food cue-induced brain activity in the orbitofrontal cortex, a major reward area. This association was independent of simultaneous alterations in insulin and glucose concentrations. The association was present in lean and overweight participants. By contrast, postprandial insulin changes were associated with orbitofrontal activations in lean individuals only.
Conclusions: The postprandial release of GLP-1 might alter reward processes in the orbitofrontal cortex and might thereby support the termination of food intake and reduce hunger. While obese persons showed brain insulin resistance, no GLP-1 resistance was observed. Our study provides novel insight into the central regulation of food intake by the incretin hormone GLP-1.[Hide abstract]