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Aims/hypothesis Drug and surgical-based therapies in type 2 diabetes are associated with altered gut microbiota architecture. Here we investigated the role of the gut microbiome in improved glucose homeostasis following bariatric surgery. Methods We carried out gut microbiome analyses in gastrectomised (by vertical sleeve gastrectomy [VSG]) rats of the Goto–Kakizaki (GK) non-obese model of spontaneously occurring type 2 diabetes, followed by physiological studies in the GK rat. Results VSG in the GK rat led to permanent improvement of glucose tolerance associated with minor changes in the gut microbiome, mostly characterised by significant enrichment of caecal Prevotella copri. Gut microbiota enrichment with P. copri in GK rats through permissive antibiotic treatment, inoculation of gut microbiota isolated from gastrectomised GK rats, and direct inoculation of P. copri, resulted in significant improvement of glucose tolerance, independent of changes in body weight. Plasma bile acids were increased in GK rats following inoculation with P. copri and P. copri-enriched microbiota from VSG-treated rats; the inoculated GK rats then showed increased liver glycogen and upregulated expression of Fxr (also known as Nr1h4), Srebf1c, Chrebp (also known as Mlxipl) and Il10 and downregulated expression of Cyp7a1. Conclusions Our data underline the impact of intestinal P. copri on improved glucose homeostasis through enhanced bile acid metabolism and farnesoid X receptor (FXR) signalling, which may represent a promising opportunity for novel type 2 diabetes therapeutics.

Dietary n-3 polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), are known to influence glucose homeostasis. We recently showed that Elovl2 expression in beta cells, which regulates synthesis of endogenous DHA, was associated with glucose tolerance and played a key role in insulin secretion. The present study aimed to examine the role of the very long chain fatty acid elongase 2 (ELOVL2)/DHA axis on the adverse effects of palmitate with high glucose, a condition defined as glucolipotoxicity, on beta cells. We detected ELOVL2 in INS-1 beta cells and mouse and human islets using quantitative PCR and western blotting. Downregulation and adenoviral overexpression of Elovl2 was carried out in beta cells. Ceramide and diacylglycerol levels were determined by radio-enzymatic assay and lipidomics. Apoptosis was quantified using caspase-3 assays and poly (ADP-ribose) polymerase cleavage. Palmitate oxidation and esterification were determined by [U-14C]palmitate labelling. We found that glucolipotoxicity decreased ELOVL2 content in rodent and human beta cells. Downregulation of ELOVL2 drastically potentiated beta cell apoptosis induced by glucolipotoxicity, whereas adenoviral Elovl2 overexpression and supplementation with DHA partially inhibited glucolipotoxicity-induced cell death in rodent and human beta cells. Inhibition of beta cell apoptosis by the ELOVL2/DHA axis was associated with a decrease in ceramide accumulation. However, the ELOVL2/DHA axis was unable to directly alter ceramide synthesis or metabolism. By contrast, DHA increased palmitate oxidation but did not affect its esterification. Pharmacological inhibition of AMP-activated protein kinase and etomoxir, an inhibitor of carnitine palmitoyltransferase 1 (CPT1), the rate-limiting enzyme in fatty acid β-oxidation, attenuated the protective effect of the ELOVL2/DHA axis during glucolipotoxicity. Downregulation of CPT1 also counteracted the anti-apoptotic action of the ELOVL2/DHA axis. By contrast, a mutated active form of Cpt1 inhibited glucolipotoxicity-induced beta cell apoptosis when ELOVL2 was downregulated. Our results identify ELOVL2 as a critical pro-survival enzyme for preventing beta cell death and dysfunction induced by glucolipotoxicity, notably by favouring palmitate oxidation in mitochondria through a CPT1-dependent mechanism.

Brain lipid sensing is necessary to regulate energy balance. Lipoprotein lipase (LPL) may play a role in this process. We tested if hippocampal LPL regulated energy homeostasis in rodents by specifically attenuating LPL activity in the hippocampus of rats and mice, either by infusing a pharmacological inhibitor (tyloxapol), or using a genetic approach (adeno-associated virus expressing Cre-GFP injected into Lpl (lox/lox) mice). Decreased LPL activity by either method led to increased body weight gain due to decreased locomotor activity and energy expenditure, concomitant with increased parasympathetic tone (unchanged food intake). Decreased LPL activity in both models was associated with increased de novo ceramide synthesis and neurogenesis in the hippocampus, while intrahippocampal infusion of de novo ceramide synthesis inhibitor myriocin completely prevented body weight gain. We conclude that hippocampal lipid sensing might represent a core mechanism for energy homeostasis regulation through de novo ceramide synthesis.

Dietary supplement may potentially help to fight obesity and other metabolic disorders such as insulin-resistance and low-grade inflammation. The present study aimed to test whether supplementation with Agaricus blazei murill (ABM) extract could have an effect on diet-induced obesity in rats. Wistar rats were fed with control diet (CD) or high-fat diet (HF) and either with or without supplemented ABM for 20 weeks. HF diet-induced body weight gain and increased fat mass compared to CD. In addition HF-fed rats developed hyperleptinemia and insulinemia as well as insulin resistance and glucose intolerance. In HF-fed rats, visceral adipose tissue also expressed biomarkers of inflammation. ABM supplementation in HF rats had a protective effect against body weight gain and all study related disorders. This was not due to decreased food intake which remained significantly higher in HF rats whether supplemented with ABM or not compared to control. There was also no change in gut microbiota composition in HF supplemented with ABM. Interestingly, ABM supplementation induced an increase in both energy expenditure and locomotor activity which could partially explain its protective effect against diet-induced obesity. In addition a decrease in pancreatic lipase activity is also observed in jejunum of ABM-treated rats suggesting a decrease in lipid absorption. Taken together these data highlight a role for ABM to prevent body weight gain and related disorders in peripheral targets independently of effect in food intake in central nervous system.

Obesity-related diseases such as diabetes and dyslipidemia result from metabolic alterations including the defective conversion, storage and utilization of nutrients, but the central mechanisms that regulate this process of nutrient partitioning remain elusive. As positive regulators of feeding behaviour, agouti-related protein (AgRP) producing neurons are indispensible for the hypothalamic integration of energy balance. Here, we demonstrate a role for AgRP-neurons in the control of nutrient partitioning. We report that ablation of AgRP-neurons leads to a change in autonomic output onto liver, muscle and pancreas affecting the relative balance between lipids and carbohydrates metabolism. As a consequence, mice lacking AgRP-neurons become obese and hyperinsulinemic on regular chow but display reduced body weight gain and paradoxical improvement in glucose tolerance on high-fat diet. These results provide a direct demonstration of a role for AgRP-neurons in the coordination of efferent organ activity and nutrient partitioning, providing a mechanistic link between obesity and obesity-related disorders.

International audience; Feeding behavior is exquisitely regulated by homeostatic and hedonic neural substrates that integrate energy demand as well as the reinforcing and rewarding aspects of food. Understanding the net contribution of homeostatic and reward-driven feeding has become critical because of the ubiquitous source of energy-dense foods and the consequent obesity epidemic. Hypothalamic agouti-related peptide-secreting neurons (AgRP neurons) provide the primary orexigenic drive of homeostatic feeding. Using models of neuronal inhibition or ablation, we demonstrate that the feeding response to a fast ghrelin or serotonin receptor agonist relies on AgRP neurons. However, when palatable food is provided, AgRP neurons are dispensable for an appropriate feeding response. In addition, AgRP-ablated mice present exacerbated stress-induced anorexia and palatable food intake--a hallmark of comfort feeding. These results suggest that, when AgRP neuron activity is impaired, neural circuits sensitive to emotion and stress are engaged and modulated by food palatability and dopamine signaling.

It is now clearly demonstrated that fatty acids (FAs) may modulate neural control of energy homeostasis and specifically affect both insulin secretion and action. Indeed, pancreatic β-cells receive rich neural innervation and FAs induce important changes in autonomic nervous activity. We previously reported that chronic infusion of lipids decreased sympathetic nervous system activity and led to exaggerated glucose-induced insulin secretion (GIIS), as would be expected from the known inhibitory effect of sympathetic splanchnic nerve activity on insulin secretion. Intracarotid infusion of lipids that do not change plasma FA concentrations also lead to increased GIIS. This effect of FAs on GIIS was prevented by inhibition of β-oxidation. It is noteworthy that a single intracarotid injection of oleic acid also induced a transient increase in plasma insulin without any change in plasma glucose, suggesting that FAs per se can regulate neural control of insulin secretion. Finally, using whole cell current clamp recordings in hypothalamic slices and calcium imaging in dissociated hypothalamic neurons, we identified a hypothalamic subpopulation of neurons either excited (13%) or inhibited (6%) by FAs. Thus, FAs per se or their metabolites modulate neuronal activity, as a means of directly monitoring ongoing fuel availability by central nervous system nutrient-sensing neurons involved in the regulation of insulin secretion.

International audience; Circulating triglycerides (TGs) normally increase after a meal but are altered in pathophysiological conditions, such as obesity. Although TG metabolism in the brain remains poorly understood, several brain structures express enzymes that process TG-enriched particles, including mesolimbic structures. For this reason, and because consumption of high-fat diet alters dopamine signaling, we tested the hypothesis that TG might directly target mesolimbic reward circuits to control reward-seeking behaviors. We found that the delivery of small amounts of TG to the brain through the carotid artery rapidly reduced both spontaneous and amphetamine-induced locomotion, abolished preference for palatable food and reduced the motivation to engage in food-seeking behavior. Conversely, targeted disruption of the TG-hydrolyzing enzyme lipoprotein lipase specifically in the nucleus accumbens increased palatable food preference and food-seeking behavior. Finally, prolonged TG perfusion resulted in a return to normal palatable food preference despite continued locomotor suppression, suggesting that adaptive mechanisms occur. These findings reveal new mechanisms by which dietary fat may alter mesolimbic circuit function and reward seeking.

Reproductive function is highly dependent on nutritional input. We recently provided evidence that the unsaturated ω6 fatty acid (FA), linoleic acid (linoleic), interferes with transcription and secretion of the gonadotropin LH, highlighting the existence of a lipid sensing in pituitary gonadotropes. Here, we show, using a combination of in vivo and in vitro models, that linoleic differentially regulates Lhb and Fshb expression. Central exposure of rats to linoleic over 7 days was associated with increase of Lhb but not Fshb transcript levels. Consistently, exposure of rat pituitary cells or LβT2 cells to linoleic increased Lhb, whereas it dramatically decreased Fshb transcript levels without affecting its stability. This effect was also induced by ω9 and ω3-polyunsaturated FA but not by saturated palmitic acid. Analysis of the underlying mechanisms in LβT2 cells using small interfering RNA revealed that early growth response protein 1 mediates linoleic stimulation of Lhb expression. Furthermore, we demonstrated that linoleic counteracts activin and bone morphogenetic protein-2 stimulation of Fshb expression. Using Western blotting and Smad-responsive reporter gene assays, linoleic was shown to decrease basal Smad2/3 phosphorylation levels as well as activin- and bone morphogenetic protein-2-dependent activation of Smad, uncovering a new FA-sensitive signaling cascade. Finally, the protein phosphatase magnesium-dependent 1A was shown to mediate linoleic inhibition of basal Smad phosphorylation and Fshb expression, identifying protein phosphatase magnesium-dependent 1A as a new target of FA in gonadotropes. Altogether, this study provides a novel mechanism by which FAs target gene expression and underlines the relevant role of pituitary gonadotropes in mediating the effects of nutritional FA on reproductive function.

Dietary supplement may potentially help to fight obesity and other metabolic disorders such as insulin-resistance and low-grade inflammation. The present study aimed to test whether supplementation with Agaricus blazei murill (ABM) extract could have an effect on diet-induced obesity in rats.Wistar rats were fed with control diet (CD) or high-fat diet (HF) and either with or without supplemented ABM for 20 weeks.HF diet-induced body weight gain and increased fat mass compared to CD. In addition HF-fed rats developed hyperleptinemia and insulinemia as well as insulin resistance and glucose intolerance. In HF-fed rats, visceral adipose tissue also expressed biomarkers of inflammation. ABM supplementation in HF rats had a protective effect against body weight gain and all study related disorders. This was not due to decreased food intake which remained significantly higher in HF rats whether supplemented with ABM or not compared to control. There was also no change in gut microbiota composition in HF supplemented with ABM. Interestingly, ABM supplementation induced an increase in both energy expenditure and locomotor activity which could partially explain its protective effect against diet-induced obesity. In addition a decrease in pancreatic lipase activity is also observed in jejunum of ABM-treated rats suggesting a decrease in lipid absorption.Taken together these data highlight a role for ABM to prevent body weight gain and related disorders in peripheral targets independently of effect in food intake in central nervous system.

International audience; Objective: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2‐arachidonoylglycerol and anandamide (N‐arachidonoylethanolamine), are up‐regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids.Research Methods and Procedures: The production of endocannabinoids by human adipocytes was investigated in a model of human white subcutaneous adipocytes in primary culture. The effects of leptin, adiponectin, and peroxisome proliferator‐activated receptor (PPAR)‐γ activation on endocannabinoid production by adipocytes were explored. Endocannabinoid levels were determined by high‐performance liquid chromatography (HPLC)‐atmospheric pressure chemical ionization (APCI)‐mass spectrometry (MS) analysis, leptin and adiponectin secretion measured by enzyme‐linked immunosorbent assay (ELISA), and PPAR‐γ protein expression examined by Western blotting.Results: We show that 2‐arachidonoylglycerol, anandamide, and both anandamide analogs, N‐palmitoylethanolamine and N‐oleylethanolamine, are produced by human white subcutaneous adipocytes in concentrations ranging from 0.042 ± 0.004 to 0.531 ± 0.048 pM/mg lipid extract. N‐palmitoylethanolamine is the most abundant cannabimimetic compound produced by human adipocytes, and its levels are significantly down‐regulated by leptin but not affected by adiponectin and PPAR‐γ agonist ciglitazone. N‐palmitoylethanolamine itself does not affect either leptin or adiponectin secretion or PPAR‐γ protein expression in adipocytes.Discussion: This study has led to the identification of human adipocytes as a new source of endocannabinoids and related compounds. The biological significance of these adipocyte cannabimimetic compounds and their potential implication in obesity should deserve further investigations.

To evaluate the relationship between the development of obesity, nervous system activity, and insulin secretion and action, we tested the effect of a 2-mo high-fat diet in rats (HF rats) on glucose tolerance, glucose-induced insulin secretion (GIIS), and glucose turnover rate compared with chow-fed rats (C rats). Moreover, we measured pancreatic and hepatic norepinephrine (NE) turnover, as assessment of sympathetic tone, and performed hypothalamic microdialysis to quantify extracellular NE turnover. Baseline plasma triglyceride, free fatty acid, insulin, and glucose concentrations were similar in both groups. After 2 days of diet, GIIS was elevated more in HF than in C rats, whereas plasma glucose time course was similar. There was a significant increase in basal pancreatic NE level of HF rats, and a twofold decrease in the fractional turnover constant was observed, indicating a change in sympathetic tone. In ventromedian hypothalamus of HF rats, the decrease in NE extracellular concentration after a glucose challenge was lower compared with C rats, suggesting changes in overall activity. After 7 days, insulin hypersecretion persisted, and glucose intolerance appeared. Later (2 mo), there was no longer insulin hypersecretion, whereas glucose intolerance worsened. At all times, HF rats also displayed hepatic insulin resistance. On day 2 of HF diet, GIIS returned to normal after treatment with oxymetazoline, an α2A-adrenoreceptor agonist, thus suggesting the involvement of a low sympathetic tone in insulin hypersecretion in response to glucose in HF rats. In conclusion, the HF diet rapidly results in an increased GIIS, at least in part related to a decreased sympathetic tone, which can be the first step of a cascade of events leading to impaired glucose homeostasis.

The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller F- ion is a stronger inhibitor than I- and Br- and that Cl- has the highest Ki value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.

Subject Areas: carotid arteries; emulsions; fatty acids; gene expression; heparin; hypothalamus; neurons; oxidation.; International audience; Variations in plasma fatty acid (FA) concentrations are detected by FA sensing neurons in specific brain areas such as the hypothalamus. These neurons play a physiological role in the control of food intake and the regulation of hepatic glucose production. Le Foll et al. previously showed in vitro that at least 50% of the FA sensing in ventromedial hypothalamic (VMH) neurons is attributable to the interaction of long chain FA with FA translocase/CD36 (CD36). The present work assessed whether in vivo effects of hypothalamic FA sensing might be partly mediated by CD36 or intracellular events such as acylCoA synthesis or β-oxidation. To that end, a catheter was implanted in the carotid artery toward the brain in male Wistar rats. After 1 wk recovery, animals were food-deprived for 5 h, then 10 min infusions of triglyceride emulsion, Intralipid +/- heparin (IL, ILH, respectively) or saline/heparin (SH) were carried out and food intake was assessed over the next 5 h. Experimental groups included: 1) Rats previously injected in ventromedian nucleus (VMN) with shRNA against CD36 or scrambled RNA; 2) Etomoxir (CPT1 inhibitor) or saline co-infused with ILH/SH; and 3) Triacsin C (acylCoA synthase inhibitor) or saline co-infused with ILH/SH. ILH significantly lowered food intake during refeeding compared to SH (p