Protective role of the ELOVL2/docosahexaenoic acid axis in glucolipotoxicity-induced apoptosis in rodent beta cells and human islets.

AIMS/HYPOTHESIS: 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. METHODS: 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. RESULTS: 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 ß-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. CONCLUSIONS/INTERPRETATION: 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.

Inhibition of central de novo ceramide synthesis restores insulin signaling in hypothalamus and enhances ß-cell function of obese Zucker rats.

OBJECTIVES: Hypothalamic lipotoxicity has been shown to induce central insulin resistance and dysregulation of glucose homeostasis; nevertheless, elucidation of the regulatory mechanisms remains incomplete. Here, we aimed to determine the role of de novo ceramide synthesis in hypothalamus on the onset of central insulin resistance and the dysregulation of glucose homeostasis induced by obesity. METHODS: Hypothalamic GT1-7 neuronal cells were treated with palmitate. De novo ceramide synthesis was inhibited either by pharmacological (myriocin) or molecular (si-Serine Palmitoyl Transferase 2, siSPT2) approaches. Obese Zucker rats (OZR) were intracerebroventricularly infused with myriocin to inhibit de novo ceramide synthesis. Insulin resistance was determined by quantification of Akt phosphorylation. Ceramide levels were quantified either by a radioactive kinase assay or by mass spectrometry analysis. Glucose homeostasis were evaluated in myriocin-treated OZR. Basal and glucose-stimulated parasympathetic tonus was recorded in OZR. Insulin secretion from islets and ß-cell mass was also determined. RESULTS: We show that palmitate impaired insulin signaling and increased ceramide levels in hypothalamic neuronal GT1-7 cells. In addition, the use of deuterated palmitic acid demonstrated that palmitate activated several enzymes of the de novo ceramide synthesis pathway in hypothalamic cells. Importantly, myriocin and siSPT2 treatment restored insulin signaling in palmitate-treated GT1-7 cells. Protein kinase C (PKC) inhibitor or a dominant-negative PKCζ also counteracted palmitate-induced insulin resistance. Interestingly, attenuating the increase in levels of hypothalamic ceramides with intracerebroventricular infusion of myriocin in OZR improved their hypothalamic insulin-sensitivity. Importantly, central myriocin treatment partially restored glucose tolerance in OZR. This latter effect is related to the restoration of glucose-stimulated insulin secretion and an increase in ß-cell mass of OZR. Electrophysiological recordings also showed an improvement of glucose-stimulated parasympathetic nerve activity in OZR centrally treated with myriocin. CONCLUSION: Our results highlight a key role of hypothalamic de novo ceramide synthesis in central insulin resistance installation and glucose homeostasis dysregulation associated with obesity.

Palatability Can Drive Feeding Independent of AgRP Neurons.

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.

Fatty acid transporter CD36 mediates hypothalamic effect of fatty acids on food intake in rats.

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 ß-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, IL(H), 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 IL(H)/S(H); and 3) Triacsin C (acylCoA synthase inhibitor) or saline co-infused with IL(H)/S(H). IL(H) significantly lowered food intake during refeeding compared to S(H) (p<0.001). Five hours after refeeding, etomoxir did not affect this inhibitory effect of IL(H) on food intake while VMN CD36 depletion totally prevented it. Triacsin C also prevented IL(H) effects on food intake. In conclusion, the effect of FA to inhibit food intake is dependent on VMN CD36 and acylCoA synthesis but does not required FA oxidation.

Dietary supplementation with Agaricus blazei murill extract prevents diet-induced obesity and insulin resistance in rats.

OBJECTIVE: 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. DESIGN AND METHODS: Wistar rats were fed with control diet (CD) or high-fat diet (HF) and either with or without supplemented ABM for 20 weeks. RESULTS: 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. CONCLUSIONS: 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.

Hypothalamic AgRP-neurons control peripheral substrate utilization and nutrient partitioning.

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.

Hypothalamic serotonin-insulin signaling cross-talk and alterations in a type 2 diabetic model.

Serotonin and insulin are key regulators of homeostatic mechanisms in the hypothalamus. However, in type 2 diabetes, the hypothalamic responsiveness to serotonin is not clearly established. We used a diabetic model, the Goto Kakizaki (GK) rats, to explore insulin receptor expression, insulin and serotonin efficiency in the hypothalamus and liver by means of Akt phosphorylation. Insulin or dexfenfluramine (stimulator of serotonin) treatment induced Akt phosphorylation in Wistar rats but not in GK rats that exhibit down-regulated insulin receptor. Studies in a neuroblastoma cell line showed that serotonin-induced Akt phosphorylation is PI3-kinase dependent. Finally, in response to food intake, hypothalamic serotonin release was reduced in GK rats, indicating impaired responsiveness of this neurotransmitter. In conclusion, hypothalamic serotonin as insulin efficiency is impaired in diabetic GK rats. The insulin-serotonin cross-talk and impairment observed is one potential key modification in the brain during the onset of diabetes.

A dietary fat excess alters metabolic and neuroendocrine responses before the onset of metabolic diseases.

Early changes in neuroendocrine pathways are essential in the development of metabolic pathologies. Thus, it is important to have a better understanding of the signals involved in their initiation. Long-term consumption of high-fat diets induces insulin resistance, obesity, diabetes. Here, we have investigated early neural and endocrine events in the hypothalamus and hippocampus induced by a short-term high fat, low carbohydrate diet in adult male Wistar rats. The release of serotonin, which is closely associated with the actions of insulin and leptin, was measured, by electrochemical detection following reverse-phase liquid chromatography (HPLC), in the extracellular space of the medial hypothalamus and the dorsal hippocampus in samples obtained from non-anesthetized animals, by microdialysis. The high-fat diet had a specific effect on the hypothalamus. Serotonin release induced by food intake was reduced after 1 week, and effectively ceased after 6 weeks of the diet. After 1 week, there was an increased gene expression of the insulin receptor and the insulin receptor substrates IRS1 and IRS2, as measured by real-time PCR. After 6 weeks of diet, insulin gene expression increased. Leptinemia increased in all cases. This new data support the concept that high-fat diets, in addition to have peripheral effects, cause a rapid alteration in specific central mechanisms involved in energy and glucose homeostasis. The changes in the gene expression of insulin and signaling elements represent possible adaptations aimed at counterbalancing the reduced responsiveness of the serotonergic system to nutritional signals and maintaining homeostasis.

Persisting neural and endocrine modifications induced by a single fat meal.

1. High-fat diets, modify the neuroendocrine response and, when prolonged, result in positive energy balance and obesity. Little is known about the effects of fat on the mechanisms operating in the initial steps of the neural and endocrine disturbances. 2. The studies reported here were designed to access the impact of the consumption of a single exclusively animal fat meal (lard), 24 h following its ingestion a) on the response of the hypothalamic serotonergic system to a standard laboratory chow meal and b) on the circulating levels of glucose, insulin, and leptin. The release of serotonin in the extracellular medial hypothalamic space (including the paraventricular-PVN and ventromedian-VMH nuclei) was determined using electrochemical detection following HPLC in samples obtained in vivo by microdialysis, in nonanesthetized adult male Wistar rats. 3. A lard meal resulted in decreased hypothalamic serotonin release postprandially and attenuated (24 h later) the hypothalamic serotonin response that normally follows a balanced meal. 4. In permanently catheterized rats, postprandial glucose and insulin levels measured in samples obtained in vivo, were either not, or only slightly, modified after a lard meal, whereas plasma leptin levels were increased. Interestingly, 24 h after a meal, insulin and leptin levels were increased in those animals eating a fat meal compared with those eating chow. Next-day glucose levels remained identical after the absorption either of a chow, or a lard meal. 5. The changes induced by the fat meal on peripheral and central regulators of energy and glucose homeostasis represent either adaptive mechanisms or early alterations that could render the organism vulnerable to further insults.

Alpha-lactalbumin-enriched diets enhance serotonin release and induce anxiolytic and rewarding effects in the rat.

Among food proteins, alpha-lactalbumin (LAC) has the highest ratio of tryptophan (Trp) over its competitor amino acids. Consequently, contrary to casein (CAS), LAC ingestion increases Trp access to the brain leading to enhanced serotonin (5-HT) synthesis. As an index of serotonergic activity, we assessed extracellular 5-HT in response to LAC ingestion, using microdialysis, and performed behavioural tests in rats in order to characterise the suggested improvements of mood observed in humans after ingestion of this protein. Rats were fed with diets enriched either in LAC or CAS as control, acutely (30 min meals) or chronically (3 and 6 days). A 30 min LAC meal significantly increased 5-HT release in the medial hypothalamus. This effect disappeared after 3 and 6 days of diet. The basal premeal 5-HT levels were increasingly enhanced by the LAC diet. Compared to a CAS meal, LAC increased the percentage of time spent on the open arms of the elevated plus maze and the number of visits to the centre of the open field, suggesting an anxiolytic-like effect. A single LAC meal decreased sucrose consumption, while 3 or 6 days diets enhanced it, reflecting an appetitive and/or rewarding action. In conclusion, LAC ingestion induces anxiolytic-like and rewarding effects possibly related to serotonergic activation. Shifting transiently, the commonly consumed CAS-enriched to LAC-enriched diets may induce beneficial effects on mood.

Behavioral responses to ingestion of different sources of fat. Involvement of serotonin?

In previous experiments, we described a decrease in extracellular hypothalamic serotonin, 5-hydroxy-tryptamine (5-HT), in response to ingestion of a lard meal. This effect was related to the low satiating potency of lard as compared with other nutrients. Since the composition in fatty acids might be important for these effects, the purpose of this study was to analyze the neurochemical and behavioral responses to ingestion of different sources of fat. Unique meals of three margarines used in human feeding were compared with a meal of lard with regard to their effects on hypothalamic 5-HT, satiety, anxiety-like behavior and sucrose consumption. A vegetable margarine with a high content in saturated fatty acids also decreased hypothalamic 5-HT while ingestion of a sunflower oil and an olive oil enriched margarines, both high in polyunsaturated fatty acids, did not affect significantly 5-HT levels. However, these two last ones were not the most satiating. The olive oil margarine induced a tendency to an anxiety-like behavior while lard increased sucrose consumption. Thus, ingestion of fats may alter specifically behavioral responses. The involvement of 5-HT is likely in the case of lard ingestion but probably not for the other sources of fat.