Serge Luquet

Hypothalamic neurons that express neuropeptide Y (NPY) and agouti-related protein (AgRP) are thought to be critical regulators of feeding behavior and body weight. To determine whether NPY/AgRP neurons are essential in mice, we targeted the human diphtheria toxin receptor to the Agrp locus, which allows temporally controlled ablation of NPY/AgRP neurons to occur after an injection of diphtheria toxin. Neonatal ablation of NPY/AgRP neurons had minimal effects on feeding, whereas their ablation in adults caused rapid starvation. These results suggest that network-based compensatory mechanisms can develop after the ablation of NPY/AgRP neurons in neonates but do not readily occur when these neurons become essential in adults.

Hepatic steatosis is present in insulin-resistant obese rodents and is concomitant with active lipogenesis.Hepatic lipogenesis depends on the insulin-induced activation of the transcription factor SREBP-1c.Despite prevailing insulin resistance, SREBP-1c is activated in the livers of genetically and diet-induced obese rodents.Recent studies have reported the presence of an ER stress response in the livers of obese ob/ob mice.To assess whether ER stress promotes SREBP-1c activation and thus contributes to lipogenesis, we overexpressed the chaperone glucose-regulated protein 78 (GRP78) in the livers of ob/ob mice using an adenoviral vector.GRP78 overexpression reduced ER stress markers and inhibited SREBP-1c cleavage and the expression of SREBP-1c and SREBP-2 target genes.Furthermore, hepatic triglyceride and cholesterol contents were reduced, and insulin sensitivity improved, in GRP78-injected mice.These metabolic improvements were likely mediated by restoration of IRS-2 expression and tyrosine phosphorylation.Interestingly, GRP78 overexpression also inhibited insulin-induced SREBP-1c cleavage in cultured primary hepatocytes.These findings demonstrate that GRP78 inhibits both insulin-dependent and ER stress-dependent SREBP-1c proteolytic cleavage and explain the role of ER stress in hepatic steatosis in obese rodents.

Huntington's disease (HD) is a fatal, dominantly inherited disorder caused by polyglutamine repeat expansion in the huntingtin (htt) gene. Here, we observe that HD mice develop hypothermia associated with impaired activation of brown adipose tissue (BAT). Although sympathetic stimulation of PPARgamma coactivator 1alpha (PGC-1alpha) was intact in BAT of HD mice, uncoupling protein 1 (UCP-1) induction was blunted. In cultured cells, expression of mutant htt suppressed UCP-1 promoter activity; this was reversed by PGC-1alpha expression. HD mice showed reduced food intake and increased energy expenditure, with dysfunctional BAT mitochondria. PGC-1alpha is a known regulator of mitochondrial function; here, we document reduced expression of PGC-1alpha target genes in HD patient and mouse striatum. Mitochondria of HD mouse brain show reduced oxygen consumption rates. Finally, HD striatal neurons expressing exogenous PGC-1alpha were resistant to 3-nitropropionic acid treatment. Altered PGC-1alpha function may thus link transcription dysregulation and mitochondrial dysfunction in HD.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors exerting several functions in development and metabolism. The physiological functions of PPARdelta remain elusive. By using a CRE-Lox recombination approach, we generated an animal model for muscle-specific PPARdelta overexpression to investigate the role of PPARdelta in this tissue. Muscle-specific PPARdelta overexpression results in a profound change in fiber composition due to hyperplasia and/or shift to more oxidative fiber and, as a consequence, leads to the increase of both enzymatic activities and genes implicated in oxidative metabolism. These changes in muscle are accompanied by a reduction of body fat mass, mainly due to a large reduction of adipose cell size. Furthermore, we demonstrate that endurance exercise promotes an accumulation of PPARdelta protein in muscle of wild-type animals. Collectively, these results suggest that PPARdelta plays an important role in muscle development and adaptive response to environmental changes, such as training exercise. They strongly support the idea that activation of PPARdelta could be beneficial in prevention of metabolic disorders, such as obesity or type 2 diabetes.

We report that astrocytic insulin signaling co-regulates hypothalamic glucose sensing and systemic glucose metabolism. Postnatal ablation of insulin receptors (IRs) in glial fibrillary acidic protein (GFAP)-expressing cells affects hypothalamic astrocyte morphology, mitochondrial function, and circuit connectivity. Accordingly, astrocytic IR ablation reduces glucose-induced activation of hypothalamic pro-opio-melanocortin (POMC) neurons and impairs physiological responses to changes in glucose availability. Hypothalamus-specific knockout of astrocytic IRs, as well as postnatal ablation by targeting glutamate aspartate transporter (GLAST)-expressing cells, replicates such alterations. A normal response to altering directly CNS glucose levels in mice lacking astrocytic IRs indicates a role in glucose transport across the blood-brain barrier (BBB). This was confirmed in vivo in GFAP-IR KO mice by using positron emission tomography and glucose monitoring in cerebral spinal fluid. We conclude that insulin signaling in hypothalamic astrocytes co-controls CNS glucose sensing and systemic glucose metabolism via regulation of glucose uptake across the BBB.

The delivery of blood-borne molecules conveying metabolic information to neural networks that regulate energy homeostasis is restricted by brain barriers. The fenestrated endothelium of median eminence microvessels and tight junctions between tanycytes together compose one of these. Here, we show that the decrease in blood glucose levels during fasting alters the structural organization of this blood-hypothalamus barrier, resulting in the improved access of metabolic substrates to the arcuate nucleus. These changes are mimicked by 2-deoxyglucose-induced glucoprivation and reversed by raising blood glucose levels after fasting. Furthermore, we show that VEGF-A expression in tanycytes modulates these barrier properties. The neutralization of VEGF signaling blocks fasting-induced barrier remodeling and significantly impairs the physiological response to refeeding. These results implicate glucose in the control of blood-hypothalamus exchanges through a VEGF-dependent mechanism and demonstrate a hitherto unappreciated role for tanycytes and the permeable microvessels associated with them in the adaptive metabolic response to fasting.

Endocannabinoids, including anandamide (arachidonoyl ethanolamide) have been implicated in the regulation of a growing number of physiological and pathological processes. Anandamide can be generated from its membrane phospholipid precursor N-arachidonoyl phosphatidylethanolamine (NAPE) through hydrolysis by a phospholipase D (NAPE-PLD). Recent evidence indicates, however, the existence of two additional, parallel pathways. One involves the sequential deacylation of NAPE by α,β-hydrolase 4 (Abhd4) and the subsequent cleavage of glycerophosphate to yield anandamide, and the other one proceeds through phospholipase C-mediated hydrolysis of NAPE to yield phosphoanandamide, which is then dephosphorylated by phosphatases, including the tyrosine phosphatase PTPN22 and the inositol 5′ phosphatase SHIP1. Conversion of synthetic NAPE to AEA by brain homogenates from wild-type and NAPE-PLD−/− mice can proceed through both the PLC/phosphatase and Abdh4 pathways, with the former being dominant at shorter (<10 min) and the latter at longer (60 min) incubations. In macrophages, the endotoxin-induced synthesis of anandamide proceeds uniquely through the phospholipase C/phosphatase pathway.

Astrocytes, microglia, and tanycytes play active roles in the regulation of hypothalamic feeding circuits. These non-neuronal cells are crucial in determining the functional interactions of specific neuronal subpopulations involved in the control of metabolism. Recent advances in biology, optics, genetics, and pharmacology have resulted in the emergence of novel and highly sophisticated approaches for studying hypothalamic neuronal-glial networks. Here we summarize the progress in the field and argue that glial-neuronal interactions provide a core hub integrating food-related cues, interoceptive signals, and internal states to adapt a complex set of physiological responses operating on different timescales to finely tune behavior and metabolism according to metabolic status. This expanding knowledge helps to redefine our understanding of the physiology of food intake and energy metabolism.

Obesity is associated with a cluster of metabolic disorders, low-grade inflammation and altered gut microbiota. Whether host metabolism is controlled by intestinal innate immune system and the gut microbiota is unknown. Here we report that inducible intestinal epithelial cell-specific deletion of MyD88 partially protects against diet-induced obesity, diabetes and inflammation. This is associated with increased energy expenditure, an improved glucose homeostasis, reduced hepatic steatosis, fat mass and inflammation. Protection is transferred following gut microbiota transplantation to germ-free recipients. We also demonstrate that intestinal epithelial MyD88 deletion increases anti-inflammatory endocannabinoids, restores antimicrobial peptides production and increases intestinal regulatory T cells during diet-induced obesity. Targeting MyD88 after the onset of obesity reduces fat mass and inflammation. Our work thus identifies intestinal epithelial MyD88 as a sensor changing host metabolism according to the nutritional status and we show that targeting intestinal epithelial MyD88 constitutes a putative therapeutic target for obesity and related disorders.

The association between altered gut microbiota, intestinal permeability, inflammation and cardiometabolic diseases is becoming increasingly clear but remains poorly understood1,2. Indoleamine 2,3-dioxygenase is an enzyme induced in many types of immune cells, including macrophages in response to inflammatory stimuli, and catalyzes the degradation of tryptophan along the kynurenine pathway. Indoleamine 2,3-dioxygenase activity is better known for its suppression of effector T cell immunity and its activation of regulatory T cells3,4. However, high indoleamine 2,3-dioxygenase activity predicts worse cardiovascular outcome5-9 and may promote atherosclerosis and vascular inflammation6, suggesting a more complex role in chronic inflammatory settings. Indoleamine 2,3-dioxygenase activity is also increased in obesity10-13, yet its role in metabolic disease is still unexplored. Here, we show that obesity is associated with an increase of intestinal indoleamine 2,3-dioxygenase activity, which shifts tryptophan metabolism from indole derivative and interleukin-22 production toward kynurenine production. Indoleamine 2,3-dioxygenase deletion or inhibition improves insulin sensitivity, preserves the gut mucosal barrier, decreases endotoxemia and chronic inflammation, and regulates lipid metabolism in liver and adipose tissues. These beneficial effects are due to rewiring of tryptophan metabolism toward a microbiota-dependent production of interleukin-22 and are abrogated after treatment with a neutralizing anti-interleukin-22 antibody. In summary, we identify an unexpected function of indoleamine 2,3-dioxygenase in the fine tuning of intestinal tryptophan metabolism with major consequences on microbiota-dependent control of metabolic disease, which suggests indoleamine 2,3-dioxygenase as a potential therapeutic target.

Consumption of dietary fat is one of the key factors leading to obesity. High-fat diet (HFD)-induced obesity is characterized by induction of inflammation in the hypothalamus; however, the temporal regulation of proinflammatory markers and their impact on hypothalamic appetite-regulating neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons remains undefined.Mice were injected with an acute lipid infusion for 24 h or fed a HFD over 8-20 weeks. Characterized mouse NPY/AgRP hypothalamic cell lines were used for in vitro experimentation. Immunohistochemistry in brain slices or quantitative real-time PCR in cell lines, was performed to determine changes in the expression of key inflammatory markers and neuropeptides.Hypothalamic inflammation, indicated by tumor necrosis factor (TNF)-α expression and astrocytosis in the arcuate nucleus, was evident following acute lipid infusion. HFD for 8 weeks suppressed TNF-α, while significantly increasing heat-shock protein 70 and ciliary neurotrophic factor, both neuroprotective components. HFD for 20 weeks induced TNF-α expression in NPY/AgRP neurons, suggesting a detrimental temporal regulatory mechanism. Using NPY/AgRP hypothalamic cell lines, we found that palmitate provoked a mixed inflammatory response on a panel of inflammatory and endoplasmic reticulum (ER) stress genes, whereas TNF-α significantly upregulated IκBα, nuclear factor (NF)-κB and interleukin-6 mRNA levels. Palmitate and TNF-α exposure predominantly induced NPY mRNA levels. Utilizing an I kappa B kinase β (IKKβ) inhibitor, we demonstrated that these effects potentially occur via the inflammatory IKKβ/NF-κB pathway.These findings indicate that acute lipid and chronic HFD feeding in vivo, as well as acute palmitate and TNF-α exposure in vitro, induce markers of inflammation or ER stress in the hypothalamic appetite-stimulating NPY/AgRP neurons over time, which may contribute to a dramatic alteration in NPY/AgRP content or expression. Acute and chronic HFD feeding in vivo temporally regulates arcuate TNF-α expression with reactive astrocytosis, which suggests a time-dependent neurotrophic or neurotoxic role of lipids.

Over the past 20 years, insights from human and mouse genetics have illuminated the central role of the brain leptin-melanocortin pathway in controlling mammalian food intake, with genetic disruption resulting in extreme obesity, and more subtle polymorphic variations influencing the population distribution of body weight. At the end of 2020, the U.S. Food and Drug Administration (FDA) approved setmelanotide, a melanocortin 4 receptor agonist, for use in individuals with severe obesity due to either pro-opiomelanocortin (POMC), proprotein convertase subtilisin/kexin type 1 (PCSK1), or leptin receptor (LEPR) deficiency.Herein, we chart the melanocortin pathway's history, explore its pharmacology, genetics, and physiology, and describe how a neuropeptidergic circuit became an important druggable obesity target.Unravelling the genetics of the subset of severe obesity has revealed the importance of the melanocortin pathway in appetitive control; coupling this with studying the molecular pharmacology of compounds that bind melanocortin receptors has brought a new obesity drug to the market. This process provides a drug discovery template for complex disorders, which for setmelanotide took 25 years to transform from a single gene into an approved drug.

A line of dopamine-deficient (DD) mice was generated to allow selective restoration of normal dopamine signaling to specific brain regions. These DD floxed stop (DDfs) mice have a nonfunctional Tyrosine hydroxylase (Th) gene because of insertion of a NeoR gene flanked by lox P sites targeted to the first intron of the Th gene. DDfs mice have trace brain dopamine content, severe hypoactivity, and aphagia, and they die without intervention. However, they can be maintained by daily treatment with l-3,4-dihydroxyphenylalanine (L-dopa). Injection of a canine adenovirus (CAV-2) engineered to express Cre recombinase into the central caudate putamen restores normal Th gene expression to the midbrain dopamine neurons that project there because CAV-2 efficiently transduces axon terminals and is retrogradely transported to neuronal cell bodies. Bilateral injection of Cre recombinase into the central caudate putamen restores feeding and normalizes locomotion in DDfs mice. Analysis of feeding behavior by using lickometer cages revealed that virally rescued DDfs mice are hyperphagic and have modified meal structures compared with control mice. The virally rescued DDfs mice are also hyperactive at night, have reduced motor coordination, and are thigmotactic compared with controls. These results highlight the critical role for dopamine signaling in the dorsal striatum for most dopamine-dependent behaviors but suggest that dopamine signaling in other brain regions is important to fine-tune these behaviors. This approach offers numerous advantages compared with previous models aimed at examining dopamine signaling in discrete dopaminergic circuits.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors primarily involved in lipid homeostasis. PPARdelta displays strong expression in tissues with high lipid metabolism, such as adipose, intestine and muscle. Its role in skeletal muscle remains largely unknown. After a 24-h starvation period, PPARdelta mRNA levels are dramatically up-regulated in gastrocnemius muscle of mice and restored to control level upon refeeding. The rise of PPARdelta is accompanied by parallel up-regulations of fatty acid translocase/CD36 (FAT/CD36) and heart fatty acid binding protein (H-FABP), while refeeding promotes down-regulation of both genes. To directly access the role of PPARdelta in muscle cells, we forced its expression and that of a dominant-negative PPARdelta mutant in C2C12 myogenic cells. Differentiated C2C12 cells responds to 2-bromopalmitate or synthetic PPARdelta agonist by induction of genes involved in lipid metabolism and increment of fatty acid oxidation. Overexpression of PPARdelta enhanced these cellular responses, whereas expression of the dominant-negative mutant exerts opposite effects. These data strongly support a role for PPARdelta in the regulation of fatty acid oxidation in skeletal muscle and in adaptive response of this tissue to lipid catabolism.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors exerting several functions in development and metabolism. PPARalpha, activated by polyunsaturated fatty acids and fibrates, is implicated in regulation of lipid metabolism, lipoprotein synthesis and metabolism and inflammatory response in liver and other tissues. PPARgamma plays important roles in regulation of proliferation and differentiation of several cell types, including adipose cells. Its activation by thiazolidinediones results in insulin sensibilization and antidiabetic action. Until recently, the physiological functions of PPARdelta remain elusive. The utilization of specific agonists and of appropriate cellular and animal models revealed that PPARdelta has an important role in metabolic adaptation of several tissues to environmental changes. Treatment of obese animals by specific PPARdelta agonists results in normalization of metabolic parameters and reduction of adiposity. The nuclear receptor appeared to be implicated in the regulation of fatty acid burning capacities of skeletal muscle and adipose tissue by controlling the expression of genes involved in fatty acid uptake, beta-oxidation and energy uncoupling. PPARdelta is also implicated in the adaptive metabolic response of skeletal muscle to endurance exercise by controlling the number of oxidative myofibers. Given the results obtained with animal models, PPARdelta agonists may have therapeutic usefulness in metabolic syndrome by increasing fatty acid consumption in skeletal muscle and adipose tissue.

The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of α-linolenic acid compared with recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid:α-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players, such as CSF3 and Nocturnin. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans. The prevalence of obesity has steadily increased over the last few decades. During this time, populations of industrialized countries have been exposed to diets rich in fat with a high content of linoleic acid and a low content of α-linolenic acid compared with recommended intake. To assess the contribution of dietary fatty acids, male and female mice fed a high-fat diet (35% energy as fat, linoleic acid:α-linolenic acid ratio of 28) were mated randomly and maintained after breeding on the same diet for successive generations. Offspring showed, over four generations, a gradual enhancement in fat mass due to combined hyperplasia and hypertrophy with no change in food intake. Transgenerational alterations in adipokine levels were accompanied by hyperinsulinemia. Gene expression analyses of the stromal vascular fraction of adipose tissue, over generations, revealed discrete and steady changes in certain important players, such as CSF3 and Nocturnin. Thus, under conditions of genome stability and with no change in the regimen over four generations, we show that a Western-like fat diet induces a gradual fat mass enhancement, in accordance with the increasing prevalence of obesity observed in humans. The prevalence of obesity and the risk of developing associated diseases have steadily increased across generations over the last few decades. In addition, these events now emerge earlier in life. This epidemic is not attributable to genetic factors as it has occurred relatively recently and is observed in a wide range of human populations. High-fat diets are considered to be obesogenic in that they produce a consistent increase in fat mass that is directly related to the content of the diet and duration of feeding. However, the contribution of dietary fats compared with an excess energy intake in increasing body weight remains controversial, as no major change in the total amount of ingested fats has occurred in the last two decades (1.Troiano R.P. Briefel R.R. Carroll M.D. Bialostosky K. Energy and fat intakes of children and adolescents in the united states: data from the national health and nutrition examination surveys.Am. J. Clin. Nutr. 2000; 72: 1343S-1353SCrossref PubMed Google Scholar, 2.Donahoo W. Wyatt H.R. Kriehn J. Stuht J. Dong F. Hosokawa P. Grunwald G.K. Johnson S.L. Peters J.C. Hill J.O. Dietary fat increases energy intake across the range of typical consumption in the United States.Obesity (Silver Spring). 2008; 16: 64-69Crossref PubMed Scopus (30) Google Scholar). In addition to caloric excess, a qualitative issue has emerged as a risk factor for obesity in rodents and possibly in humans; i.e., the disequilibrium in polyunsaturated fatty acid (PUFA) metabolism with a high ratio of linoleic acid (C18:2 ω6, LA) versus α-linolenic acid (C18:3 ω3, LNA) (3.Ailhaud G. Guesnet P. Cunnane S.C. An emerging risk factor for obesity: does disequilibrium of polyunsaturated fatty acid metabolism contribute to excessive adipose tissue development?.Br. J. Nutr. 2008; 100: 461-470Crossref PubMed Scopus (105) Google Scholar). Notably, in rodents, reducing this ratio from 59 to 2 under isolipidic, isoenergetic conditions (40% energy as fat) by inclusion of dietary LNA counteracted the enhancing effects of LA on body weight and fat mass, which then became similar to that observed with a chow diet (4.Massiera F. Saint-Marc P. Seydoux J. Murata T. Kobayashi T. Narumiya S. Guesnet P. Amri E.Z. Negrel R. Ailhaud G. Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?.J. Lipid Res. 2003; 44: 271-279Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). ω6 PUFAs were more potent than ω3 PUFAs in promoting adipogenesis (5.Gaillard D. Negrel R. Lagarde M. Ailhaud G. Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.Biochem. J. 1989; 257: 389-397Crossref PubMed Scopus (163) Google Scholar, 6.Vassaux G. Gaillard D. Ailhaud G. Negrel R. Prostacyclin is a specific effector of adipose cell differentiation. Its dual role as a cAMP- and Ca(2+)-elevating agent.J. Biol. Chem. 1992; 267: 11092-11097Abstract Full Text PDF PubMed Google Scholar, 7.Kim H.K. Della-Fera M. Lin J. Baile C.A. Docosahexaenoic acid inhibits adipocyte differentiation and induces apoptosis in 3T3–L1 preadipocytes.J. Nutr. 2006; 136: 2965-2969Crossref PubMed Scopus (186) Google Scholar). When combined with high carbohydrate content, a linoleic acid-enriched diet was found to be pro-adipogenic in vivo through cAMP-dependent signaling (8.Madsen L. Pedersen L.M. Liaset B. Ma T. Petersen R.K. van den Berg S. Pan J. Muller-Decker K. Dulsner E.D. Kleemann R. et al.cAMP-dependent signaling regulates the adipogenic effect of n-6 polyunsaturated fatty acids.J. Biol. Chem. 2008; 283: 7196-7205Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). LA acts through arachidonic acid (C20:4 ω6, ARA) and prostacyclin, as pups from mice invalidated for the prostacyclin receptor (IP-R) and fed a LA-rich diet exhibit reduced body weight and fat mass compared with wild-type mice fed the same diet (4.Massiera F. Saint-Marc P. Seydoux J. Murata T. Kobayashi T. Narumiya S. Guesnet P. Amri E.Z. Negrel R. Ailhaud G. Arachidonic acid and prostacyclin signaling promote adipose tissue development: a human health concern?.J. Lipid Res. 2003; 44: 271-279Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). Overall, these results emphasize the importance of adipose tissue development in rodents of a high-fat diet combined with a high LA:LNA ratio. Therefore, by analogy to humans, where consumers have been continuously exposed—from in utero to old age—to dietary fats with a high LA content and a low LNA content and in which the prevalence of overweight and obesity has increased within a few generations (9.Ailhaud G. Massiera F. Weill P. Legrand P. Alessandri J.M. Guesnet P. Temporal changes in dietary fats: role of n-6 polyunsaturated fatty acids in excessive adipose tissue development and relationship to obesity.Prog. Lipid Res. 2006; 45: 203-236Crossref PubMed Scopus (368) Google Scholar), we decided to set up a nutritional model mimicking a human situation. For this purpose, male and female mice were chronically exposed over four generations to a single Western-like fat diet; i.e., 35% energy as fat with a LA/LNA ratio similar to that found in the most consumed foods. The results show that this condition was sufficient to trigger gradual transgenerational enhancement of the fat mass observed at early and adult ages. In addition to changes in insulin and adipokine circulating levels and to changes in cellularity of adipose tissue, gene expression profiling over generations was used to highlight the major molecular events favoring adipose tissue hyperplasia and metabolic imprinting that lead to an obese phenotype. A colony of pure inbred C57BL6/J mice was established by mating four males and four females from the same litter (Charles River, France) that was fed a chow diet. At weaning, pups were either maintained on a chow diet (STD mice) or fed a high-fat diet (ω6HFD) (HF mice). Both diets contained 21.4% proteins, 3.9% fiber, and 5.7% minerals and ash. The fatty acid composition of the ω6HFD (35% energy as fat) and chow diet is detailed in supplementary Table I. The ω6HFD contained a 3.7-fold higher amount of LA (7.9 g per 100 g versus 2.2 g per 100 g) but the same amount of LNA (0.24–0.26 g/100 g) as the chow diet. As shown in Table 1, male and female mice were first fed ω6HFD at weaning and later mated to generate HF0 mice, whereas HF0–HF4 male and female mice were fed ω6HFD. In this way, HF0–HF4 mice were continuously exposed over generations to the isocaloric, isolipidic diet. The whole population of male and female HF0 adults was mated randomly at 10 weeks of age to give HF1, the first generation (Table 1). Among several possibilities, pups could have been derived from a low body fat gainer male crossed with a low body fat gainer female, or from a high body fat gainer male crossed with a high body fat gainer female. Many additional combinations were also possible. Random mating was chosen on purpose to exclude any selection process. After lactation by HF0 dams, HF1 pups were fed at weaning on ω6HFD and mated randomly as described above to generate HF2 mice. The same protocol was used to establish generations HF3 and HF4. Reversion experiments were performed by switching HF1, HF3, and HF4 pups to a chow diet at weaning; these mice were respectively termed revHF1, revHF3, and revHF4 mice. Adult male and female revHF4 mice then fed a chow diet were mated randomly to give birth to the fifth generation of pups that at weaning were fed either the chow diet (termed std5 mice) or ω6HFD (termed hf5 mice) (Table 1). Mice were housed five per cage and body weight was measured weekly for each group of mice from birth to 22 weeks of age. All other measurements were performed with adult mice, i.e., from 8 to 22 weeks of age. At 8 weeks of age, food intake was measured daily for one week. Plasma extraction and adipose tissue dissection were performed as described previously (10.Massiera F. Seydoux J. Geloen A. Quignard-Boulange A. Turban S. Saint-Marc P. Fukamizu A. Negrel R. Ailhaud G. Teboul M. Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity.Endocrinology. 2001; 142: 5220-5225Crossref PubMed Scopus (135) Google Scholar). All experimental animal protocols were performed in accordance with the recommendations of the French and European Accreditation of Laboratory Animal Care and were approved by the local experimentation committee.TABLE 1Experimental protocol to obtain generations of chow diet (STD)- and ω6HFD-fed miceSTDHF0HF1HF2HF3HF4revHF1revHF3revHF4hf5std5Number of generations on a ω6HFD00123413444Diet of parents before and during breeding, pregnancy, and lactationChowChowHFDHFDHFDHFDHFDHFDHFDChowChowDiet after weaningChowHFDHFDHFDHFDHFDChowChowChowHFDChowAbbreviations: HF0–HF4, HFD-fed mice; hf5, ω6HFD-fed fifth-generation offspring of revHF4; HFD, high-fat diet (ω6HFD); revHF1, HF1 pups fed STD diet at weaning; revHF3, HF3 pups fed STD diet at weaning; revHF4, HF4 pups fed STD diet at weaning; STD, chow diet-fed mice; std5, STD-fed fifth-generation offspring of revHF4. Open table in a new tab Abbreviations: HF0–HF4, HFD-fed mice; hf5, ω6HFD-fed fifth-generation offspring of revHF4; HFD, high-fat diet (ω6HFD); revHF1, HF1 pups fed STD diet at weaning; revHF3, HF3 pups fed STD diet at weaning; revHF4, HF4 pups fed STD diet at weaning; STD, chow diet-fed mice; std5, STD-fed fifth-generation offspring of revHF4. After a 12 h fast, blood was collected from 14- and 22-week-old mice (n = 4). Plasma and adipose tissue were analyzed for the fatty acid content by direct transesterification as described (11.Lepage G. Roy C.C. Specific methylation of plasma nonesterified fatty acids in a one-step reaction.J. Lipid Res. 1988; 29: 227-235Abstract Full Text PDF PubMed Google Scholar). Indirect measurement was performed by collecting the stomach content of breast-fed HF1 pups (n = 4 for each group). Total lipids of the stomach content were extracted in the presence of 0.02% butylhydroxytoluene before performing gas chromatography (11.Lepage G. Roy C.C. Specific methylation of plasma nonesterified fatty acids in a one-step reaction.J. Lipid Res. 1988; 29: 227-235Abstract Full Text PDF PubMed Google Scholar). Blood was collected in 22-week-old mice (n ≥ 4) by heart puncture after a 14 h fast. Then plasma leptin, insulin, total adiponectin, interleukin (IL)-6, tumor necrosis factor (TNF)-α, resistin, plasminogen activator inhibitor (PAI)-1, and monocyte chemotactic protein (MCP)-1 concentrations were assessed with a Lincoplex assay (Millipore, St Quentin en Yvelines, France). Epididymal fat pads from 10-week-old mice (n ≥ 3) were isolated and weighed before cell dissociation for 30 min with collagenase type IV (Sigma Chem.). After filtration (250 μm), the infranatant was removed by catheter aspiration. In each condition, three pictures of isolated adipocytes per mouse were taken with a light microscope. Measurement of the cell diameter and number was performed as described previously (10.Massiera F. Seydoux J. Geloen A. Quignard-Boulange A. Turban S. Saint-Marc P. Fukamizu A. Negrel R. Ailhaud G. Teboul M. Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity.Endocrinology. 2001; 142: 5220-5225Crossref PubMed Scopus (135) Google Scholar). Total RNA was extracted from stromal vascular fraction (SVF) cells prepared from periepididymal adipose tissues of 10-week-old mice (n ≥ 3 per group) using TRI-REAGENT (Euromedex, Souffelweyersheim, France) according to the manufacturer's instructions, and then used for cDNA microarray and quantitative PCR. Biological experiments used for expression profiling were independently performed twice. Hybridization was dye balanced to reduce the effects of using two distinct dyes, Cy3 and Cy5. The quality of total RNA was controlled on an Agilent Bioanalyzer 2100 as previously described (12.Moreilhon C. Gras D. Hologne C. Bajolet O. Cottrez F. Magnone V. Merten M. Groux H. Puchelle E. Barbry P. Live Staphylococcus aureus and bacterial soluble factors induce different transcriptional responses in human airway cells.Physiol. Genomics. 2005; 20: 244-255Crossref PubMed Scopus (68) Google Scholar). Pangenomic microarrays were printed using the mouse RNG/MRC oligonucleotide collection (corresponding to 25,299 distinct probes) as previously described (http://www.microarray.fr/). Experimental data and associated microarray designs have been deposited in the National Center for Biotechnology Information (NBCI) Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) under series GSE16613 and GSE16636, and under platform record GPL1456. All calculations were performed with the Bioconductor packages (13.Gentleman R.C. Carey V.J. Bates D.M. Bolstad B. Dettling M. Dudoit S. Ellis B. Gautier L. Ge Y. Gentry J. et al.Bioconductor: open software development for computational biology and bioinformatics.Genome Biol. 2004; 5: R80Crossref PubMed Google Scholar) limma (14.Wettenhall J.M. Smyth G.K. limmaGUI: a graphical user interface for linear modeling of microarray data.Bioinformatics. 2004; 20: 3705-3706Crossref PubMed Scopus (567) Google Scholar) and topGO (15.Alexa A. Rahnenfuhrer J. Lengauer T. Improved scoring of functional groups from gene expression data by decorrelating GO graph structure.Bioinformatics. 2006; 22: 1600-1607Crossref PubMed Scopus (1290) Google Scholar). Differentially expressed genes were selected using a Benjamini-Hochberg correction of the P value for multiple tests, based on a P value of 0.05 or below. Additional parameters corresponded to an average log2(Signal) above 6, a log2(Fold change) above 0.7 (absolute value) in one of the comparisons. Reverse transcriptase reactions and quantitative RT-PCR assays were performed as described (16.Zaragosi L.E. Ailhaud G. Dani C. Autocrine fibroblast growth factor 2 signaling is critical for self-renewal of human multipotent adipose-derived stem cells.Stem Cells. 2006; 24: 2412-2419Crossref PubMed Scopus (210) Google Scholar). The expression of selected genes was normalized to that of 18S rRNAs and quantified using the comparative-ΔCt method. Oligonucleotide sequences, designed using Primer Express software (PerkinElmer Life Sciences, Courtaboeuf, France), are available upon request. Data are presented as the mean ± SEM. Measurements were compared via the two-tailed t-test or Kruskal and Wallis nonparametric rank-sum test. Body weight curves were compared by ANOVA analysis for repeated measures using XLstat software, followed by either Bonferroni (for pairwise comparison) or Dunnet (for comparison to STD) post hoc analysis. Microarray experiments were analyzed using the limma package from Bioconductor. The scripts used for the analysis are available in the supplementary data. As shown in Table 1 and supplementary Table I, four male and four female C57BL/6J mice from the same litter were mated randomly. At weaning, their pups were fed an LA-enriched diet [termed ω6 high-fat diet (ω6HFD)], which contained 35% energy as fat with a LA:LNA ratio ∼28 (see supplementary data). From weaning up to 8 weeks old, body weight was not affected by the diet. However, when the adult male and female HF0 mice on the ω6HFD were mated randomly and produced HF1 pups, the body weight of the male mice at weaning became significantly higher (10.9 ± 0.2 versus 9.6 ± 0.6 g, P < 0.05; n ≥ 6 for each generation) than that of the HF0 mice; this weight difference persisted at the adult age. Suckling HF1 pups from both sexes were fed the ω6HFD at weaning and then mated as above. The difference in body weight both at weaning and at 8 weeks was further increased in HF2 male pups and adults compared with HF1 mice, although no significant weight difference persisted between HF1 and HF2 mice at the adult age (supplementary Fig. IA). Regarding fat mass, a large difference in the weight of the epididymal fat pad was already observed across generations at 8 weeks for HF0, HF1, and HF2 mice (Fig. 1A; n ≥ 6 for each generation). This difference became more evident when comparing HF0 and HF2 mice at 14 (Fig. 1B) and 22 weeks (Fig. 1C), consistent with the obvious difference in the morphological appearance of STD and HF4 mice at 22 weeks (Fig. 1D). Most importantly, the number of pups per litter was not statistically different: 5.5 ± 0.6, 5.6 ± 0.5, 5.0 ± 0.4, 4.5 ± 0.5, and 4.7 ± 0.4 for STD/HF0, HF1, HF2, HF3, and HF4 mice, respectively. This finding showed that the ω6HFD had no impact upon reproduction, thus excluding the possibility that litter size could explain an indirect effect of the diet across generations by increasing the energy intake of suckling pups. When assessed at 8 weeks, no significant difference in the caloric intake could be observed among STD, HF0, HF1, HF2, and HF4 mice (n ≥ 5; supplementary Table II). Moreover, when HF4 pups were transferred at weaning to a chow diet (Table 1), the food intake of revHF4 mice (n = 3) remained similar to that of HF4 and previous generations. Thus, these results exclude the possibility that changes in food and fat intake—across generations early and late in life—explain the transgenerational increase in body weight and fat mass observed at the adult age, suggesting that other mechanisms are implicated. Interestingly, body weight at birth appeared to decrease across generations, but this trend was not statistically significant (supplementary Fig. IB). The major effect of the ω6HFD on body weight of STD/HF0 versus HF3 mice took place between the second and third week, suggesting a similar differential effect on fat mass that was not accurately measurable at this very early age (supplementary Fig. IC). PUFA metabolism in the mothers' milk lipids was altered in response to the linoleic acid-enriched diet but then remained similar across generations. The ω6HFD altered the PUFA composition of milk lipids of HF0 dams. It strongly increased the content of LA and to a lesser extent that of ARA (supplementary Fig. IIA, B; n = 4 for each group). In contrast, it decreased significantly the content of long-chain polyunsaturated fatty acids (LC-PUFA) of the ω3 series, i.e., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), thus increasing the ARA:DHA and ARA:DHA+EPA ratio from 1.3 to 2.2 and from 1.0 to 1.5, respectively (supplementary Fig. IIA, C). As for mothers' milk, the ω6HFD induced an increase in the ω6 content (LA and ARA) and a decrease in LNA, EPA, and DHA in plasma and adipose tissue lipids at 14 weeks (Fig. 2 and supplementary Table III). All these observations are in agreement with human studies showing that an increase in LA intake leads to stimulation of ARA and/or inhibition of EPA and DHA synthesis (17.Guesnet P. Pugo-Gunsam P. Maurage C. Pinault M. Giraudeau B. Alessandri J.M. Durand G. Antoine J.M. Couet C. Blood lipid concentrations of docosahexaenoic and arachidonic acids at birth determine their relative postnatal changes in term infants fed breast milk or formula.Am. J. Clin. Nutr. 1999; 70: 292-298Crossref PubMed Scopus (65) Google Scholar). The PUFA profile was not different between HF0 and HF4 mice (n = 4), showing that no further changes occurred once STD-fed mice had been switched to the ω6HFD (Fig. 2). Taken together, these results show that the PUFA profile was altered by the diet fat content and that a steady state was then observed across generations. As expected, changes in the PUFA content induced by the ω6HFD were reversed in a single generation of mice fed the chow diet (revHF4; n = 3). Palmitoleate (C16:1 ω7) has recently been reported to be an insulin-sensitizing, adipose tissue-derived hormone that improves glucose metabolism (18.Cao H. Gerhold K. Mayers J.R. Wiest M.M. Watkins S.M. Hotamisligil G.S. Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism.Cell. 2008; 134: 933-944Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). Palmitoleate levels were regulated in plasma and adipose tissue with a 2- to 3-fold reduction upon initial ω6HFD exposure, but no further change occurred across generations (supplementary Table III). The decrease in the ω3 LC-PUFA status in mice fed the ω6HFD had no impact on the DHA concentration in brain phospholipids (supplementary Table III). This finding excludes an essential fatty acid deficiency arising across generations, which would have altered various physiological functions and subsequently affected white adipose tissue (WAT) development. To assess whether further transgenerational effects of the ω6HFD could be reversed, HF4 mice were switched at weaning to the chow diet. In plasma, the percentage of ω6 PUFAs of revHF4 mice was also similar at 14 weeks old to that of STD mice. The ARA:EPA+DHA ratio increased more than 2-fold in HF4 mice and reversed completely in revHF4 mice. A reversible pattern was also observed for the ω6 and ω3 PUFA composition of adipose tissue lipids (Fig. 2 and supplementary Table III). Under these conditions, the body weight of revHF4 mice returned 5 weeks later to that of HF0 mice (Fig. 3A; n ≥ 4). Importantly, a similar but incomplete reversal was observed for the epididymal fatpad weight (Fig. 3B; n ≥ 6). Therefore adult male and female revHF4 mice were subsequently mated randomly to give birth to the 5th generation of pups (Table 1). When the litters were fed at weaning either the chow diet (std5 mice) or the ω6HFD (hf5 mice; n = 5 for each group), not only were std5 mice heavier at weaning than STD mice, but a large difference in the rate of weight gain could be observed in hf5 mice compared with std5, as early as the first week after weaning and maintained thereafter (Fig. 3C). It is striking that such a difference was not observed for HF0 mice for which the body weight was similar to that of STD mice from weaning until 8 weeks (supplementary Fig. IA). The incomplete reversal of the adipose phenotype at later generations suggested that some transgenerational memory had been acquired, allowing revHF4 mice to respond more rapidly than STD mice to the ω6HFD. This point was further examined at the physiological and cellular levels. Compared with mice fed a chow diet, those fed the ω6HFD for 19 weeks after weaning exhibited an increase in the plasma level of most parameters traditionally associated with the metabolic syndrome (i.e., TNFα, resistin, insulin, leptin, and MCP-1) in the first, second, and third generations, while adiponectin and IL-6 levels remained rather constant. Surprisingly, this inflammatory signature was almost reversed at the fourth generation. TNFα, resistin, IL-6, MCP-1, and leptin levels became dramatically reduced compared with those observed in HF3 mice and became similar to those of STD mice (Fig. 4; n ≥ 4). Fasting insulin levels followed a pattern similar to that of cytokines but remained significantly higher than those of HF0 mice. These observations indicate that, despite the fact that glycemia in HF4 mice appeared normal at 22 weeks old (151 ± 30 mg/dl for HF4 versus 170 ± 30 mg/dl for STD mice), continuous exposure to the ω6HFD led to a sustained increase in plasma insulin levels, which strongly suggests the emergence of insulin resistance of adult animals at later generations. Exposing pups at weaning to the ω6HFD led within seven weeks to changes in adipose tissue cellularity (Fig. 5; n ≥ 3). HF0 mice exhibited an increase in the percentage of very small adipocytes (∼20 μm) and a slight increase in that of large adipocytes (40–70 μm). This finding suggests recruitment of adipocyte precursors from the SVF and a trend toward adipocyte hypertrophy. The percentage of small adipocytes (20–30 μm) increased in HF1 mice, suggesting further recruitment of adipocyte precursors. However, in HF3 mice, a substantial change in cell hypertrophy occurred, with a shift to large-sized adipocytes (50–70 μm) and the emergence of a population of severely hypertrophied adipocytes (80–100 μm). Cell hypertrophy became dramatic for HF4 mice, in which two additional populations of large-size adipocytes, 50–70 and 70–130 μm, could be observed. A fairly large proportion of small adipocytes (20–40 μm) remained detectable in HF4 compared with HF0 mice, suggesting that adipocyte recruitment was still taking place at the fourth generation. When HF4 mice were switched at weaning to a chow diet, the pattern of adipocyte size distribution of revHF4 mice was dramatically altered after seven weeks as both populations of large-size adipocytes could no longer be observed. The values for STD, HF0, HF4, and revHF4 mice were 35.4 ± 15.4, 35.8 ± 17.9, 48.8 ± 29.1 and 41 ± 11.5 µm for the mean adipocyte size, whereas the percentage of cells above 50 µm in diameter was 12%, 20%, 40%, and 22%, respectively, suggesting “remnant” adipocyte hypertrophy. To gain insight into recruitment of adipocyte precursor cells, we analyzed by qRT-PCR the expression levels of preadipocyte markers. No significant enrichment was found in the stromal vascular fraction of adipose tissue of HF0–HF3 mice compared with STD mice with respect to Pref-1, PPARβ/δ, C/EBPβ, FAT/CD36, or aP2/FABP4 (not shown). These results suggest that the hyperplastic growth of fat tissue implicated progenitor cells were more immature than preadipocytes and able to replenish the pool of preadipocytes. To characterize progenitor cell populations that could ultimately contribute to the formation of new adipocytes, a comparative FACS analysis of stromal vascular cells of epididymal fat tissue was carried out in STD, HF0, HF1, and HF2 mice (n = 8) at 4, 8, and 22 weeks of age by using cell surface-specific markers of various cell lineages known to be present in this cell fraction. No difference could be found in the proportion of Sca1+/CD34+ cells representative of stem cells and immature progenitors or in that of CD45− and CD31− cells representative of the hematopoietic and endothelial lineages, respectively (not shown). Clearly, we cannot exclude changes in the proportion of the minor subpopulation of CD34+/Sca1+/CD24+ cells, which have been shown very recently to behave in vivo as early adipocyte progenitors (19.Rodeheffer M.S. Birsoy K. Friedman J.M. Identification of white adipocyte progenitor cells in vivo.Cell. 2008; 135: 240-249Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar). Expression profiles of the SVF of adipose tissue were established for the different generations of mice fed the ω6HFD (n ≥ 3 per group). Two independent experiments were carried out, and the identified probes were kept for subsequent analysis. To rule out possible contamination by adjacent tissues, a preliminary experiment was performed to directly compare the profile of SVF with those of testis and epidydimis. This led to a list of 2,366 distinct SVF-specific probes, which was subsequently used for further analysis. We then selected from this list about 108 transcripts divided into four groups (supplementary Table IV), based on a linear model proposed by Smyth in the limma package from Bioconductor (20.Smyth G.K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments.Stat. Appl. Genet. Mol. Biol. 2004; 3 (Article 3.)Crossref PubMed Scopus (9103) Google Scholar). Probes were selected based on average log2(Sig

Abstract Obesity is a pandemic disease associated with many metabolic alterations and involves several organs and systems. The endocannabinoid system (ECS) appears to be a key regulator of energy homeostasis and metabolism. Here we show that specific deletion of the ECS synthesizing enzyme, NAPE-PLD, in adipocytes induces obesity, glucose intolerance, adipose tissue inflammation and altered lipid metabolism. We report that Napepld -deleted mice present an altered browning programme and are less responsive to cold-induced browning, highlighting the essential role of NAPE-PLD in regulating energy homeostasis and metabolism in the physiological state. Our results indicate that these alterations are mediated by a shift in gut microbiota composition that can partially transfer the phenotype to germ-free mice. Together, our findings uncover a role of adipose tissue NAPE-PLD on whole-body metabolism and provide support for targeting NAPE-PLD-derived bioactive lipids to treat obesity and related metabolic disorders.

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.

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.

Variations in N-acylethanolamines (NAE) levels are associated with obesity and metabolic comorbidities. Their role in the gut remains unclear. Therefore, we generated a mouse model of inducible intestinal epithelial cell (IEC)-specific deletion of N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD), a key enzyme involved in NAE biosynthesis (Napepld∆IEC). We discovered that Napepld∆IEC mice are hyperphagic upon first high-fat diet (HFD) exposure, and develop exacerbated obesity and steatosis. These mice display hypothalamic Pomc neurons dysfunctions and alterations in intestinal and plasma NAE and 2-acylglycerols. After long-term HFD, Napepld∆IEC mice present reduced energy expenditure. The increased steatosis is associated with higher gut and liver lipid absorption. Napepld∆IEC mice display altered gut microbiota. Akkermansia muciniphila administration partly counteracts the IEC NAPE-PLD deletion effects. In conclusion, intestinal NAPE-PLD is a key sensor in nutritional adaptation to fat intake, gut-to-brain axis and energy homeostasis and thereby constitutes a novel target to tackle obesity and related disorders.

The arcuate nucleus (ARC) of the hypothalamus is particularly regarded as a critical platform that integrates circulating signals of hunger and satiety reflecting energy stores and nutrient availability. Among ARC neurons, pro-opiomelanocortin (POMC) and agouti-related protein and neuropeptide Y (NPY/AgRP neurons) are considered as two opposing branches of the melanocortin signaling pathway. Integration of circulating signals of hunger and satiety results in the release of the melanocortin receptor ligand α-melanocyte-stimulating hormone (αMSH) by the POMC neurons system and decreases feeding and increases energy expenditure. The orexigenic/anabolic action of NPY/AgRP neurons is believed to rely essentially on their inhibitory input onto POMC neurons and second-orders targets. Recent updates in the field have casted a new light on the role of the ARC neurons in the coordinated regulation of peripheral organs involved in the control of nutrient storage, transformation and substrate utilization independent of food intake.

A leading hypothesis of N-acyl ethanolamine (NAE) biosynthesis, including the endogenous cannabinoid anandamide (AEA), is that it depends on hydrolysis of N-acyl-phosphatidylethanolamines (NAPE) by a NAPE-specific phospholipase D (NAPE-PLD). Thus, deletion of NAPE-PLD should attenuate NAE levels. Previous analyses of two different NAPE-PLD knockout (KO) strains produced contradictory data on the importance of NAPE-PLD to AEA biosynthesis. Here, we examine this hypothesis with a strain of NAPE-PLD KO mice whose lipidome is uncharacterized. Using HPLC/MS/MS, over 70 lipids, including the AEA metabolite, N-arachidonoyl glycine (NAGly), the endocannabinoid 2-arachidonyl glycerol (2-AG) and prostaglandins (PGE(2) and PGF(2α)), and over 60 lipoamines were analyzed in 8 brain regions of KO and wild-type (WT) mice. Lipidomics analysis of this third NAPE-PLD KO strain shows a broad range of lipids that were differentially affected by lipid species and brain region. Importantly, all 6 NAEs measured were significantly reduced, though the magnitude of the effect varied by fatty acid saturation length and brain region. 2-AG levels were only impacted in the brainstem, where levels were significantly increased in KO mice. Correspondingly, levels of arachidonic acid were significantly decreased exclusively in brainstem. NAGly levels were significantly increased in 4 brain regions and levels of PGE(2) increased in 6 of 8 brain regions in KO mice. These data indicate that deletion of NAPE-PLD has far broader effects on the lipidome than previously recognized. Therefore, behavioral characteristics of suppressing NAPE-PLD activity may be due to a myriad of effects on lipids and not simply due to reduced AEA biosynthesis.

<h2>Summary</h2> There is a general consensus that overconsumption of sugar-sweetened beverages contributes to the prevalence of obesity and related comorbidities such as type 2 diabetes (T2D). Whether a similar relationship exists for no- or low-calorie "diet" drinks is a subject of intensive debate and controversy. Here, we demonstrate that consuming seven sucralose-sweetened beverages with, but not without, a carbohydrate over 10 days decreases insulin sensitivity in healthy human participants, an effect that correlates with reductions in midbrain, insular, and cingulate responses to sweet, but not sour, salty, or savory, taste as assessed with fMRI. Taste perception was unaltered and consuming the carbohydrate alone had no effect. These findings indicate that consumption of sucralose in the presence of a carbohydrate rapidly impairs glucose metabolism and results in longer-term decreases in brain, but not perceptual sensitivity to sweet taste, suggesting dysregulation of gut-brain control of glucose metabolism.

Liraglutide, an anti-diabetic drug and agonist of the glucagon-like peptide one receptor (GLP1R), has recently been approved to treat obesity in individuals with or without type 2 diabetes. Despite its extensive metabolic benefits, the mechanism and site of action of liraglutide remain unclear. Here, we demonstrate that liraglutide is shuttled to target cells in the mouse hypothalamus by specialized ependymoglial cells called tanycytes, bypassing the blood-brain barrier. Selectively silencing GLP1R in tanycytes or inhibiting tanycytic transcytosis by botulinum neurotoxin expression not only hampers liraglutide transport into the brain and its activation of target hypothalamic neurons, but also blocks its anti-obesity effects on food intake, body weight and fat mass, and fatty acid oxidation. Collectively, these striking data indicate that the liraglutide-induced activation of hypothalamic neurons and its downstream metabolic effects are mediated by its tanycytic transport into the mediobasal hypothalamus, strengthening the notion of tanycytes as key regulators of metabolic homeostasis.

The hypothalamus is key in the control of energy balance. However, strategies targeting hypothalamic neurons have failed to provide viable options to treat most metabolic diseases. Conversely, the role of astrocytes in systemic metabolic control has remained largely unexplored. Here, we show that obesity promotes anatomically restricted remodeling of hypothalamic astrocyte activity. In the paraventricular nucleus (PVN) of the hypothalamus, chemogenetic manipulation of astrocytes results in bidirectional control of neighboring neuron activity, autonomic outflow, glucose metabolism, and energy balance. This process recruits a mechanism involving the astrocytic control of ambient glutamate levels, which becomes defective in obesity. Positive or negative chemogenetic manipulation of PVN astrocyte Ca2+ signals, respectively, worsens or improves metabolic status of diet-induced obese mice. Collectively, these findings highlight a yet unappreciated role for astrocytes in the direct control of systemic metabolism and suggest potential targets for anti-obesity strategy.

Animals respond to hypoglycemia by eating and by stimulating gluconeogenesis. These responses to glucose deprivation are initiated by glucose-sensing neurons in the brain, but the neural circuits that control feeding behavior are not well established. Neurons in the arcuate region of the hypothalamus that express neuropeptide Y (NPY) and agouti-related protein (AgRP) have been implicated in mediating the feeding response to glucoprivation. We devised a method to selectively ablate these neurons in neonatal mice and then tested adult mice for their feeding responses to fasting, mild hypoglycemia, 2-deoxy-d-glucose and a ghrelin receptor agonist. Whereas the feeding response to the ghrelin receptor agonist was completely abrogated, the feeding response to glucoprivation was normal. The feeding response after a fast was attenuated when standard chow was available but normal with more palatable solid or liquid diet. We conclude that NPY/AgRP neurons are not necessary for generating or mediating the orexigenic response to glucose deficiency, but they are essential for the feeding response to ghrelin and refeeding on standard chow after a fast.

Background— Abnormal glucose metabolism is a central feature of disorders with increased rates of cardiovascular disease. Low levels of high-density lipoprotein (HDL) are a key predictor for cardiovascular disease. We used genetic mouse models with increased HDL levels (apolipoprotein A-I transgenic [apoA-I tg]) and reduced HDL levels (apoA-I–deficient [apoA-I ko]) to investigate whether HDL modulates mitochondrial bioenergetics in skeletal muscle. Methods and Results— ApoA-I ko mice exhibited fasting hyperglycemia and impaired glucose tolerance test compared with wild-type mice. Mitochondria isolated from gastrocnemius muscle of apoA-I ko mice displayed markedly blunted ATP synthesis. Endurance capacity during exercise exhaustion test was impaired in apoA-I ko mice. HDL directly enhanced glucose oxidation by increasing glycolysis and mitochondrial respiration rate in C2C12 muscle cells. ApoA-I tg mice exhibited lower fasting glucose levels, improved glucose tolerance test, increased lactate levels, reduced fat mass, associated with protection against age-induced decline of endurance capacity compared with wild-type mice. Circulating levels of fibroblast growth factor 21, a novel biomarker for mitochondrial respiratory chain deficiencies and inhibitor of white adipose lipolysis, were significantly reduced in apoA-I tg mice. Consistent with an increase in glucose utilization of skeletal muscle, genetically increased HDL and apoA-I levels in mice prevented high-fat diet–induced impairment of glucose homeostasis. Conclusions— In view of impaired mitochondrial function and decreased HDL levels in type 2 diabetes mellitus, our findings indicate that HDL-raising therapies may preserve muscle mitochondrial function and address key aspects of type 2 diabetes mellitus beyond cardiovascular disease.

Fatty acid (FA) -sensitive neurons are present in the brain, especially the hypothalamus, and play a key role in the neural control of energy and glucose homeostasis including feeding behavior, secretion insulin and action. Subpopulations of neurons in the arcuate and ventromedial hypothalamic nuclei are selectively either activated or inhibited by FA. Molecular effectors of these FA effects include ion channels such as chloride, potassium or calcium. In addition, at least half of the responses in the hypothalamic ventromedial FA neurons are mediated through interaction with the FA translocator/receptor, FAT/CD36, that does not require metabolism to activate intracellular signaling downstream. Recently, an important role of lipoprotein lipase in FA detection has also been demonstrated not only in the hypothalamus, but also in the hippocampus and striatum. Finally, FA could overload energy homeostasis via increased hypothalamic ceramide synthesis which could, in turn, contribute to the pathogenesis of diabetes of obesity and/or type 2 in predisposed individuals by disrupting the endocrine signaling pathways of insulin and/or leptin.

Myostatin (Mstn) participates in the regulation of skeletal muscle size and has emerged as a regulator of muscle metabolism. Here, we hypothesized that lack of myostatin profoundly depresses oxidative phosphorylation-dependent muscle function. Toward this end, we explored Mstn −/− mice as a model for the constitutive absence of myostatin and AAV-mediated overexpression of myostatin propeptide as a model of myostatin blockade in adult wild-type mice. We show that muscles from Mstn −/− mice, although larger and stronger, fatigue extremely rapidly. Myostatin deficiency shifts muscle from aerobic toward anaerobic energy metabolism, as evidenced by decreased mitochondrial respiration, reduced expression of PPAR transcriptional regulators, increased enolase activity, and exercise-induced lactic acidosis. As a consequence, constitutively reduced myostatin signaling diminishes exercise capacity, while the hypermuscular state of Mstn −/− mice increases oxygen consumption and the energy cost of running. We wondered whether these results are the mere consequence of the congenital fiber-type switch toward a glycolytic phenotype of constitutive Mstn −/− mice. Hence, we overexpressed myostatin propeptide in adult mice, which did not affect fiber-type distribution, while nonetheless causing increased muscle fatigability, diminished exercise capacity, and decreased Pparb/d and Pgc1a expression. In conclusion, our results suggest that myostatin endows skeletal muscle with high oxidative capacity and low fatigability, thus regulating the delicate balance between muscle mass, muscle force, energy metabolism, and endurance capacity.

Neuronal circuits in the brain help to control feeding behavior and systemic metabolism in response to afferent nutrient and hormonal signals. Although astrocytes have historically been assumed to have little relevance for such neuroendocrine control, we investigated whether lipid uptake via lipoprotein lipase (LPL) in astrocytes is required to centrally regulate energy homeostasis. Ex vivo studies with hypothalamus-derived astrocytes showed that LPL expression is upregulated by oleic acid, whereas it is decreased in response to palmitic acid or triglycerides. Likewise, astrocytic LPL deletion reduced the accumulation of lipid droplets in those glial cells. Consecutive in vivo studies showed that postnatal ablation of LPL in glial fibrillary acidic protein–expressing astrocytes induced exaggerated body weight gain and glucose intolerance in mice exposed to a high-fat diet. Intriguingly, astrocytic LPL deficiency also triggered increased ceramide content in the hypothalamus, which may contribute to hypothalamic insulin resistance. We conclude that hypothalamic LPL functions in astrocytes to ensure appropriately balanced nutrient sensing, ceramide distribution, body weight regulation, and glucose metabolism.

Energy-dense food alters dopaminergic (DA) transmission in the mesocorticolimbic (MCL) system and can promote reward dysfunctions, compulsive feeding, and weight gain. Yet the mechanisms by which nutrients influence the MCL circuitry remain elusive. Here, we show that nutritional triglycerides (TGs), a conserved post-prandial metabolic signature among mammals, can be metabolized within the MCL system and modulate DA-associated behaviors by gating the activity of dopamine receptor subtype 2 (DRD2)-expressing neurons through a mechanism that involves the action of the lipoprotein lipase (LPL). Further, we show that in humans, post-prandial TG excursions modulate brain responses to food cues in individuals carrying a genetic risk for reduced DRD2 signaling. Collectively, these findings unveil a novel mechanism by which dietary TGs directly alter signaling in the reward circuit to regulate behavior, thereby providing a new mechanistic basis by which energy-rich diets may lead to (mal)adaptations in DA signaling that underlie reward deficit and compulsive behavior.

The regulation of food intake, a sine qua non requirement for survival, thoroughly shapes feeding and energy balance by integrating both homeostatic and hedonic values of food. Unfortunately, the widespread access to palatable food has led to the development of feeding habits that are independent from metabolic needs. Among these, binge eating (BE) is characterized by uncontrolled voracious eating. While reward deficit seems to be a major contributor of BE, the physiological and molecular underpinnings of BE establishment remain elusive. Here, we combined a physiologically relevant BE mouse model with multiscale in vivo approaches to explore the functional connection between the gut-brain axis and the reward and homeostatic brain structures. Our results show that BE elicits compensatory adaptations requiring the gut-to-brain axis which, through the vagus nerve, relies on the permissive actions of peripheral endocannabinoids (eCBs) signaling. Selective inhibition of peripheral CB1 receptors resulted in a vagus-dependent increased hypothalamic activity, modified metabolic efficiency, and dampened activity of mesolimbic dopamine circuit, altogether leading to the suppression of palatable eating. We provide compelling evidence for a yet unappreciated physiological integrative mechanism by which variations of peripheral eCBs control the activity of the vagus nerve, thereby in turn gating the additive responses of both homeostatic and hedonic brain circuits which govern homeostatic and reward-driven feeding. In conclusion, we reveal that vagus-mediated eCBs/CB1R functions represent an interesting and innovative target to modulate energy balance and counteract food-reward disorders.

Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown.This study aimed to assess in vivo the physiological role of sEH-P.CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity.The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury.Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.

Growing evidence demonstrates the role of the striatal dopamine system in the regulation of glucose metabolism. Treatment with dopamine antagonists is associated with insulin resistance and hyperglycemia, while dopamine agonists are used in treatment of type 2 diabetes. The mechanism underlying striatal dopamine effects in glucose metabolism, however is not fully understood. Here, we provide mechanistic insights into the role of nucleus accumbens shell (sNAc) dopaminergic signaling in systemic glucose metabolism.Endogenous glucose production (EGP), blood glucose and mRNA expression in the lateral hypothalamic area (LHA) in male Wistar rats were measured following infusion of vanoxerine (VNX, dopamine reuptake inhibitor) in the sNAc. Thereafter, we analyzed projections from sNAc Drd1-expressing neurons to LHA using D1-Cre male Long-Evans rats, Cre-dependent viral tracers and fluorescence immunohistochemistry. Brain slice electrophysiology in adult mice was used to study spontaneous excitatory postsynaptic currents of sNAc Drd1-expressing neurons following VNX application. Finally, we assessed whether GABAergic LHA activity and hepatic vagal innervation were required for the effect of sNAc-VNX on glucose metabolism by combining infusion of sNAc-VNX with LHA-bicuculline, performing vagal recordings and combining infusion of sNAc-VNX with hepatic vagal denervation.VNX infusion in the sNAc strongly decreased endogenous glucose production, prevented glucose increases over time, reduced Slc17A6 and Hcrt mRNA in LHA, and increased vagal activity. Furthermore, sNAc Drd1-expressing neurons increased spontaneous firing following VNX application, and viral tracing of sNAc Drd1-expressing neurons revealed direct projections to LHA with on average 67 % of orexin cells directly targeted by sNAc Drd1-expressing neurons. Importantly, the sNAc-VNX-induced effect on glucose metabolism was dependent on GABAergic signaling in the LHA and on intact hepatic vagal innervation.We show that sNAc dopaminergic signaling modulates hepatic glucose metabolism through GABAergic inputs to glutamatergic LHA cells and hepatic vagal innervation. This demonstrates that striatal control of glucose metabolism involves a dopaminergic sNAc-LHA-liver axis and provides a potential explanation for the effects of dopamine agonists and antagonists on glucose metabolism.

Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPARγ or PPARδ promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPARδ in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPARδ mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPARδ enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPARγ and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPARδ mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPARδ plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPARδ expression or activity could affect adaptive responses of white adipose tissue to nutritional changes. Fatty acids have been postulated to regulate adaptation of adipose mass to nutritional changes by controlling expression of genes implicated in lipid metabolism via activation of nuclear receptors. Ectopic expression of the nuclear receptors PPARγ or PPARδ promotes adipogenesis in fibroblastic cells exposed to thiazolidinediones or long-chain fatty acids. To investigate the role of PPARδ in fatty acid regulation of gene expression and adipogenesis in a preadipose cellular context, we studied the effects of overexpressing the native receptor or the dominant-negative PPARδ mutant in Ob1771 and 3T3-F442A cells. Overexpression of PPARδ enhanced fatty acid induction of the adipose-related genes for fatty acid translocase, adipocyte lipid binding protein, and PPARγ and fatty acid effects on terminal differentiation. A transactivation-deficient form of PPARδ mutated in the AF2 domain severely reduced these effects. Findings are similar in Ob1771 or 3T3-F442A preadipose cells. These data demonstrate that PPARδ plays a central role in fatty acid-controlled differentiation of preadipose cells. Furthermore, they suggest that modulation of PPARδ expression or activity could affect adaptive responses of white adipose tissue to nutritional changes. long-chain fatty acid adipocyte lipid binding protein 2-bromopalmitate fatty acid transporter glyceraldehyde-3-phosphate dehydrogenase glycerophosphate dehydrogenase peroxisome proliferator-activated receptor PPAR responsive element retinoid X receptor kilobase(s) Dietary long-chain fatty acids (LCFA)1 control adipose tissue mass by regulating both the number and the size, i.e.the lipid accumulation, of adipocytes. This was illustrated in vivo by the findings that high fat diets promote hyperplastic and hypertrophic development of adipose tissue and massive obesity in adult rodents (1Faust I.M. Johnson P.R. Stern J.S. Hirsch J. Am. J. Physiol. 1978; 235: E279-E286Crossref PubMed Google Scholar, 2Klyde J.B. Hirsch J. J. Lipid Res. 1979; 20: 705-715Abstract Full Text PDF PubMed Google Scholar, 3Shillabeer G. Lau D.C. J. Lipid Res. 1994; 35: 592-600Abstract Full Text PDF PubMed Google Scholar). Adipogenic effects of LCFA have also been documentedin vitro by demonstrating that exposure of preadipose cells to native or non-metabolized fatty acids, such as 2-bromopalmitate (2BrP), increased the number of cells committed to differentiate as well as expression levels of adipose-related genes (4Amri E.Z. Ailhaud G. Grimaldi P.A. J. Lipid Res. 1994; 35: 930-937Abstract Full Text PDF PubMed Google Scholar). Cellular effects of fatty acids and some of their metabolites are related, at least in part, to activation of nuclear receptors called PPARs. Two different PPAR subtypes, δ and γ, are expressed in preadipose and adipose cells. PPARγ has been shown to play a central role in the control of gene expression and adipogenesis (5Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar, 6Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 7Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (411) Google Scholar, 8Gurnell M. Wentworth J.M. Agostine M. Adams M. Collingwood T.N. Provenzano C. Browne P.O. Rajanayagam O. Burris T.P. Schwabe J.W. Lazar M.A. Chatterjee V.K.K. J. Biol. Chem. 2000; 275: 5754-5759Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). Synthetic and naturally occurring PPARγ activators, such as thiazolidinediones or 15-deoxy-Δ12–14-prostaglandin J2, are potent stimulators of terminal differentiation of cultured preadipose cells (9Lehman J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3469) Google Scholar, 10Forman B.M. Tontonoz P. Chen J. Brun R.P. Spiegelman B.M. Evans R.M. Cell. 1995; 83: 803-812Abstract Full Text PDF PubMed Scopus (2740) Google Scholar). We have recently proposed that PPARδ acts as an early player in LCFA induction of terminal differentiation by promoting PPARγ expression. Fibroblasts ectopically expressing PPARδ respond to LCFA by transcriptional activation of genes for fatty acid translocase (FAT/CD36), adipocyte lipid binding protein (ALBP), and PPARγ. Although treatment with fatty acids alone was not sufficient to trigger adipogenesis, exposure to a combination of PPARδ and PPARγ activators, for example 2BrP and thiazolidinedione, or to a pan-PPAR activator, such as prostacyclin, promotes the expression of a typical adipose differentiation program and adipogenesis (11Bastie C. Holst D. Gaillard D. Jehl-Pietri C. Grimaldi P.A. J. Biol. Chem. 1999; 274: 21920-21925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). These experiments, which documented the role of PPARδ in the adipogenic action of LCFA, also illustrated a major difference between fibroblasts and preadipose cells. Fibroblasts expressing PPARδ strictly require exposure to strong PPARγ activators to trigger terminal differentiation, whereas preadipose cells do not. A similar situation had already been described for PPARγ-expressing fibroblasts (5Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar). This discrepancy may reflect the ability of differentiating preadipose cells to synthesize and to accumulate enough endogenous PPARγ activator to undergo terminal differentiation. Because many other differences could exist between preadipose cells and PPAR-expressing fibroblasts, it is crucial to examine the role of these transcription factors in a preadipose cellular context to confirm their role in adipocyte differentiation. Therefore, we have investigated the effects of overexpression of PPARδ and dominant-negative PPARδ mutant on the control by LCFA of gene expression and terminal differentiation in Ob1771 preadipose cells. The dominant-negative PPARδ was generated by substitution of a glutamate residue by a proline in the loop preceding the AF-2 domain. This was based on numerous studies (12Schulman I.G. Juguillon H. Evans R.M. Mol. Cell. Biol. 1996; 16: 3807-3813Crossref PubMed Scopus (117) Google Scholar, 13Fritsch M.C. Leary C.M. Furlow J.D. Ahrens H. Schuh T.J. Mueller G.C. Gorski J. Biochemistry. 1992; 31: 5303-5311Crossref PubMed Scopus (55) Google Scholar, 14Voegel J.J. Heine M.J.S. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (953) Google Scholar, 15Tate B.F. Grippo J.F. J. Biol. Chem. 1995; 270: 20258-20263Crossref PubMed Scopus (22) Google Scholar, 16Baniahmad A. Leng X. Burris T.P. Tsai S.Y. Tsai M.J. O'Maley B.W. Mol. Cell. Biol. 1995; 15: 76-86Crossref PubMed Google Scholar) with various members of the nuclear receptor superfamily showing that the AF-2 domain is important for activity. Receptors mutated in, or near, the AF-2 region, fail to either release corepressors or interact with coactivators and are inactive. Mutations in the C-terminal region of RARα, i.e. close to the AF-2 domain, were also reported to yield dominant-negative mutant receptors (17Durand B. Saunders M. Gaudon C. Roy B. Losson R. Chambon P. EMBO J. 1994; 13: 5370-5382Crossref PubMed Scopus (316) Google Scholar). The crystal structures of some nuclear receptors, including PPARδ (18Xu H.E. Lambert M.H. Montana V.G. Parks D.J. Blanchard S.G. Brown P.J. Sternbach D.D. Lehmann J.M. Wisely G.B. Willson T.M. Kliexer S.A. Milburn M.V. Mol. Cell. 1999; 3: 397-403Abstract Full Text Full Text PDF PubMed Scopus (970) Google Scholar), strongly support the hypothesis that ligand binding promotes a conformational change resulting in release of corepressors and interaction with coactivators, permitting the transition from an inactive to an active transcriptional complex (reviewed in Ref. 19Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (600) Google Scholar). We report that overexpression of PPARδ enhances LCFA responsiveness of preadipose cells in terms of maximal response and sensitivity and promotes terminal adipose differentiation. By opposition, expression of a PPARδ mutant, that exerts a dominant-negative action, strongly decreases both the short term, i.e. activation of gene expression, and long term, i.e. adipogenesis, responses to LCFA. The retroviral constructs containing PPARδ cDNA or PPARδE411P mutant cDNA were derived from pSG5-FAAR (20Amri E.Z. Bonino F. Ailhaud G. Abumrad N.A. Grimaldi P.A. J. Biol. Chem. 1995; 270: 2367-2371Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar) and cloned into the BamHI site of pBizeoneo retroviral vector (Dr. K. Kristiansen, University of Odense, Denmark). The PPARδE411P was obtained by the site-directed mutagenesis method of Viville (21Viville S. Methods Mol. Biol. 1995; 31: 57-65Google Scholar) using the 5′-CATCATCTGGGCGTGTGGGGTGACCAGCTG-3′ oligonucleotide, and the construct was verified by sequencing. Cells were grown in Dulbecco's modified Eagle's medium complemented with 8% fetal calf serum, 200 units/ml penicillin, 50 μg/ml streptomycin, 33 μm biotin, 17 nm calcium pantothenate (standard medium). For differentiation, cells were shifted after confluence to a standard medium supplemented with 17 nm insulin and 1 nmtriiodothyronine (differentiation medium). Medium was changed every other day. Oil Red O staining was performed as described previously (22Green H. Kehinde O. Cell. 1994; 1: 113-116Abstract Full Text PDF Scopus (746) Google Scholar). BOSC23 cells were transfected at 50–70% of confluence by lipofection (Fugene 6, Roche Molecular Biochemicals) by 8 μg of pBizeoneo or pBizeoneoPPARδ or pBizeoneoPPARδE411P expression vectors. After 8 h, cells were re-fed with fresh standard medium and viral supernatants were collected 48 h later. Ob1771 (23Amri E.Z. Dani C. Doglio A. Etienne J. Grimaldi P. Ailhaud G. Biochem. J. 1986; 238: 115-122Crossref PubMed Scopus (49) Google Scholar) or 3T3F442A (24Green H. Kehinde O. Cell. 1994; 7: 105-113Abstract Full Text PDF Scopus (616) Google Scholar) cells grown in standard medium were infected with equal titers of recombinant virus for 6 h and then maintained for 48 h in fresh standard medium and then replated with a 1:5 dilution in standard medium containing 0.4 mg/ml Geneticin. Stable populations were obtained after 7–10 days of selection. HEK-293 cells were grown in standard medium and plated in 24-well plates. At 80% confluence, cells were transfected by Fugene 6 with 0.5 μg/well 3xACOPPRE-Tk luciferase reporter vector, 25 ng/well pCMV-RXRα expression vector, 15 ng/well pCMV-βGalactosidase vector, and various amounts of pBizeoneoPPARδ or pBizeoneoPPARδE411P expression vectors. After 6 h, cells were re-fed with fresh standard medium with or without 50 μm2BrP. A 50 mm stock solution of 2BrP and dilutions were prepared in Me2SO. Luciferase and galactosidase activities were analyzed 48 h later using the luciferase assay system (Promega France), and the Galacto-Light assay system (Tropix, PerkinElmer, France), respectively. Each transfection was performed in triplicate, and the fluorescence of the samples was measured using a 1450 Micro Beta luminometer (Wallac, Finland). Total RNA was prepared and analyzed by Northern blotting as described previously (4Amri E.Z. Ailhaud G. Grimaldi P.A. J. Lipid Res. 1994; 35: 930-937Abstract Full Text PDF PubMed Google Scholar). Probes were labeled with [α-32P]dCTP using the random priming kit from Stratagene, and hybridizations were performed at 42 °C in a 50% formamide buffer. Blots were subjected to digital imaging (FujixBAS1000). GAPDH mRNA, which is not affected by adipose differentiation, was monitored as the internal standard. Total cell extracts were prepared from the various virally infected Ob1771 and 3T3F-442A populations, in a buffer containing 50 mm Tris (pH 7, 4), 250 mm NaCl, 5 mm EDTA, 1 mm vanadate, 0,5 mmphenylmethylsulfonyl fluoride, 0,1% Nonidet P-40. The extracts were separated on 10% polyacrylamide SDS gels and blotted to nitrocellulose membranes. PPARδ and PPARδE441P mutant proteins were detected using a polyclonal antiserum raised against the A/B domain of mouse PPARδ and which recognizes native and mutated PPARδ. Immunodetection was performed by chemiluminescence using an ECL reagent from Amersham Pharmacia Biotech (France). Glycerophosphate dehydrogenase (GPDH) activity, which provides the glycerol 3-phosphate required for triglyceride synthesis and is induced during terminal differentiation, was assayed spectrophotometrically as described previously (25Negrel R. Grimaldi P. Ailhaud G. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 6054-6058Crossref PubMed Scopus (204) Google Scholar). Enzyme activity was expressed in milliunits,i.e. nanomoles of product formed per min/mg of protein. Protein content of samples was determined according to Lowry et al. (26Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 173-181Google Scholar) using bovine serum albumin as standard. Cellular triglyceride content was measured by using the Sigma Triglycerides 320-UV kit according to the manufacturer's instructions. Triolein was used as standard. Culture media, fetal calf serum, and Geneticin were from Life Technologies, Inc. (France). 2BrP and other chemical products were purchased from Sigma and Aldrich (France). Radioactive materials and nylon membranes were from Amersham Pharmacia Biotech (France). Rosiglitazone was a kind gift from SmithKline Beecham Pharmaceuticals (United Kingdom). The PPARδE411P mutant was obtained by replacing Glu-411 by a proline residue. This mutated PPARδ was assayed for activation of a PPAR-responsive reporter gene and for dominant-negative activity against either native PPARδ or PPARγ. HEK-293 cells were transfected with a PPAR-responsive luciferase reporter (27Kliewer S.A. Umesono K. Noonan D.J. Heyman R. Evans R.M. Nature. 1992; 258: 771-774Crossref Scopus (1525) Google Scholar) and an expression vector for the obligate partner RXRα. As shown in Fig. 1, treatment of these cells with 2BrP, a non-metabolized long-chain fatty acid (28Grimaldi P.A. Knobel S.M. Whitesell R.R. Abumrad N.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10930-10934Crossref PubMed Scopus (118) Google Scholar) and potent activator of PPARδ (20Amri E.Z. Bonino F. Ailhaud G. Abumrad N.A. Grimaldi P.A. J. Biol. Chem. 1995; 270: 2367-2371Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar) resulted in a very moderate induction of luciferase. Cells transfected with the PPARδ expression vector showed a 12-fold 2BrP-dependent activation. In contrast, no activation was observed in cells transfected with the mutated PPARδ, indicating that this mutant is inactive.Figure 1Characterization of a dominant-negative mutation of PPAR δ. A, HEK-293 cells were transfected as described under “Experimental Procedures” with luciferase and galactosidase plasmids, 25 ng of RXRα expression vector, and the indicated amounts in nanograms of PPARδ and PPARδE411P expression vectors. Cells were then maintained for 48 h in the absence (open bars) or presence (solid bars) of 50 μm 2BrP prior to determination of luciferase and galactosidase activities. B, HEK-293 cells were treated as in A but transfected with a PPARγ expression vector instead PPARδ expression vector and exposed (hatched bars) or not (open bars) to 50 μm rosiglitazone for 48 h prior to enzymatic assays. Results are presented by taking as 1 the value obtained for cells maintained in control medium and transfected with PPARδ (A) or PPARγ (B) vectors and are the mean ± S.D. of three separate experiments.View Large Image Figure ViewerDownload (PPT) The dominant-negative activity of PPARδE411P was then investigated by transfection of HEK-293 cells with a constant amount of PPARδ (Fig. 1 A) or PPARγ (Fig. 1 B) expression vector and increasing amounts of PPARδE411P expression vector. These experiments revealed that PPARδE411P inhibits, in a dose-dependent manner, the PPARδ-mediated transactivation. Luciferase induction was decreased by 50% and 85% when the mutated PPARδ was used in a 4- and 20-fold excess, respectively (Fig. 1 A). In contrast, PPARδE411P did not inhibit rosiglitazone-induced and PPARγ-mediated transactivation of the reporter gene (Fig. 1 B). Taken together, these observations indicated that the PPARδE411P exerts a dominant-negative activity specifically on the PPARδ-mediated transactivation. Expression of either PPARδ or dominant-negative mutant PPARδE411P was accomplished in Ob1771 by retroviral infection (11Bastie C. Holst D. Gaillard D. Jehl-Pietri C. Grimaldi P.A. J. Biol. Chem. 1999; 274: 21920-21925Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). As shown in Fig. 2 A, Ob1771Biz cells expressed at day 1 post-confluence the endogenous PPARδ mRNA at the expected size of 3.5 kb. A stronger signal was found at about 5 kb corresponding to the viral transcript in both Ob1771PPARδ and Ob1771PPARδE411P. Further experiments revealed that the level of endogenous PPARδ mRNA increased by 4-fold during the first week after confluence in all cell populations, whereas expression of BizeoneoPPARδ and BizeoneoPPARδE411P mRNA remained unchanged (not shown). Western blot analysis, performed with an antiserum directed against the PPARδ A/B domain and thus cross-reacting with both native and mutated proteins, revealed that at day 1 post-confluence Ob1771PPARδ and Ob1771PPARδE411P cells contained, respectively, 8- and 6-fold more PPARδ protein than did control Ob1771Biz cells (Fig. 2 B). To investigate the effects of PPARδ and the dominant-negative PPARδ mutant on responsiveness to LCFA-induced transcription, Ob1771Biz, Ob1771PPARδ, and Ob1771PPARδE411P cell populations were grown to confluence and then exposed for 2 days to increasing concentrations of 2BrP. As described previously for parental Ob1771 cells (29Amri E.Z. Bertrand B. Ailhaud G. Grimaldi P. J. Lipid Res. 1991; 32: 1449-1456Abstract Full Text PDF PubMed Google Scholar), control Ob1771Biz cells showed a fatty acid dose-dependent induction of the fatty acid transporter FAT and ALBP mRNA (Fig. 3). Induction of these genes was markedly enhanced in Ob1771 overexpressing PPARδ. These cells were also more sensitive to 2BrP than control cells (EC50 of about 10 μm in Ob1771PPARδversus greater than 30 μm in Ob1771Biz). In contrast, Ob1771 expressing the dominant-negative PPARδ displayed a reduced induction of FAT and ALBP mRNA even at higher fatty acid concentrations. Interestingly, the mutated protein did not completely abolish the transcriptional response to 2BrP. Taken together, the data indicate that a change in the PPARδ activity, i.e. overexpression or inhibition, modulates the action of LCFA on gene expression in preadipose cells. To promote adipogenesis, cells were maintained after confluence in differentiation medium and treated for the first 5 days with increasing concentrations of 2BrP. Adipogenesis was estimated by Oil Red O staining (Fig. 4) and by determination of cellular triglyceride amounts (Fig. 5) at day 14 post-confluence and measurements of GPDH activity at days 9 and 14 (Fig. 6).Figure 6Effects of PPARδ and dominant-negative PPARδ on GPDH expression in Ob1771 cells. Ob1771Biz (▪), Ob1771PPARδ (●), and Ob1771PPARδE411P (▴) cells were maintained as in Fig. 4 and GPDH specific activity was determined as described under “Experimental Procedures” at days 9 and 14 post-confluence. Results are the mean ± S.D. from three separate experiments.View Large Image Figure ViewerDownload (PPT) As described previously for the original Ob1771 cell line (4Amri E.Z. Ailhaud G. Grimaldi P.A. J. Lipid Res. 1994; 35: 930-937Abstract Full Text PDF PubMed Google Scholar), 2BrP treatment during the preadipose state enhanced terminal differentiation in Ob1771Biz cells in a dose-dependent manner, as shown by the tremendous increase of triglyceride accumulation (Figs. 4 and 5). The adipogenic effect of the fatty acid was also illustrated by the induction of GPDH activity during the course of differentiation (day 9) or at the end of the process (day 14). Compared with control cells, Ob1771PPARδ cells displayed an increased ability to differentiate and enhanced fatty acid sensitivity. This is illustrated by greater increases of both triglyceride accumulation (Figs. 4 and 5) and GPDH activity (Fig. 6) occurring at lower 2BrP concentrations. Interestingly, as shown by the Oil Red O staining, adipose differentiation was nearly complete when the cells were treated by 25 μm fatty acid, which suggests that PPARδ overexpression dramatically promoted the commitment of preadipose cells to terminal differentiation. The decrease of PPARδ activity in Ob1771, expressing the dominant-negative form of this nuclear receptor, resulted in a significant reduction of adipose differentiation. Indeed, Ob1771PPARδE411P cells did not accumulate lipids when maintained in low 2BrP concentrations. At high concentrations of 2BrP, these cells displayed moderate adipogenesis (Fig. 4) and contained less triglyceride than control cells (Fig. 5). This is confirmed by the significantly lower GPDH activities measured in Ob1771PPARδE411P than in control cells under all conditions assayed (Fig. 6). Because PPARγ plays a crucial role in terminal differentiation, we investigated its expression pattern in the three cell populations treated with 25 μm 2BrP during the first 5 days after confluence. As described for the original Ob1771 line (20Amri E.Z. Bonino F. Ailhaud G. Abumrad N.A. Grimaldi P.A. J. Biol. Chem. 1995; 270: 2367-2371Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar), in Ob1771Biz cells, PPARγ mRNA emerged after confluence and gradually increased until terminal differentiation (Fig. 7). In Ob1771PPARδ, PPARγ mRNA was already detected at confluence and accumulated thereafter to reach a maximal value at day 6. Noteworthy, at the end of adipose differentiation, i.e. day 14, PPARγ mRNA amounts were nearly similar in the two cell populations. In contrast, expression of the dominant-negative PPARδ resulted in a marked down-regulation of PPARγ mRNA. Expression levels remained relatively low and were, at day 14 post-confluence, 6-fold lower than those measured in control cells. The effects of dominant-negative PPARδ were also investigated in the 3T3-F442A preadipose cell line, which was established from mouse embryo (24Green H. Kehinde O. Cell. 1994; 7: 105-113Abstract Full Text PDF Scopus (616) Google Scholar). Cell populations were obtained after infection with retroviral pBizeoneo vector with or without the dominant-negative PPARδ coding sequence. Fig. 8 A shows that 442Abiz cells expressed a significant level of PPARδ at confluence, whereas PPARδ protein was 8-fold more abundant in confluent 442APPARδE441P cells. Exposure for 4 days after confluence to 10 μm 2BrP was associated with low adipogenesis in 442APPARδE411P cells, indicating that the dominant-negative PPARδ mutant strongly repressed the process. This is evidenced by the marked reduction of Oil Red O staining in 442APPARδE411P population. Although treatment of these cells with 2BrP significantly increased lipid accumulation, levels still remained lower than those observed in 442Abiz cells maintained in control medium without 2BrP (Fig. 8 B). The time course of PPARγ gene expression was investigated in 442Abiz and 442APPARδE411P cells exposed to 2BrP. In 442Abiz, PPARγ mRNA emerged at day 2 post-confluence and then accumulated rapidly to reach a maximal level at day 5. On the other hand, the induction of PPARγ mRNA was seriously delayed and reduced in 442APPARδE411P cells,i.e. emerging only at day 5 and remaining lower than in control cells at all times of determination (Fig. 8 C). Consistent with the Oil Red O staining, 442APPARδE411P cells expressed lower amounts of ALBP, FAT, and GPDH mRNAs than control cells when maintained in the absence or presence of 2BrP treatment (Fig. 8 D). These findings demonstrate that PPARδ is important for LCFA regulation of 3T3-F442A differentiation. This study is a first examination of the role of PPARδ in mediating LCFA regulation of gene expression and adipogenesis in preadipose cells. Two complementary approaches have been used in the study, i.e. overexpression of the native nuclear receptor and expression of a dominant-negative PPARδ mutant. As would be predicted based on studies with other nuclear receptors (17Durand B. Saunders M. Gaudon C. Roy B. Losson R. Chambon P. EMBO J. 1994; 13: 5370-5382Crossref PubMed Scopus (316) Google Scholar), the substitution of a glutamate residue by a proline at position 411 in PPARδ generates a protein without transcriptional activity and which exerts an inhibitory effect on the endogenous PPARδ. With other nuclear receptors, such mutations yield proteins that bind the ligand and the DNA-responsive element but that constitutively remain in a repressed form (19Glass C.K. Rose D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1997; 9: 222-232Crossref PubMed Scopus (600) Google Scholar). Consequently, the inhibitory action of the constitutively repressed PPARδE411P mutant may reflect its competition with endogenous native PPARδ for binding to the PPRE. However, regardless of the underlying mechanism behind the dominant-negative activity of PPARδE411P, it is worth noting that the mutant did not exert a complete inhibition on the native receptor even when used in high excess (Fig. 1 A) and did not affect the transcriptional activity of PPARγ (Fig. 1 B). The lack of effect on PPARγ probably relates to the higher affinity of the γ isoform for the PPRE as compared with that of PPARδ. Such a difference in binding affinities between the two isoforms for several natural PPRE has been previously documented by electrophoretic mobility shift assay experiments (30Juge-Aubry C. Pernin A. Favez T. Burger A.G. Wahli W. Meier C.A. Desvergne B. J. Biol. Chem. 1997; 272: 25252-25259Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). This work clearly demonstrates that a change in PPARδ level or activity strongly alters the response of Ob1771 cells to LCFA and that this nuclear receptor acts early in the adipose differentiation time course. Preadipose Ob1771PPARδ cells, in which the PPARδ level was increased by retroviral infection, display a magnified response to fatty acids as shown by higher induction of FAT and ALBP gene expression at low fatty acid concentrations (Fig. 3). The adipogenic action of fatty acids in promoting terminal differentiation also occurs at lower concentration in Ob1771PPARδ as shown by Oil Red O staining (Fig. 4), by GPDH activity (Fig. 5), and by triglyceride accumulation (Fig. 6). Furthermore, the almost complete terminal differentiation, shown by the homogenous Oil Red O staining observed in Ob1771PPARδ exposed for only the first 5 days to 2BrP (Fig. 4), strongly supports a crucial role of LCFA activated-PPARδ during early confluent phase in promoting the commitment of preadipocytes to adipogenesis. In addition to enhancing the effects of added fatty acids, the increase in PPARδ expression also exerts a potent action on terminal adipose differentiation of cells maintained in control medium. Ob1771PPARδ cells accumulate significantly more lipids (Fig. 5) and express more GPDH activity (Fig. 6) than do control cells. The considerable increase of terminal differentiation observed in the absence of added 2BrP could be explained by increased sensitivity to the effects of LCFA from the serum or from endogenous origin or to other naturally occurring activators such as prostacyclin, a potent PPARδ activator (7Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (411) Google Scholar) synthesized by early confluent Ob1771 cells (31Negrel R. Ailhaud G. Biochem. Cell Biol. Commun. 1981; 98: 768-777Google Scholar). The role of PPARδ as a nuclear mediator of LCFA effects on gene transcription and adipose differentiation is confirmed by the demonstration that expressing the dominant-negative mutant in Ob1771 cells considerably attenuates 2BrP-induced transcriptions of FAT and ALBP genes in preadipose cells (Fig. 3) and 2BrP enhancement of adipogenesis (Fig. 4). However, the cells remain able to respond to the fatty acid derivative, as shown by exposure to high concentrations of 2BrP. Morphological (Fig. 4) and biochemical investigations (Figs. 5and 6) indicate that expression of the dominant-negative mutant, by partially inhibiting action of the endogenous PPARδ, dramatically impairs terminal differentiation of Ob1771 cells. Ob1771PPARδE411P cells do not undergo terminal differentiation when maintained in medium containing low concentrations of activator but display adipogenesis when high concentrations of 2BrP are used. Residual responses during short term or long term exposure to high concentrations of LCFA are not surprising, because transactivation experiments revealed that the dominant-negative PPARδ mutant, even when used at a high excess, does not completely suppress fatty acid activation of the native nuclear receptor (Fig. 1). The amount of the mutated protein expressed in Ob1771PPARδE411P at day 1 after confluence was estimated to be approximately 6-fold higher than the endogenous PPAR (Fig. 2 B). Thus, in Ob1771PPARδE411P cells exposed to high concentrations of 2BrP, the amount of activated native PPARδ may be high enough to effectively compete with the mutated repressed receptor for binding to the PPRE and to allow transcription of LCFA-responsive genes. It is possible that, at high concentrations of LCFA, there is accumulation of ligand-activated native PPARδ, which favors the binding of the active transcription factor to the PPRE of LCFA-responsive gene promoters. The respective positive or negative actions of either native or dominant-negative PPARδ on terminal differentiation are a likely consequence of the alterations of PPARγ expression. Although overexpression of the native nuclear receptor did not significantly change the maximal expression of PPARγ, it resulted in an earlier induction of its expression when compared with control cells. Expression of the dominant-negative mutant led to an impairment of PPARγ expression during the differentiation phase (Fig. 7). Thus, the pattern of PPARγ expression in the three different Ob1771-derived cell lines used in this study supports the interpretation that LCFA-activated PPARδ controls PPARγ gene expression and that PPARγ is crucial for the induction of genes related to terminal differentiation. These findings were applicable to both Ob1771 and 3T3F442A cells and indicated that they are not dependent on nor specific to a particular cell line. For example, despite the fact that terminal differentiation of 3T3-F442A cells is less dependent on fatty acid supply as shown for 442Abiz cells (Fig. 8, A and C), expression of the dominant-negative PPARδ in 3T3-F442A cells severely reduces adipogenesis in a way similar to Ob1771 cells. In summary, the observations reported in this study, clearly establish the role for PPARδ as nuclear mediator of LCFA-mediated transcriptional and adipogenic actions in a preadipose cell context. The findings suggest that changes in the amount or activity of this nuclear receptor in preadipose cells may have important functional consequences with respect to the response of adipose mass to nutritional changes. Possibly, up-regulation of the receptor would result in hypersensitivity to LCFA effects in increasing adipose tissue mass, whereas down-regulation may confer resistance to LCFA. PPARδ is also expressed in various tissues, including heart, skeletal muscle, and intestine, and it would be of interest to characterize what tissue-specific effects up- or down-regulation of this nuclear receptor would have in the whole animal. The dominant-negative PPARδ mutant described in this study can be used to selectively inhibit LCFA-induced transcriptional activation in particular tissues. The construction of transgenic animals expressing the PPARδE411P mutant in a tissue-specific manner would provide valuable information on the role of PPARδ in various tissues. We thank Nada A. Abumrad (Stony Brook, NY) and Ellen Van Obberghen-Schilling (Nice, France) for critical comments and review of the manuscript and Delphine Brignon for expert technical assistance.

Peroxisome proliferator-activated receptors (PPARs) are lipid-activated transcription factors playing important regulatory functions in development and metabolism. PPARα and PPARγ are the most extensively examined and characterized, mainly because they are activated by marketed hypolipidemic and insulin sensitizer compounds, such as fibrates and thiazolidinediones. It has been established that the third member of the family, PPARδ is implicated in developmental regulations, but until recently, its role in metabolism remained unclear. The availability of specific PPARδ agonists and of appropriate cellular and animal models revealed that PPARδ plays a crucial role in fatty acid metabolism in several tissues. Treatment of obese animals with PPARδ agonists results in normalization of metabolic parameters and reduction of adiposity. Activation of the nuclear receptor promotes fatty acid burning in skeletal muscle and adipose tissue by upregulation of fatty acid uptake, β-oxidation and energy uncoupling. PPARδ is also involved in the adaptive metabolic responses of skeletal muscle to environmental changes, such as long-term fasting or physical exercise, by controlling the number of oxidative myofibers. These observations strongly suggest that PPARδ agonists may have therapeutic usefulness in metabolic syndrome by increasing fatty acid consumption and decreasing obesity.

The arcuate nucleus of the hypothalamus contains at least two populations of neurons that continuously monitor signals reflecting energy status and promote the appropriate behavioral and metabolic responses to changes in energy demand. Activation of neurons making pro-opiomelanocortin (POMC) decreases food intake and increases energy expenditure through activation of G protein-coupled melanocortin receptors via the release of α-melanocyte-stimulating hormone. Until recently, the prevailing idea was that the neighboring neurons [agouti-related protein (AgRP) neurons] co-expressing the orexigenic neuropeptides, AgRP, and neuropeptide Y increase feeding by opposing the anorexigenic actions of the POMC neurons. However, it has now been demonstrated that only AgRP neurons activation - not POMC neurons inhibition - is necessary and sufficient to promote feeding. Projections of AgRP-expressing axons innervate mesolimbic, midbrain, and pontine structures where they regulate feeding and feeding-independent functions such as reward or peripheral nutrient partitioning. AgRP neurons also make gamma aminobutyric acid , which is now thought to mediate many of critical functions of these neurons in a melanocortin-independent manner and on a timescale compatible with neuromodulation.

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.

Osteoporosis and overweight/obesity constitute major worldwide public health burdens that are associated with aging. A high proportion of women develop osteoporosis and increased intraabdominal adiposity after menopause. which leads to bone fractures and metabolic disorders. There is no efficient treatment without major side effects for these 2 diseases. We previously showed that the administration of oxytocin (OT) normalizes ovariectomy-induced osteopenia and bone marrow adiposity in mice. Ovariectomized mice, used as an animal model mimicking menopause, were treated with OT or vehicle. Trabecular bone parameters and fat mass were analyzed using micro-computed tomography. Herein, we show that this effect on trabecular bone parameters was mediated through the restoration of osteoblast/osteoclast cross talk via the receptor activator of nuclear factor-κB ligand /osteoprotegerin axis. Moreover, the daily administration of OT normalized body weight and intraabdominal fat depots in ovariectomized mice. Intraabdominal fat mass is more sensitive to OT that sc fat depots, and this inhibitory effect is mediated through inhibition of adipocyte precursor's differentiation with a tendency to lower adipocyte size. OT treatment did not affect food intake, locomotors activity, or energy expenditure, but it did promote a shift in fuel utilization favoring lipid oxidation. In addition, the decrease in fat mass resulted from the inhibition of the adipose precursor's differentiation. Thus, OT constitutes an effective strategy for targeting osteopenia, overweight, and fat mass redistribution without any detrimental effects in a mouse model mimicking the menopause.

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.

Identification of new adipokines that potentially link obesity to insulin resistance represents a major challenge. We recently showed that NOV/CCN3, a multifunctional matricellular protein, is synthesized and secreted by adipose tissue, with plasma levels highly correlated with BMI. NOV involvement in tissue repair, fibrotic and inflammatory diseases, and cancer has been previously reported. However, its role in energy homeostasis remains unknown. We investigated the metabolic phenotype of NOV−/− mice fed a standard or high-fat diet (HFD). Strikingly, the weight of NOV−/− mice was markedly lower than that of wild-type mice but only on an HFD. This was related to a significant decrease in fat mass associated with an increased proportion of smaller adipocytes and to a higher expression of genes involved in energy expenditure. NOV−/− mice fed an HFD displayed improved glucose tolerance and insulin sensitivity. Interestingly, the absence of NOV was associated with a change in macrophages profile (M1-like to M2-like), in a marked decrease in adipose tissue expression of several proinflammatory cytokines and chemokines, and in enhanced insulin signaling. Conversely, NOV treatment of adipocytes increased chemokine expression. Altogether, these results show that NOV is a new adipocytokine that could be involved in obesity-associated insulin-resistance.

Scientific Report12 October 2016free access Source DataTransparent process Central CCL2 signaling onto MCH neurons mediates metabolic and behavioral adaptation to inflammation Ophélia Le Thuc Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Céline Cansell Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Miled Bourourou Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Raphaël GP Denis Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, Paris, France Search for more papers by this author Katharina Stobbe Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Nadège Devaux Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Alice Guyon Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Julie Cazareth Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Catherine Heurteaux Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author William Rostène Institut de la Vision, UMRS 968-Université Pierre et Marie Curie, Paris, France Search for more papers by this author Serge Luquet Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, Paris, France Search for more papers by this author Nicolas Blondeau Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Jean-Louis Nahon Corresponding Author [email protected] orcid.org/0000-0001-9572-7779 Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Station de Primatologie, UPS846 CNRS, Rousset-sur-Arc, France Search for more papers by this author Carole Rovère Corresponding Author [email protected] orcid.org/0000-0001-9400-7972 Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Ophélia Le Thuc Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Céline Cansell Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Miled Bourourou Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Raphaël GP Denis Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, Paris, France Search for more papers by this author Katharina Stobbe Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Nadège Devaux Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Alice Guyon Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Julie Cazareth Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Catherine Heurteaux Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author William Rostène Institut de la Vision, UMRS 968-Université Pierre et Marie Curie, Paris, France Search for more papers by this author Serge Luquet Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, Paris, France Search for more papers by this author Nicolas Blondeau Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Jean-Louis Nahon Corresponding Author [email protected] orcid.org/0000-0001-9572-7779 Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Station de Primatologie, UPS846 CNRS, Rousset-sur-Arc, France Search for more papers by this author Carole Rovère Corresponding Author [email protected] orcid.org/0000-0001-9400-7972 Université Côte d'Azur, Nice, France CNRS, IPMC, Sophia Antipolis, France Search for more papers by this author Author Information Ophélia Le Thuc1,2, Céline Cansell1,2, Miled Bourourou1,2, Raphaël GP Denis3, Katharina Stobbe1,2, Nadège Devaux1,2, Alice Guyon1,2, Julie Cazareth1,2, Catherine Heurteaux1,2, William Rostène4, Serge Luquet3, Nicolas Blondeau1,2,‡, Jean-Louis Nahon *,1,2,5,‡ and Carole Rovère *,1,2,‡ 1Université Côte d'Azur, Nice, France 2CNRS, IPMC, Sophia Antipolis, France 3Univ Paris Diderot, Sorbonne Paris Cité, Unité de Biologie Fonctionnelle et Adaptative, CNRS UMR 8251, Paris, France 4Institut de la Vision, UMRS 968-Université Pierre et Marie Curie, Paris, France 5Station de Primatologie, UPS846 CNRS, Rousset-sur-Arc, France ‡These co-authors share last author position *Corresponding author. Tel: +33 93957741; E-mail: [email protected] *Corresponding author. Tel: +33 93957753; E-mail: [email protected] EMBO Rep (2016)17:1738-1752https://doi.org/10.15252/embr.201541499 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Sickness behavior defines the endocrine, autonomic, behavioral, and metabolic responses associated with infection. While inflammatory responses were suggested to be instrumental in the loss of appetite and body weight, the molecular underpinning remains unknown. Here, we show that systemic or central lipopolysaccharide (LPS) injection results in specific hypothalamic changes characterized by a precocious increase in the chemokine ligand 2 (CCL2) followed by an increase in pro-inflammatory cytokines and a decrease in the orexigenic neuropeptide melanin-concentrating hormone (MCH). We therefore hypothesized that CCL2 could be the central relay for the loss in body weight induced by the inflammatory signal LPS. We find that central delivery of CCL2 promotes neuroinflammation and the decrease in MCH and body weight. MCH neurons express CCL2 receptor and respond to CCL2 by decreasing both electrical activity and MCH release. Pharmacological or genetic inhibition of CCL2 signaling opposes the response to LPS at both molecular and physiologic levels. We conclude that CCL2 signaling onto MCH neurons represents a core mechanism that relays peripheral inflammation to sickness behavior. Synopsis CCL2/CCR2 signaling is a central relay for the behavioral and metabolic changes associated with sickness behavior in response to inflammation. Enhanced CCL2/CCR2 signaling inhibits the activity of MCH neurons in the hypothalamus and induces a decrease in body weight and food intake. LPS treatment enhances CCL2/CCR2 signaling, which can lead to inhibition of MCH neurons. LPS-induced weight loss is mediated through CCL2/CCR2 signaling. CCR2 signaling on MCH neurons relays inflammatory signals from the periphery to the CNS to mediate sickness behavior. Introduction Sickness behavior refers to the broad metabolic and behavioral changes that develop over the course of illness in response to inflammatory stimuli. Among these, pro-inflammatory cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), as well as chemokines, can directly alters mood, food intake, and body weight through local action onto central hypothalamic network regulation energy balance and stress response 1234. In visceral injuries, local production of cytokines can rapidly signal to the brain via a direct action onto primary afferent nerves, including the vagus and the trigeminal nerves. Following bacterial infection, activation of resident macrophages of the choroid plexus and of the circumventricular organs, devoid of blood–brain barrier (BBB), induces synthesis of pro-inflammatory cytokines that directly enter the brain. Peripheral cytokines may also cross the BBB using saturable transport systems or transitory local openings. Cytokine activation of perivascular macrophages and brain endothelial cells generates prostaglandins E2 that act on the hypothalamo–pituitary–adrenal axis to regulate stress responses 5. Finally, the central nervous system (CNS) may synthesize de novo cytokines following systemic or central inflammation 6. However, the cellular and molecular basis by which peripheral inflammation is integrated centrally to adapt food intake and body weight remains largely unknown. Lipopolysaccharide (LPS), when injected peripherally, triggers all the key features of sickness behavior, that is, peripheral and central inflammation, suppression of appetite, and weight loss 7. While the molecular underpinnings remain unknown, it has been shown that central, rather than peripheral, inflammation is mediating LPS-induced appetite and weight loss 8, and interestingly, CNS inflammation can be triggered independently from systemic cytokines, by non-hematopoietic cells of the brain expressing the LPS receptor: the Toll-like receptor 4 (TLR4) 9. Central neural substrate regulating feeding and energy expenditure is composed of several neuropeptidergic circuits primarily located in the hypothalamus and brainstem. Among these, the “first-order” neuronal populations that lie close to circumventricular organs integrate circulating signals of hunger and satiety and represent also a target for peripheral inflammatory signals. They include both orexigenic/anabolic producing neuropeptide Y (NPY)/agouti-related polypeptide (AgRP) and anorectic/catabolic neurons that produce pro-opiomelanocortin (POMC)/cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus (ARC). Once ARC neurons have integrated peripheral signals, they, in turn, project to “second-order” neurons located in various brain regions, including hypothalamic areas such as the lateral hypothalamic area (LHA), the ventromedial hypothalamic area (VMH), and the paraventricular nucleus (PVN) 1011. The hypothalamus–brainstem structure is referred as to the homeostatic circuitry and operates adaptive metabolic and behavioral responses that regulate body weight. 12. Thus, it was tempting to speculate that central inflammatory signaling may temporarily alter “first-order” and “second-order” neurons to promote LPS-provoked weight loss. While few interleukins receptors were found on hypothalamic neurons 1314, they are particularly lacking in the LHA neurons, whereas chemokine receptors are widely expressed and functional onto hypothalamic neurons 15, particularly the orexigenic melanin-concentrating hormone (MCH) 1617181920 and the hypocretins/orexins (ORX) neurons 21. We thus hypothesized that chemokines could be crucial intermediates connecting peripheral inflammation to the neuronal substrate mediating metabolic changes and anorexia associated with sickness behavior. Among chemokines, we identified the monocyte chemoattractant protein 1 MCP-1/C-C motif chemokine ligand 2 (CCL2) as a potential key player because CCL2 is expressed in glial cells and discrete neuronal populations 22, is consistently increased in the CNS following peripheral inflammation 23, and has been shown to be crucially involved in LPS-mediated brain inflammation 24. Using genetic, electrophysiological, and pharmacological approaches, we demonstrate that CCL2/CCR2 signaling onto MCH neurons is the core mechanism by which peripheral inflammatory response is centrally relayed to operate the behavioral and metabolic changes associated with sickness behavior. Results Systemic injection of LPS induces neuroinflammation and activates gene expression of neuropeptides involved in feeding behavior/energy balance In order to fully characterize the sequence of molecular events that occurs at the central level in response to peripheral inflammation, we first studied the dose–response relationship of peripheral LPS injection on sickness behavior response. Intraperitoneal (ip) injection of 5 μg of LPS per mouse recapitulated most of the characteristic response seen in sickness behavior and was therefore selected for subsequent molecular analyses (Fig EV1A). We next performed a time-course study using targeted transcription profiling by PCR analysis study of pro-inflammatory cytokine genes (IL-1β, IL-6, TNF-α) and selected neuropeptide-encoding genes involved in feeding behavior to precise the molecular events that occur at hypothalamic level after ip LPS injection. As expected 25, ip LPS injection induced a very early (1–3 after post-injection) overexpression of the pro-inflammatory cytokine mRNAs (IL-1β, IL-6, TNF-α), with specific profiles (Fig EV1B). Ip LPS challenge also induced, 1 h after injection, a transitory, yet massive (up to 20-fold), induction of all the energy-related neuropeptide mRNA-encoding genes expressed in the ARC (POMC, CART, NPY, AgRP) (Fig EV1D). Strikingly, ip LPS injection induced a delayed and sustained decrease in MCH and ORX mRNA expression 24 and 48 h after injection (Fig EV1E). Click here to expand this figure. Figure EV1. Study of weight loss and gene expression of the hypothalamic expression of cytokines, chemokines, and neuropeptides in ip LPS-injected miceSee also Figs 1 and 3. A. Dose–response relationship between ip LPS injection and mice weight loss (n = 6). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; color-coded asterisks indicate a significant difference from the experimental condition assigned to the respective color-coded curve. B–E. Real-time PCR analysis of the genes coding for the pro-inflammatory cytokines IL-1β, IL-6, TNF-α (B), the CCL2 chemokine (C), the hypothalamic peptides POMC, CART, NPY, AgRP (D), and MCH and ORX (E) in ip LPS-injected mice at different times after injection (from 1 to 48 h), normalized to values in ip saline-injected mice (n = 6 per group). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; LPS condition vs. saline condition. Data information: Data were analyzed by Student's unpaired two-tailed t-test (A–E). Download figure Download PowerPoint Central inflammation mediated by LPS activates the expression of pro-inflammatory cytokines and CCL2 chemokine family A dose of 500 ng of LPS per mouse injected centrally was selected based on its ability to recapitulate the time course of weight loss observed after peripheral injection (Fig 1A). Hypothalamic mRNA coding for cytokines and chemokines were quantified in a time window that precedes the first wave of increased cytokine gene expression and before downregulation of MCH or ORX gene expression, that is, 6 h after intracerebroventricular (icv) LPS or saline injection. LPS induced a sixfold to eightfold increase in mRNA-encoding IL-1β, IL-17A, and several other pro-inflammatory cytokines, including TNF-α (Fig 1B). Strikingly, the most robust activation of expression was found for genes encoding the CCL chemokines that bind to the CCR2 and/or CCR5 receptors (CCL2, CCL3, CCL4, CCL5, and CCL7 (Fig 1C)). As CCL2 plays a major role in brain inflammation following peripheral injection of LPS and selectively interacts with CCR2 in rodent brains 242627, we focused on the CCL2/CCR2 signaling as a potential key mechanism relaying centrally the action of peripheral inflammation. Figure 1. Analysis of inflammatory marker expression in icv LPS-injected miceSee also Fig EV1. A. Dose–response relationship between icv LPS injection and mice weight loss (n = 3). Data are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001; color-coded asterisks indicate a significant difference between the control saline condition and the experimental condition assigned to the respective color-coded curve. B, C. Analysis by real-time PCR. Arrays of interleukins, cytokines (B), and CC chemokine ligands and receptors (C) gene expression in the hypothalamus 6 h after an acute icv injection of LPS vs. saline in WT mice (n = 4–6 per group). LPS-induced fold upregulation vs. saline condition was calculated using the ΔΔCT method according to the manufacturer's protocol. Data are expressed as means ± SEM. *P < 0.05; **P < 0.01, and ***P < 0.001. Data information: Data were analyzed by Student's unpaired two-tailed t-test (A–C). Source data are available online for this figure. Source Data for Figure 1 [embr201541499-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint Central CCR2 signaling is required to operate metabolic and behavioral changes induced by LPS Brain injection of 500 ng of LPS in WT mice induced a long-lasting decrease in body weight compared to saline-injected WT mice (Fig 2A and Appendix Fig S2). Pharmacologic or genetic blockade of CCL2/CCR2 signaling was achieved through either central CCR2 antagonist injection or in the genetic context of CCR2 KO mice. CCR2 antagonist-injected mice and genetic impairment of CCR2 signaling prevented LPS-induced weight loss with a maximum effect at the early times (6–8 h) and a late partial recovery (Fig 2A–C). As shown in the Figs EV2A and B and 2A and B, icv administration of CCR2 antagonist INCB3344 or genetic invalidation of CCR2 expression in mice induced a decrease in the percentage of weight loss of similar magnitude upon peripheral injection of LPS (Fig EV2A and B and Appendix Fig S1) or following icv LPS injection (Fig 2A and B) in mice. Figure 2. LPS decreases body weight and food intake and increases fat oxidation activity through a CCR2-dependent mechanismSee also Figs EV2 and EV3, Appendix Fig S2 and Appendix Table S2. Weight variation (%) compared to initial body weight at different times (from 2 to 48 h) after acute icv injection of saline (black curve), INCB3344 (gray curve), LPS (red curve) or LPS+INCB3344 (blue curve) in WT mice (n = 6–12). Fold increase in weight loss compared to appropriate saline-injected control 26 h after acute icv injection of LPS (red bar) or LPS+INCB3344 (blue bar) in WT mice or LPS in CCR2 KO mice (striped bar) (n = 6). Fold increase in weight loss compared to appropriate saline-injected control 26 h after acute icv injection of CCL2 in WT mice (orange bar) or in CCR2 KO mice (striped bar) (n = 6). Variation of food intake recorded for 45 h during light and dark period (gray area) after acute icv injection of saline (black curve), LPS (red curve), or LPS+INCB3344 (blue curve) in WT mice (n = 5–8). Food intake average over four periods of 12 h after acute icv injection of saline (black bars), LPS (red bars), or LPS+INCB3344 (blue bars) in WT mice (n = 5–8). Variation of fat oxidation recorded for 45 h during light and dark period (gray area) after acute icv injection of saline (black curve), LPS (red curve), or LPS+INCB3344 (blue curve) in WT mice (n = 5–8). Fat oxidation average over four periods of 12 h after acute icv injection of saline (black bars), LPS (red bars), or LPS+INCB3344 (blue bars) in WT mice (n = 5–8). Data information: Data are expressed as means ± SEM. In (A–D and F), data were analyzed by Student's unpaired two-tailed t-test. *,$,#P < 0.05; **,$$,##P < 0.01 and ***,$$$,###P < 0.001. * compares saline and LPS conditions; $ compares saline and LPS+INCB3344 conditions; and # compares LPS and LPS+INCB3344 conditions. In (E and G), analyses of variances were performed followed by a Tukey's post hoc test with the appropriate parameters and their interaction as factor. Data with different superscript letters (a, b, c) differ significantly (P < 0.05). Source data are available online for this figure. Source Data for Figure 2 [embr201541499-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Effect of ip LPS on body weight and MCH expression decreases through a CCR2-dependent mechanismSee also Figs 2 and 3, Appendix Fig S1 and Appendix Table S1. Weight variation (%) compared to initial body weight at different times (from 2 to 72 h) after ip injection of saline (black curve), INCB3344 (gray curve), LPS (red curve), or LPS+INCB3344 (blue curve) in WT mice (n = 6–12). Fold increase in weight loss compared to appropriate saline-injected control 32 h after ip injection of LPS (red bar) or LPS+INCB3344 (blue bar) in WT mice or LPS in CCR2 KO mice (striped bar) (n = 6). The decrease in MCH mRNA levels observed 24 h after ip LPS injection is abolished by the CCR2 antagonist INCB3344 (10 μM) and in CCR2 KO mice, as shown by real-time PCR analysis for MCH in the hypothalamus. Results were normalized to values in saline-injected WT mice (n = 6 per group). Data information: Data are expressed as means ± SEM. $P < 0.05, **,$$P < 0.01, ***,$$$P < 0.001. * saline vs. ip LPS injection in WT mice. $ ip LPS injection in WT mice vs. ip LPS+icv INCB3344 injection in WT mice or ip LPS injection in CCR2 KO mice. Data were analyzed by Student's unpaired two-tailed t-test (A–C). Download figure Download PowerPoint Integrated analysis of energy homeostasis using indirect calorimetry coupled with activity and feeding measurement was used to fully characterize the physiologic implication of CCL2/CCR2 signaling in LPS-induced metabolic changes. Body weight loss following central LPS delivery was associated with a sharp decrease in food intake, energy expenditure, locomotor activity, and a metabolic shift toward lipid oxidation profile as indicated by both fat oxidation and respiratory quotient analysis (Figs 2 and EV3). Pharmacologically opposing CCR2 signaling through central delivery of the selective CCR2 antagonist INCB3344 28 affected various aspects of LPS-induced weight loss. First, CCR2 antagonist counteracted the anorectic response initiated by LPS (Fig 2D and E) and mitigated the acute decrease in energy expenditure (Fig EV3C and D). Peripheral substrate utilization was calculated based on respiratory exchange ratio (VCO2/VO2: RER = 1 indicative of carbohydrate oxidation and RER = 0.7 indicative of lipid oxidation). Icv LPS injection induced a sharp shift in lipid oxidation profile (Figs 2F and G, and EV3E and F) and CCR2 signaling blockade protected fat stores while opposing this shift (Figs 2F and G, and EV3E and F). Importantly, the action of both LPS and CCR2 signaling could only be partially correlated to change in food intake and locomotor activity (Figs 2D and E, and EV3A and B). Those results indicate that central inflammation not only affects feeding but also peripheral nutrient partitioning in a CCR2-dependent manner. Click here to expand this figure. Figure EV3. Injection of CCR2 antagonist reverses LPS effects on locomotor activity and respiratory quotientSee also Fig 2. A–F. Variation of locomotor activity (A), energy expenditure (C), and respiratory quotient (E) recorded for 45 h during light and dark period (gray bar) after acute icv injection of saline (black curve), LPS (red curve), or LPS+INCB3344 (blue curve) in WT mice (n = 5–8) (left). Locomotor activity (B), energy expenditure (D), and respiratory quotient (F) average for periods of 12 h after acute icv injection of saline (black bars), LPS (red bars), or LPS+INCB3344 (blue bars) in WT mice (n = 5–8) (right). Data information: Data are expressed as means ± SEM. In (A, C and E), data were analyzed by Student's unpaired two-tailed t-test. *,$,#P < 0.05. * compares saline and LPS conditions; $ compares saline and LPS+INCB3344 conditions, and # compares LPS and LPS+INCB3344 conditions. In (B, D and F), analyses of variances were performed followed by a Tukey's post hoc test with the appropriate parameters and their interaction as factor. Data with different superscript letters (a, b, c) differ significantly (P < 0.05). Download figure Download PowerPoint Brain-injected LPS reduces MCH mRNA and peptide expression through CCR2 signaling To study the molecular mechanisms involved in inflammation-induced weight loss and associated changes in energy balance at the brain level, and particularly at the hypothalamic level, we determined levels of mRNA-encoding cytokines (IL-1β, IL-6, TNF-α), 1, 3, 6, 18, and 24 h after icv injection of LPS, to cover primary responses, and 18 and 24 h after injection, to analyze secondary and counteractive responses. As shown in Fig 3A, IL-1β mRNA expression levels displayed two distinct waves that peaked at 3 and 18 h (23-fold) post-injection while IL-6 mRNA (16-fold) and TNF-α mRNA (sevenfold) levels were transitorily elevated at 3 h in the LPS-treated group as compared to the saline controls. We also determined the temporal patterns of CCL2 expression induced by icv injection of LPS and confirmed the strong upregulation of both gene and protein, 3 and 6 h after icv LPS (Fig 3B and C) and ip (Fig EV1C) injection, respectively. Figure 3. Brain-injected LPS induced differential variations in hypothalamic expression of cytokines and of the orexigenic neuropeptide MCHSee also Figs EV4 and EV5. A. Real-time PCR analysis of the genes coding for the pro-inflammatory cytokines IL-1β, IL-6, TNF-α in the hypothalamus of icv LPS-injected mice at different times after injection (from 1 h to 18 h), normalized to values in icv saline-injected mice (n = 6 per group). B–E. Study of gene and protein hypothalamic expression of the chemokine CCL2 (B, C) and MCH peptide (D, E) in icv LPS-injected mice. Real-time PCR analysis for CCL2 (B) and MCH (D) at different times after injection (from 1 to 24 h), normalized to values in control icv saline-injected mice (n = 6 per group). Measurement of CCL2 (C) and MCH (E) concentrations by EIA after icv LPS injection (black bars) or icv saline injection (gray bars) in mice at 1 h, 6 h and 18 h after injection. Cerebellum was used as negative control (3 independent experiments, n = 6 per group in each experiment). F. The decrease in MCH mRNA levels observed 18 h after icv LPS injection is partly abolished by the CCR2 antagonist INCB3344 (10 μM) and in CCR2 KO mice, as shown by real-time PCR analysis for MCH in the hypothalamus. Results were normalized to values in icv saline-injected WT mice (n = 6 per group). Data are expressed as means ± SEM. **,$$P < 0.01. * saline vs. icv LPS injection in WT mice. $ icv LPS injection in WT mice vs. LPS+INCB3344 icv injection in WT mice or icv LPS injection in CCR2 KO mice. Data information: Data in (A–E) are expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, icv LPS injection vs. icv saline injection. Data were analyzed by Student's unpaired two-tailed t-test (A–F). Download figure Download PowerPoint For the neuropeptide-encoding mRNAs, there are differentiable responses following LPS icv injection. Robust (up to 12-fold) and transitory peak of induction was found for POMC (but not CART; Fig EV4A and B), whereas NPY (and also AgRP) mRNA expression was downregulated (twofold) at 1 h post-injection (Fig EV4C and D). In contrast, a late and sustained downregulation occurred for MCH mRNA (Fig 3D) and ORX (Fig EV5A) 18 and 24 h after LPS injection. We therefore characterized the protein levels of MCH and ORX and found that they followed the mRNA patterns observed at 1, 6, 18 h LPS post-injection. MCH (53.5 ± 5.2 ng/mg of proteins) (Fig 3E) and ORX (83.1 ± 2.1 ng/mg of proteins; Fig EV5B) dropped down compared to saline group at 18 h post-LPS. No change was observed for both neuropeptides in the cerebellum extracts used as negative controls (Figs 3E and EV5B). Click here to expand this figure. Figure EV4. Study of gene expression of the ARC peptides in icv LPS-injected miceSee also Figs 1 and 3. A–D. Real-time PCR analysis of the genes coding for the hypothalamic peptides POMC (A), CART (B), NPY (C), and AgRP (D) in icv LPS-injected mice at different times after injection (from 1 to 24 h), normalized to values in icv saline-injected mice (n = 5 per group). *P < 0.05, **P < 0.01, ***P < 0.001; LPS condition vs. saline condition. Data are expressed as means ± SEM. Data information: Data were analyzed by Student's unpaired two-tailed t-test (A–D). Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Expression and effect of CCL2 on ORX neuronsSee also Figs 3, 4, 5. A–D. Study of gene and protein expression of the hypothalamic peptide ORX in icv LPS- (A, B) or CCL2-injected (C, D) mice. Real-time PCR analysis for ORX in icv LPS- (A) or CCL2-injected (C) mice at different times after injection (from 1 to 24 h), normalized to values in control icv saline-injected mice (n = 6 per group); measurement of ORX concentration by EIA after icv LPS (B) or CCL2 (D) injection (black bars) or icv saline injection (gray bars) in mice at 1, 6, and 18 h after injection (three independent experiments, n = 6 per group in each experiment). Cerebellum was used as negative control. *P < 0.05, **P < 0.01; CCL2 condition vs. saline condition. Data are expressed as means ± SEM. E. Immunohistochemistry experiments in the LHA of mice expressing CFP under the promoter of ORX. CCR2 is not expressed by ORX neurons. F. Perifusion of hypothalamic tissues. Application of CCL2 had no effect on KCl-induced ORX release (3–5 independent experiments, three chambers per

DA integrates metabolic signals with circuits regulating behavior. The Ankk1 and Fto gene variants interact to influence DA-dependent functions. Recent work highlights the importance of genetic variants that influence brain structure and function in conferring risk for polygenic obesity. The neurotransmitter dopamine (DA) has a pivotal role in energy balance by integrating metabolic signals with circuits supporting cognitive, perceptual, and appetitive functions that guide feeding. It has also been established that diet and obesity alter DA signaling, leading to compulsive-like feeding and neurocognitive impairments. This raises the possibility that genetic variants that influence DA signaling and adaptation confer risk for overeating and cognitive decline. Here, we consider the role of two common gene variants, FTO and TaqIA rs1800497 in driving gene × environment interactions promoting obesity, metabolic dysfunction, and cognitive change via their influence on DA receptor subtype 2 (DRD2) signaling. Recent work highlights the importance of genetic variants that influence brain structure and function in conferring risk for polygenic obesity. The neurotransmitter dopamine (DA) has a pivotal role in energy balance by integrating metabolic signals with circuits supporting cognitive, perceptual, and appetitive functions that guide feeding. It has also been established that diet and obesity alter DA signaling, leading to compulsive-like feeding and neurocognitive impairments. This raises the possibility that genetic variants that influence DA signaling and adaptation confer risk for overeating and cognitive decline. Here, we consider the role of two common gene variants, FTO and TaqIA rs1800497 in driving gene × environment interactions promoting obesity, metabolic dysfunction, and cognitive change via their influence on DA receptor subtype 2 (DRD2) signaling. a molecule that binds to, and activates, a receptor. alternative forms of the same gene. a type of cell in the central nervous system that was traditionally believed to perform support and maintenance for neurons, but is now believed to have a more active role in cell signaling and computation. the cognitive phenomenon where the relative value of a reward decreases as the delay in receiving it increases. a type of signaling molecule that regulates the function of other cells. heritable behavioral and/or neurocognitive traits that are associated with, or convey risk for, a syndrome. a form of interaction between genes, where the presence of a certain allele for one gene modifies or masks the expression of a different gene. a computer-based data mining-based method for determining proteome-wide protein-protein interactions (PPIs). This method is based on the ability of a program to predict, based on the literature and known sequence of the protein, the possible physical interaction between proteins. This method is typically used to narrow down potential partners involved in a signaling cascade. a series of cellular events that are triggered by an immune reaction and that result in pathological inflammation. the tendency of certain alleles of different genes to be inherited together at a rate greater than chance. a class of hydrophobic organic molecules that encompasses fats and oils. a protein complex that acts as a master transcriptional regulator in the cellular response to various stimuli, including nutrients. It is fundamental for the control of immune responses and regulates cell survival/death, cytokine production, and response to free radical damage. It also mediates the inflammatory response to nutrient overload in the brain [122,142–144] and directly controls DRD2 expression [145]. type of inheritance where the contributions of multiple genes determine a single characteristic. a variation in a genetic location of interest that can be revealed by the digestion of the gene with restriction enzymes. Different gene variations are reflected by different lengths of the DNA fragments after digestion. a variation in a single nucleotide at a specific position in the genome. a family of membrane receptors that recognize structures such as microbes and mediate immune responses. In the central nervous system, TLR are expressed on neurons and glia and mediate diet-induced inflammation through various mechanism, including direct binding of fatty acids [148–151].

Unbalanced energy partitioning participates in the rise of obesity, a major public health concern in many countries. Increasing basal energy expenditure has been proposed as a strategy to fight obesity yet raises efficiency and safety concerns. Here, we show that mice deficient for a muscle-specific enzyme of very-long-chain fatty acid synthesis display increased basal energy expenditure and protection against high-fat diet-induced obesity. Mechanistically, muscle-specific modulation of the very-long-chain fatty acid pathway was associated with a reduced content of the inner mitochondrial membrane phospholipid cardiolipin and a blunted coupling efficiency between the respiratory chain and adenosine 5'-triphosphate (ATP) synthase, which was restored by cardiolipin enrichment. Our study reveals that selective increase of lipid oxidative capacities in skeletal muscle, through the cardiolipin-dependent lowering of mitochondrial ATP production, provides an effective option against obesity at the whole-body level.

A large body of evidence highlights the importance of genetic variants in the development of psychiatric and metabolic conditions. Among these, the TaqIA polymorphism is one of the most commonly studied in psychiatry. TaqIA is located in the gene that codes for the ankyrin repeat and kinase domain containing 1 kinase (Ankk1) near the dopamine D2 receptor (D2R) gene. Homozygous expression of the A1 allele correlates with a 30% to 40% reduction of striatal D2R, a typical feature of addiction, overeating, and other psychiatric pathologies. The mechanisms by which the variant influences dopamine signaling and behavior are unknown.Here, we used transgenic and viral-mediated strategies to reveal the role of Ankk1 in the regulation of activity and functions of the striatum.We found that Ankk1 is preferentially enriched in striatal D2R-expressing neurons and that Ankk1 loss of function in the dorsal and ventral striatum leads to alteration in learning, impulsivity, and flexibility resembling endophenotypes described in A1 carriers. We also observed an unsuspected role of Ankk1 in striatal D2R-expressing neurons of the ventral striatum in the regulation of energy homeostasis and documented differential nutrient partitioning in humans with or without the A1 allele.Overall, our data demonstrate that the Ankk1 gene is necessary for the integrity of striatal functions and reveal a new role for Ankk1 in the regulation of body metabolism.

Despite numerous experiments showing that administration of neuropeptide Y (NPY) to rodents stimulates feeding and obesity, whereas acute interference with NPY signaling disrupts feeding and promotes weight loss, NPY-null mice have essentially normal body weight regulation. These conflicting observations suggest that chronic lack of NPY during development may lead to compensatory changes that normalize regulation of food intake and energy expenditure in the absence of NPY. To test this idea, we used gene targeting to introduce a doxycycline (Dox)-regulated cassette into the Npy locus, such that NPY would be expressed until the mice were given Dox, which blocks transcription. Compared with wild-type mice, adult mice bearing this construct expressed ≈4-fold more Npy mRNA, which fell to ≈20% of control values within 3 days after treatment with Dox. NPY protein also fell ≈20-fold, but the half-life of ≈5 days was surprisingly long. The biological effectiveness of these manipulations was demonstrated by showing that overexpression of NPY protected against kainate-induced seizures. Mice chronically overexpressing NPY had normal body weight, and administration of Dox to these mice did not suppress feeding. Furthermore, the refeeding response of these mice after a fast was normal. We conclude that, if there is compensation for changes in NPY levels, then it occurs within the time it takes for Dox treatment to deplete NPY levels. These observations suggest that pharmacological inhibition of NPY signaling is unlikely to have long-lasting effects on body weight.

Western diet is characterized by a hypercaloric and hyperlipidic intake, enriched in saturated fats, that is associated with the increased occurrence of metabolic diseases. To cope with this overload of dietary lipids, the intestine, which delivers dietary lipids to the body, has to adapt its capacity in lipid absorption and lipoprotein synthesis. We have studied the early effects of a high-fat diet (HFD) on intestinal lipid metabolism in mice. After 7 days of HFD, mice displayed normal fasting triglyceridemia but postprandial hypertriglyceridemia. HFD induced a decreased number of secreted chylomicrons with increased associated triglycerides. Secretion of larger chylomicrons was correlated with increased intestinal microsomal triglyceride transfer protein (MTP) content and activity. Seven days of HFD induced a repression of genes involved in fatty acid synthesis (FAS, ACC) and an increased expression of genes involved in lipoprotein assembly (apoB, MTP, and apoA-IV), suggesting a coordinated control of intestinal lipid metabolism to manage a high-fat loading. Of note, the mature form of the transcription factor SREBP-1c was increased and translocated to the nucleus, suggesting that it could be involved in the coordinated control of gene transcription. Activation of SREBP-1c was partly independent of LXR. Moreover, HFD induced hepatic insulin resistance whereas intestine remained insulin sensitive. Altogether, these results demonstrate that a short-term HFD is sufficient to impact intestinal lipid metabolism, which might participate in the development of dyslipidemia and metabolic diseases.

Lipids are essential components of a living organism as energy source but also as constituent of the membrane lipid bilayer.In addition fatty acid (FA) derivatives interact with many signaling pathways.FAs have amphipathic properties and therefore require being associated to protein for both transport and intracellular trafficking.Here we will focus on several FA handling proteins, among which the fatty acid translocase/CD36 (FAT/CD36), members of fatty acid transport proteins (FATPs), and lipid chaperones fatty acid-binding proteins (FABPs).A decade of extensive studies has helped decipher the mechanism of action of these proteins in peripheral tissue with high lipid metabolism.However, considerably less information is available regarding their role in the brain, despite the high lipid content of this tissue.This review will primarily focus on the recent studies that have highlighted the crucial role of lipid handling proteins in brain FA transport, neuronal differentiation and development, cognitive processes and brain diseases.Finally a special focus will be made on the recent studies that have revealed the role of FAT/CD36 in brain lipid sensing and nervous control of energy balance.

Central melanocortin pathways are well-established regulators of energy balance. However, scant data exist about the role of systemic melanocortin peptides. We set out to determine if peripheral α-melanocyte stimulating hormone (α-MSH) plays a role in glucose homeostasis and tested the hypothesis that the pituitary is able to sense a physiological increase in circulating glucose and responds by secreting α-MSH.We established glucose-stimulated α-MSH secretion using humans, non-human primates, and mouse models. Continuous α-MSH infusions were performed during glucose tolerance tests and hyperinsulinemic-euglycemic clamps to evaluate the systemic effect of α-MSH in glucose regulation. Complementary ex vivo and in vitro techniques were employed to delineate the direct action of α-MSH via the melanocortin 5 receptor (MC5R)-PKA axis in skeletal muscles. Combined treatment of non-selective/selective phosphodiesterase inhibitor and α-MSH was adopted to restore glucose tolerance in obese mice.Here we demonstrate that pituitary secretion of α-MSH is increased by glucose. Peripheral α-MSH increases temperature in skeletal muscles, acts directly on soleus and gastrocnemius muscles to significantly increase glucose uptake, and enhances whole-body glucose clearance via the activation of muscle MC5R and protein kinase A. These actions are absent in obese mice, accompanied by a blunting of α-MSH-induced cAMP levels in skeletal muscles of obese mice. Both selective and non-selective phosphodiesterase inhibition restores α-MSH induced skeletal muscle glucose uptake and improves glucose disposal in obese mice.These data describe a novel endocrine circuit that modulates glucose homeostasis by pituitary α-MSH, which increases muscle glucose uptake and thermogenesis through the activation of a MC5R-PKA-pathway, which is disrupted in obesity.

Widely used for their anti-inflammatory and immunosuppressive properties, glucocorticoids are nonetheless responsible for the development of diabetes and lipodystrophy. Despite an increasing number of studies focused on the adipocyte glucocorticoid receptor (GR), its precise role in the molecular mechanisms of these complications has not been elucidated. In keeping with this goal, we generated a conditional adipocyte-specific murine model of GR invalidation (AdipoGR knockout [KO] mice). Interestingly, when administered a corticosterone treatment to mimic hypercorticism conditions, AdipoGR-KO mice exhibited an improved glucose tolerance and insulin sensitivity. This was related to the adipose-specific activation of the insulin-signaling pathway, which contributed to fat mass expansion, as well as a shift toward an anti-inflammatory macrophage polarization in adipose tissue of AdipoGR-KO animals. Moreover, these mice were protected against ectopic lipid accumulation in the liver and displayed an improved lipid profile, contributing to their overall healthier phenotype. Altogether, our results indicate that adipocyte GR is a key factor of adipose tissue expansion and glucose and lipid metabolism control, which should be taken into account in the further design of adipocyte GR-selective modulators.

Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.

Background and Aims Genetically modified mice have been used extensively to study human disease. However, the data gained are not always translatable to humans because of major species differences. Liver‐humanized mice (LHM) are considered a promising model to study human hepatic and systemic metabolism. Therefore, we aimed to further explore their lipoprotein metabolism and to characterize key hepatic species‐related, physiological differences. Approach and Results Fah−/− , Rag2−/− , and Il2rg−/− knockout mice on the nonobese diabetic (FRGN) background were repopulated with primary human hepatocytes from different donors. Cholesterol lipoprotein profiles of LHM showed a human‐like pattern, characterized by a high ratio of low‐density lipoprotein to high‐density lipoprotein, and dependency on the human donor. This pattern was determined by a higher level of apolipoprotein B100 in circulation, as a result of lower hepatic mRNA editing and low‐density lipoprotein receptor expression, and higher levels of circulating proprotein convertase subtilisin/kexin type 9. As a consequence, LHM lipoproteins bind to human aortic proteoglycans in a pattern similar to human lipoproteins. Unexpectedly, cholesteryl ester transfer protein was not required to determine the human‐like cholesterol lipoprotein profile. Moreover, LHM treated with GW3965 mimicked the negative lipid outcomes of the first human trial of liver X receptor stimulation (i.e., a dramatic increase of cholesterol and triglycerides in circulation). Innovatively, LHM allowed the characterization of these effects at a molecular level. Conclusions LHM represent an interesting translatable model of human hepatic and lipoprotein metabolism. Because several metabolic parameters displayed donor dependency, LHM may also be used in studies for personalized medicine.

Forebrain dopamine-sensitive (dopaminoceptive) neurons play a key role in movement, action selection, motivation, and working memory. Their activity is altered in Parkinson's disease, addiction, schizophrenia, and other conditions, and drugs that stimulate or antagonize dopamine receptors have major therapeutic applications. Yet, similarities and differences between the various neuronal populations sensitive to dopamine have not been systematically explored. To characterize them, we compared translating mRNAs in the dorsal striatum and nucleus accumbens neurons expressing D1 or D2 dopamine receptor and prefrontal cortex neurons expressing D1 receptor. We identified genome-wide cortico-striatal, striatal D1/D2 and dorso/ventral differences in the translating mRNA and isoform landscapes, which characterize dopaminoceptive neuronal populations. Expression patterns and network analyses identified novel transcription factors with presumptive roles in these differences. Prostaglandin E2 (PGE2) was a candidate upstream regulator in the dorsal striatum. We pharmacologically explored this hypothesis and showed that misoprostol, a PGE2 receptor agonist, decreased the excitability of D2 striatal projection neurons in slices, and diminished their activity in vivo during novel environment exploration. We found that misoprostol also modulates mouse behavior including by facilitating reversal learning. Our study provides powerful resources for characterizing dopamine target neurons, new information about striatal gene expression patterns and regulation. It also reveals the unforeseen role of PGE2 in the striatum as a potential neuromodulator and an attractive therapeutic target.

The mass of pancreatic beta-cells varies according to increases in insulin demand. It is hypothesized that functionally heterogeneous beta-cell subpopulations take part in this process. Here we characterized two functionally distinct groups of beta-cells and investigated their physiological relevance in increased insulin demand conditions in rats.Two rat beta-cell populations were sorted by FACS according to their PSA-NCAM surface expression, i.e. beta(high) and beta(low)-cells. Insulin release, Ca(2+) movements, ATP and cAMP contents in response to various secretagogues were analyzed. Gene expression profiles and exocytosis machinery were also investigated. In a second part, beta(high) and beta(low)-cell distribution and functionality were investigated in animal models with decreased or increased beta-cell function: the Zucker Diabetic Fatty rat and the 48 h glucose-infused rat.We show that beta-cells are heterogeneous for PSA-NCAM in rat pancreas. Unlike beta(low)-cells, beta(high)-cells express functional beta-cell markers and are highly responsive to various insulin secretagogues. Whereas beta(low)-cells represent the main population in diabetic pancreas, an increase in beta(high)-cells is associated with gain of function that follows sustained glucose overload.Our data show that a functional heterogeneity of beta-cells, assessed by PSA-NCAM surface expression, exists in vivo. These findings pinpoint new target populations involved in endocrine pancreas plasticity and in beta-cell defects in type 2 diabetes.

How circulating signals of hunger and satiety enter the brain to reach neurons that govern energy balance has long remained a matter of controversy and speculation. Balland et al. (2014) now elucidate molecular mechanisms by which a highly specialized hypothalamic glial cell regulates transport of leptin across the blood-brain barrier.

AgRP neurons control peripheral substrate utilization and nutrient partitioning during conditions of energy deficit and nutrient replenishment, although the molecular mechanism is unknown. We examined whether carnitine acetyltransferase (Crat) in AgRP neurons affects peripheral nutrient partitioning. Crat deletion in AgRP neurons reduced food intake and feeding behavior and increased glycerol supply to the liver during fasting, as a gluconeogenic substrate, which was mediated by changes to sympathetic output and peripheral fatty acid metabolism in the liver. Crat deletion in AgRP neurons increased peripheral fatty acid substrate utilization and attenuated the switch to glucose utilization after refeeding, indicating altered nutrient partitioning. Proteomic analysis in AgRP neurons shows that Crat regulates protein acetylation and metabolic processing. Collectively, our studies highlight that AgRP neurons require Crat to provide the metabolic flexibility to optimize nutrient partitioning and regulate peripheral substrate utilization, particularly during fasting and refeeding.

Dlx5 and Dlx6 encode two homeobox transcription factors expressed by developing and mature GABAergic interneurons. During development, Dlx5/6 play a role in the differentiation of certain GABAergic subclasses. Here we address the question of the functional role of Dlx5/6 in the mature central nervous system. First, we demonstrate that Dlx5 and Dlx6 are expressed by all subclasses of adult cortical GABAergic neurons. Then we analyze VgatΔDlx5-6 mice in which Dlx5 and Dlx6 are simultaneously inactivated in all GABAergic interneurons. VgatΔDlx5-6 mice present a behavioral pattern suggesting reduction of anxiety-like behavior and obsessive-compulsive activities, and a lower interest in nest building. Twenty-month-old VgatΔDlx5-6 animals have the same size as their normal littermates, but present a 25% body weight reduction associated with a marked decline in white and brown adipose tissue. Remarkably, both VgatΔDlx5-6/+ and VgatΔDlx5-6 mice present a 33% longer median survival. Hallmarks of biological aging such as motility, adiposity and coat conditions are improved in mutant animals. Our data imply that GABAergic interneurons can regulate healthspan and lifespan through Dlx5/6-dependent mechanisms. Understanding these regulations can be an entry point to unravel the processes through which the brain affects body homeostasis and, ultimately, longevity and healthy aging.

Abstract Excessive glucose production by the liver is a key factor in the hyperglycemia observed in type 2 diabetes mellitus (T2DM). Here, we highlight a novel role of liver kinase B1 (Lkb1) in this regulation. We show that mice with a hepatocyte-specific deletion of Lkb1 have higher levels of hepatic amino acid catabolism, driving gluconeogenesis. This effect is observed during both fasting and the postprandial period, identifying Lkb1 as a critical suppressor of postprandial hepatic gluconeogenesis. Hepatic Lkb1 deletion is associated with major changes in whole-body metabolism, leading to a lower lean body mass and, in the longer term, sarcopenia and cachexia, as a consequence of the diversion of amino acids to liver metabolism at the expense of muscle. Using genetic, proteomic and pharmacological approaches, we identify the aminotransferases and specifically Agxt as effectors of the suppressor function of Lkb1 in amino acid-driven gluconeogenesis.

The abundance of energy-dense and palatable diets in the modern food environment tightly contributes to the obesity pandemic. The reward circuit participates to the regulation of body homeostasis by integrating energy-related signals with neural substrates encoding cognitive and motivational components of feeding behaviors. Obesity and lipid-rich diets alter dopamine (DA) transmission leading to reward dysfunctions and food overconsumption. Recent reports indicate that dietary lipids can act, directly and indirectly, as functional modulators of the DA circuit. This raises the possibility that nutritional or genetic conditions affecting 'lipid sensing' mechanisms might lead to maladaptations of the DA system. Here, we discuss the most recent findings connecting dietary lipid sensing with DA signaling and its multimodal influence on circuits regulating food-reward processes.

Therapies based on glucagon-like peptide-1 (GLP-1) long-acting analogs and insulin are often used in the treatment of metabolic diseases. Both insulin and GLP-1 receptors are expressed in metabolically relevant brain regions, suggesting a cooperative action. However, the mechanisms underlying the synergistic actions of insulin and GLP-1R agonists remain elusive. In this study, we show that insulin-induced hypoglycemia enhances GLP-1R agonists entry in hypothalamic and area, leading to enhanced whole-body fat oxidation. Mechanistically, this phenomenon relies on the release of tanycyctic vascular endothelial growth factor A, which is selectively impaired after calorie-rich diet exposure. In humans, low blood glucose also correlates with enhanced blood-to-brain passage of insulin, suggesting that blood glucose gates the passage other energy-related signals in the brain. This study implies that the preventing hyperglycemia is important to harnessing the full benefit of GLP-1R agonist entry in the brain and action onto lipid mobilization and body weight loss.

The CD36 scavenger receptor is involved in the uptake and transport of fatty acids, as well as the phagocytosis process in macrophages. We show here that the CD36 protein is expressed by Sertoli cells in the seminiferous epithelium, mainly during the stages where phagocytosis takes place. Using a Sertoli-derived cell line, we show that addition of germ cells and residual bodies triggers a re-localization of CD36 from the cytoplasm to the plasma membrane of the cells, while latex beads do not. Moreover, Sertoli cell phagocytosis of germ cells, but not of latex beads, is reduced by the presence of fatty acids in the culture medium. In the testis, CD36 plays a key role in both phagocytosis and lipid recycling, for constant production of mature spermatozoa.

Energy homoeostasis is maintained through a complex interplay of nutrient intake and energy expenditure. The central nervous system is an essential component of this regulation, as it integrates circulating signals of hunger and satiety to develop adaptive responses at the behavioural and metabolic levels, while the hypothalamus is regarded as a particularly crucial structure in the brain in terms of energy homoeostasis. The arcuate nucleus (ARC) of the hypothalamus contains at least two intermingled neuronal populations: the neurons that produce neuropeptide Y (NPY); and the Agouti-related protein (AgRP) produced by AgRP/NPY neurons situated below the third ventricle in close proximity to proopiomelanocortin (POMC)-producing neurons. POMC neurons exert their catabolic and anorectic actions by releasing α-melanocyte-stimulating hormone (α-MSH), while AgRP neurons oppose this action by exerting tonic GABAergic inhibition of POMC neurons and releasing the melanocortin receptor inverse agonist AgRP. The release of neurotransmitters and neuropeptides by second-order AgRP neurons appears to take place on a multiple time scale, thereby allowing neuromodulation of preganglionic neuronal activity and subsequent control of nutrient partitioning - in other words, the coordinated regulation of conversion, storage and utilization of carbohydrates vs. lipids. This suggests that the function of AgRP neurons extends beyond the strict regulation of feeding to the regulation of efferent organ activity, such that AgRP neurons may now be viewed as an important bridge between central detection of nutrient availability and peripheral nutrient partitioning, thus providing a mechanistic link between obesity and obesity-related disorders.

The importance of B-isoform of leptin receptor (LEPR-B) signaling in the hypothalamus, pancreas, or liver has been well characterized, but in the intestine, a unique site of entry for dietary nutrition into the body, it has been relatively ignored. To address this question, we characterized a mouse model deficient for LEPR-B specifically in intestinal epithelial cells (IECs). IECLEPR-B-knockout (KO) and wild-type (WT) mice were generated by Cre-Lox strategy and fed a normal or high-fat diet (HFD). The analyses of the animals involved histology and immunohistochemistry of intestinal mucosa, indirect calorimetric measurements, whole-body composition, and expression and activities of nutrient transporters. IECLEPR-B-KO mice exhibited a 2-fold increase in length of jejunal villi and have normal growth on a normal diet but were less susceptible (P<0.01) to HFD-induced obesity. No differences occurred in energy intake and expenditure between IECLEPR-B-WT and -KO mice, but IECLEPR-B-KO mice fed an HFD showed increased excreted fats (P<0.05). Activities of the Na+/glucose cotransporter SGLT-1 and GLUT2 were unaffected in LEPR-B-KO jejunum, while GLUT5-mediated fructose transport and PepT1-mediated peptide transport were substantially reduced (P<0.01). These data demonstrate that intestinal LEPR-B signaling is important for the onset of diet-induced obesity. They suggest that intestinal LEPR-B could be a potential per os target for prevention against obesity.—Tavernier, A., Cavin, J.-B., Le Gall, M., Ducroc, R., Denis, R. G. P., Cluzeaud, F., Guilmeau, S., Sakar, Y., Barbot, L., Kapel, N., Le Beyec, J., Joly, F., Chua, S., Luquet, S., Bado, A. Intestinal deletion of leptin signaling alters activity of nutrient transporters and delayed the onset of obesity in mice. FASEB J. 28, 4100-4110 (2014). www.fasebj.org

The meso-cortico-limbic system, via dopamine release, encodes the rewarding and reinforcing properties of natural rewards. It is also activated in response to abused substances and is believed to support drug-related behaviors. Dysfunctions of this system lead to several psychiatric conditions including feeding disorders and drug addiction. These disorders are also largely influenced by environmental factors and in particular stress exposure. Stressors activate the corticotrope axis ultimately leading to glucocorticoid hormone (GCs) release. GCs bind the glucocorticoid receptor (GR) a transcription factor ubiquitously expressed including within the meso-cortico-limbic tract. While GR within dopamine-innervated areas drives cocaine's behavioral responses, its implication in responses to other psychostimulants such as amphetamine has never been clearly established. Moreover, while extensive work has been made to uncover the role of this receptor in addicted behaviors, its contribution to the rewarding and reinforcing properties of food has yet to be investigated. Using mouse models carrying GR gene inactivation in either dopamine neurons or in dopamine-innervated areas, we found that GR in dopamine responsive neurons is essential to properly build amphetamine-induced conditioned place preference and locomotor sensitization. c-Fos quantification in the nucleus accumbens further confirmed defective neuronal activation following amphetamine injection. These diminished neuronal and behavioral responses to amphetamine may involve alterations in glutamate transmission as suggested by the decreased MK801-elicited hyperlocomotion and by the hyporeactivity to glutamate of a subpopulation of medium spiny neurons. In contrast, GR inactivation did not affect rewarding and reinforcing properties of food suggesting that responding for natural reward under basal conditions is preserved in these mice.

Energy homeostasis is tightly regulated by the central nervous system which responds to nervous and circulating inputs to adapt food intake and energy expenditure. However, the rewarding and motivational aspect of food is tightly dependent of dopamine (DA) release in mesocorticolimbic (MCL) system and could be operant in uncontrolled caloric intake and obesity. Accumulating evidence indicate that manipulating the microbiota-gut-brain axis through prebiotic supplementation can have beneficial impact of the host appetite and body weight. However, the consequences of manipulating the implication of the microbiota-gut-brain axis in the control motivational and hedonic/reinforcing aspects of food are still underexplored. In this study, we investigate whether and how dietary prebiotic fructo-oligosaccharides (FOS) could oppose, or revert, the change in hedonic and homeostatic control of feeding occurring after a 2-months exposure to high-fat high-sugar (HFHS) diet. The reinforcing and motivational components of food reward were assessed using a two-food choice paradigm and a food operant behavioral test in mice exposed to FOS either during or after HFHS exposure. We also performed mRNA expression analysis for key genes involved in limbic and hypothalamic control of feeding. We show in a preventive-like approach, FOS addition of HFHS diet had beneficial impact of hypothalamic neuropeptides, and decreased the operant performance for food but only after an overnight fast while it did not prevent the imbalance in mesolimbic markers for DA signaling induced by palatable diet exposure nor the spontaneous tropism for palatable food when given the choice. However, when FOS was added to control diet after chronic HFHS exposure, although it did not significantly alter body weight loss, it greatly decreased palatable food tropism and consumption and was associated with normalization of MCL markers for DA signaling. We conclude that the nature of the diet (regular chow or HFHS) as well as the timing at which prebiotic supplementation is introduced (preventive or curative) greatly influence the efficacy of the gut-microbiota-brain axis. This crosstalk selectively alters the hedonic or motivational drive to eat and triggers molecular changes in neural substrates involved in the homeostatic and non-homeostatic control of body weight.

Diverse metabolic disorders have been associated with an alteration of N-acylethanolamine (NAE) levels. These bioactive lipids are synthesized mainly by N-acylphosphatidylethanolamine-selective phospholipase D (NAPE-PLD) and influence host metabolism. We have previously discovered that NAPE-PLD in the intestine and adipose tissue is connected to the pathophysiology of obesity. However, the physiological function of NAPE-PLD in the liver remains to be deciphered. To study the role of liver NAPE-PLD on metabolism, we generated a new mouse model of inducible Napepld hepatocyte-specific deletion (Napepld∆Hep mice). In this study, we report that Napepld∆Hep mice develop a high-fat diet-like phenotype, characterized by an increased fat mass gain, hepatic steatosis and we show that Napepld∆Hep mice are more sensitive to liver inflammation. We also demonstrate that the role of liver NAPE-PLD goes beyond the mere synthesis of NAEs, since the deletion of NAPE-PLD is associated with a marked modification of various bioactive lipids involved in host homeostasis such as oxysterols and bile acids. Collectively these data suggest that NAPE-PLD in hepatocytes is a key regulator of liver bioactive lipid synthesis and a dysregulation of this enzyme leads to metabolic complications. Therefore, deepening our understanding of the regulation of NAPE-PLD could be crucial to tackle obesity and related comorbidities.

The hypothalamic melanocortin system--the melanocortin receptor of type 4 (MC4R) and its ligands: α-melanin-stimulating hormone (α-MSH, agonist, inducing hypophagia), and agouti-related protein (AgRP, antagonist, inducing hyperphagia)--is considered to play a central role in the control of food intake. We tested its implication in the mediation of the hunger-curbing effects of protein-enriched diets (PED) in mice. Whereas there was a 20% decrease in food intake in mice fed on the PED, compared to mice fed on an isocaloric starch-enriched diet, there was a paradoxical decrease in expression of the hypothalamic proopiomelanocortin gene, precursor of α-MSH, and increase in expression of the gene encoding AgRP. The hypophagia effect of PED took place in mice with invalidation of either MC4R or POMC, and was even strengthened in mice with ablation of the AgRP-expressing neurons. These data strongly suggest that the hypothalamic melanocortin system does not mediate the hunger-curbing effects induced by changes in the macronutrient composition of food. Rather, the role of this system might be to defend the body against the variations in food intake generated by the nutritional environment.

Fatty acid (FA)-sensitive neurons are present in the brain, especially the hypothalamus, and play a key role in the neural control of energy homeostasis. Through neuronal output, FA may modulate feeding behaviour as well as insulin secretion and action. Subpopulations of neurons in the ventromedial and arcuate hypothalamic nuclei are selectively either inhibited or activated by FA. Molecular effectors of these FA effects probably include chloride or potassium ion channels. While intracellular metabolism and activation of the ATP-sensitive K⁺ channel appear to be necessary for some of the signalling effects of FA, at least half of the FA responses in ventromedial hypothalamic neurons are mediated by interaction with FAT/CD36, an FA transporter/receptor that does not require intracellular metabolism to activate downstream signalling. Thus, FA or their metabolites can modulate neuronal activity as a means of directly monitoring ongoing fuel availability by brain nutrient-sensing neurons involved in the regulation of energy and glucose homeostasis. Recently, the role of lipoprotein lipase in FA sensing has also been shown in animal models not only in hypothalamus, but also in hippocampus and striatum. Finally, FA overload might impair neural control of energy homeostasis through enhanced ceramide synthesis and may contribute to obesity and/or type 2 diabetes pathogenesis in predisposed subjects.

Regulation of energy balance involves the participation of many factors, including nutrients, among which are circulating lipids, acting as peripheral signals informing the central nervous system of the energy status of the organism. It has been shown that neuronal lipoprotein lipase (LPL) participates in the control of energy balance by hydrolysing lipid particles enriched in triacylglycerols. Here, we tested the hypothesis that LPL in the mediobasal hypothalamus (MBH), a well-known nucleus implicated in the regulation of metabolic homeostasis, could also contribute to the regulation of body weight and glucose homeostasis.We injected an adeno-associated virus (AAV) expressing Cre-green fluorescent protein into the MBH of Lpl-floxed mice (and wild-type mice) to specifically decrease LPL activity in the MBH. In parallel, we injected an AAV overexpressing Lpl into the MBH of wild-type mice. We then studied energy homeostasis and hypothalamic ceramide content.The partial deletion of Lpl in the MBH in mice led to an increase in body weight compared with controls (37.72 ± 0.7 g vs 28.46 ± 0.12, p < 0.001) associated with a decrease in locomotor activity. These mice developed hyperinsulinaemia and glucose intolerance. This phenotype also displayed reduced expression of Cers1 in the hypothalamus as well as decreased concentration of several C18 species of ceramides and a 3-fold decrease in total ceramide intensity. Conversely, overexpression of Lpl specifically in the MBH induced a decrease in body weight.Our study shows that LPL in the MBH is an important regulator of body weight and glucose homeostasis.

Recent genome-wide association studies (GWAS) identified DUSP8, encoding a dual-specificity phosphatase targeting mitogen-activated protein kinases, as a type 2 diabetes (T2D) risk gene. Here, we reveal that Dusp8 is a gatekeeper in the hypothalamic control of glucose homeostasis in mice and humans. Male, but not female, Dusp8 loss-of-function mice, either with global or corticotropin-releasing hormone neuron-specific deletion, had impaired systemic glucose tolerance and insulin sensitivity when exposed to high-fat diet (HFD). Mechanistically, we found impaired hypothalamic-pituitary-adrenal axis feedback, blunted sympathetic responsiveness, and chronically elevated corticosterone levels driven by hypothalamic hyperactivation of Jnk signaling. Accordingly, global Jnk1 ablation, AAV-mediated Dusp8 overexpression in the mediobasal hypothalamus, or metyrapone-induced chemical adrenalectomy rescued the impaired glucose homeostasis of obese male Dusp8-KO mice, respectively. The sex-specific role of murine Dusp8 in governing hypothalamic Jnk signaling, insulin sensitivity, and systemic glucose tolerance was consistent with functional MRI data in human volunteers that revealed an association of the DUSP8 rs2334499 risk variant with hypothalamic insulin resistance in men. Further, expression of DUSP8 was increased in the infundibular nucleus of T2D humans. In summary, our findings suggest the GWAS-identified gene Dusp8 as a novel hypothalamic factor that plays a functional role in the etiology of T2D.

Our objective was to explore the physiological role of the intestinal endocannabinoids in the regulation of appetite upon short-term exposure to high-fat-diet (HFD) and understand the mechanisms responsible for aberrant gut-brain signaling leading to hyperphagia in mice lacking Napepld in the intestinal epithelial cells (IECs). We generated a murine model harboring an inducible NAPE-PLD deletion in IECs ( Napepld ΔIEC ). After an overnight fast, we exposed wild-type (WT) and Napepld ΔIEC mice to different forms of lipid challenge (HFD or gavage), and we compared the modification occurring in the hypothalamus, in the vagus nerve, and at endocrine level 30 and 60 min after the stimulation. Napepld ΔIEC mice displayed lower hypothalamic levels of N-oleoylethanolamine (OEA) in response to HFD. Lower mRNA expression of anorexigenic Pomc occurred in the hypothalamus of Napepld ΔIEC mice after lipid challenge. This early hypothalamic alteration was not the consequence of impaired vagal signaling in Napepld ΔIEC mice. Following lipid administration, WT and Napepld ΔIEC mice had similar portal levels of glucagon-like peptide-1 (GLP-1) and similar rates of GLP-1 inactivation. Administration of exendin-4, a full agonist of GLP-1 receptor (GLP-1R), prevented the hyperphagia of Napepld ΔIEC mice upon HFD. We conclude that in response to lipid, Napepld ΔIEC mice displayed reduced OEA in brain and intestine, suggesting an impairment of the gut-brain axis in this model. We speculated that decreased levels of OEA likely contributes to reduce GLP-1R activation, explaining the observed hyperphagia in this model. Altogether, we elucidated novel physiological mechanisms regarding the gut-brain axis by which intestinal NAPE-PLD regulates appetite rapidly after lipid exposure.

The reward system involved in hedonic food intake presents neuronal and behavioral dysregulations during obesity. Moreover, gut microbiota dysbiosis during obesity promotes low-grade inflammation in peripheral organs and in the brain contributing to metabolic alterations. The mechanisms underlying reward dysregulations during obesity remain unclear. We investigated if inflammation affects the striatum during obesity using a cohort of control-fed or diet-induced obese (DIO) male mice. We tested the potential effects of specific gut bacteria on the reward system during obesity by administrating Akkermansia muciniphila daily or a placebo to DIO male mice. We showed that dysregulations of the food reward are associated with inflammation and alterations in the blood-brain barrier in the striatum of obese mice. We identified Akkermansia muciniphila as a novel actor able to improve the dysregulated reward behaviors associated with obesity, potentially through a decreased activation of inflammatory pathways and lipid-sensing ability in the striatum. These results open a new field of research and suggest that gut microbes can be considered as an innovative therapeutic approach to attenuate reward alterations in obesity. This study provides substance for further investigations of Akkermansia muciniphila-mediated behavioral improvements in other inflammatory neuropsychiatric disorders.

Neuropeptide Y (NPY) is one of the most potent orexigenic peptides. The hypothalamic paraventricular nucleus (PVN) is a major locus where NPY exerts its effects on energy homeostasis. We investigated how NPY exerts its effect within the PVN. Patch clamp electrophysiology and Ca2+ imaging were used to understand the involvement of Ca2+ signaling and retrograde transmitter systems in the mediation of NPY induced effects in the PVN. Immuno-electron microscopy were performed to elucidate the subcellular localization of the elements of nitric oxide (NO) system in the parvocellular PVN. In vivo metabolic profiling was performed to understand the role of the endocannabinoid and NO systems of the PVN in the mediation of NPY induced changes of energy homeostasis. We demonstrated that NPY inhibits synaptic inputs of parvocellular neurons in the PVN by activating endocannabinoid and NO retrograde transmitter systems via mobilization of Ca2+ from the endoplasmic reticulum, suggesting that NPY gates the synaptic inputs of parvocellular neurons in the PVN to prevent the influence of non-feeding-related inputs. While intraPVN administered NPY regulates food intake and locomotor activity via NO signaling, the endocannabinoid system of the PVN selectively mediates NPY-induced decrease in energy expenditure. Thus, within the PVN, NPY stimulates the release of endocannabinoids and NO via Ca2+-influx from the endoplasmic reticulum. Both transmitter systems appear to have unique roles in the mediation of the NPY-induced regulation of energy homeostasis, suggesting that NPY regulates food intake, energy expenditure, and locomotor activity through different neuronal networks of this nucleus.

Leptin, the product of the Ob(Lep) gene, is a peptide hormone that plays a major role in maintaining the balance between food intake and energy expenditure. In the brain, leptin receptors are expressed by hypothalamic cells but also in the olfactory bulb, the first central structure coding for odors, suggesting a precise function of this hormone in odor-evoked activities. Although olfaction plays a key role in feeding behavior, the ability of the olfactory bulb to integrate the energy-related signal leptin is still missing. Therefore, we studied the fate of odor-induced activity in the olfactory bulb in the genetic context of leptin deficiency using the obese ob/ob mice. By means of an odor discrimination task with concomitant local field potential recordings, we showed that ob/ob mice perform better than wild-type (WT) mice in the early stage of the task. This behavioral gain of function is associated in parallel with profound changes in neuronal oscillations in the olfactory bulb. The distribution of the peaks in the gamma frequency range is shifted towards higher frequencies in ob/ob mice compared to WT mice before learning. More notably, beta oscillatory activity, which was shown previously to be correlated with olfactory discrimination learning, was longer and stronger in expert ob/ob mice after learning. Since oscillations in the olfactory bulb emerge from mitral to granule cell interactions, our results suggest that cellular dynamics in the olfactory bulb are deeply modified in ob/ob mice in the context of olfactory learning.

Hunger-sensing agouti-related peptide (AgRP) neurons ensure survival by adapting metabolism and behavior to low caloric environments. This adaption is accomplished by consolidating food intake, suppressing energy expenditure, and maximizing fat storage (nutrient partitioning) for energy preservation. The intracellular mechanisms responsible are unknown. Here we report that AgRP carnitine acetyltransferase (Crat) knockout (KO) mice exhibited increased fatty acid utilization and greater fat loss after 9 d of calorie restriction (CR). No differences were seen in mice with ad libitum food intake. Eleven days ad libitum feeding after CR resulted in greater food intake, rebound weight gain, and adiposity in AgRP Crat KO mice compared with wild-type controls, as KO mice act to restore pre-CR fat mass. Collectively, this study highlights the importance of Crat in AgRP neurons to regulate nutrient partitioning and fat mass during chronically reduced caloric intake. The increased food intake, body weight gain, and adiposity in KO mice after CR also highlights the detrimental and persistent metabolic consequence of impaired substrate utilization associated with CR. This finding may have significant implications for postdieting weight management in patients with metabolic diseases.-Reichenbach, A., Stark, R., Mequinion, M., Lockie, S. H., Lemus, M. B., Mynatt, R. L., Luquet, S., Andrews, Z. B. Carnitine acetyltransferase (Crat) in hunger-sensing AgRP neurons permits adaptation to calorie restriction.

Astrocytes are a major type of glial cell in the mammalian brain, essentially regulating neuronal development and function. Quantitative imaging represents an important approach to study astrocytic signaling in neural circuits. Focusing on astrocytic Ca2+ activity, a key pathway implicated in astrocye-neuron interaction, we here report a strategy combining fast light sheet fluorescence microscopy (LSFM) and correlative screening-based time series analysis, to map activity domains in astrocytes in living mammalian nerve tissue. Light sheet of micron-scale thickness enables wide-field optical sectioning to image astrocytes in acute mouse brain slices. Using both chemical and genetically encoded Ca2+ indicators, we demonstrate the complementary advantages of LSFM in mapping Ca2+ domains in astrocyte populations as compared to epifluorescence and two-photon microscopy. Our approach then revealed distinct kinetics of Ca2+ signals between cortical and hypothalamic astrocytes in resting conditions and following the activation of adrenergic G protein coupled receptor (GPCR). This observation highlights the activity heterogeneity across regionally distinct astrocyte populations, and indicates the potential of our method for investigating dynamic signals in astrocytes.

Ghrelin is a stomach-derived peptide hormone with salient roles in the regulation of energy balance and metabolism. Notably, ghrelin is recognized as the most powerful known circulating orexigenic hormone. Here, we systematically investigated the effects of ghrelin on energy homeostasis and found that ghrelin primarily induces a biphasic effect on food intake that has indirect consequences on energy expenditure and nutrient partitioning. We also found that ghrelin-induced biphasic effect on food intake requires the integrity of Agouti-related peptide/neuropeptide Y-producing neurons of the hypothalamic arcuate nucleus, which seem to display a long-lasting activation after a single systemic injection of ghrelin. Finally, we found that different autonomic, hormonal and metabolic satiation signals transiently counteract ghrelin-induced food intake. Based on our observations, we propose a heuristic model to describe how the orexigenic effect of ghrelin and the anorectic food intake-induced rebound sculpt a timely constrain feeding response to ghrelin.

The synchronization of circadian clock depends on a central pacemaker located in the suprachiasmatic nuclei. However, the potential feedback of peripheral signals on the central clock remains poorly characterized. To explore whether peripheral organ circadian clocks may affect the central pacemaker, we used a chimeric model in which mouse hepatocytes were replaced by human hepatocytes. Liver humanization led to reprogrammed diurnal gene expression and advanced the phase of the liver circadian clock that extended to muscle and the entire rhythmic physiology. Similar to clock-deficient mice, liver-humanized mice shifted their rhythmic physiology more rapidly to the light phase under day feeding. Our results indicate that hepatocyte clocks can affect the central pacemaker and offer potential perspectives to apprehend pathologies associated with altered circadian physiology.

The vagus nerve serves as an interoceptive relay between the body and the brain. Despite its well-established role in feeding behaviors, energy metabolism, and cognitive functions, the intricate functional processes linking the vagus nerve to the hippocampus and its contribution to learning and memory dynamics remain still elusive. Here, we investigated whether and how the gut-brain vagal axis contributes to hippocampal learning and memory processes at behavioral, functional, cellular, and molecular levels. Our results indicate that the integrity of the vagal axis is essential for long-term recognition memories, while sparing other forms of memory. In addition, by combing multi-scale approaches, our findings show that the gut-brain vagal tone exerts a permissive role in scaling intracellular signaling events, gene expressions, hippocampal dendritic spines density as well as functional long-term plasticities (LTD and LTP). These results highlight the critical role of the gut-brain vagal axis in maintaining the spontaneous and homeostatic functions of hippocampal ensembles and in regulating their learning and memory functions. In conclusion, our study provides comprehensive insights into the multifaceted involvement of the gut-brain vagal axis in shaping time-dependent hippocampal learning and memory dynamics. Understanding the mechanisms underlying this interoceptive body-brain neuronal communication may pave the way for novel therapeutic approaches in conditions associated with cognitive decline, including neurodegenerative disorders.

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes. It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation. PPARgamma, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis. We have recently demonstrated that PPARdelta, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARgamma, thereby inducing adipose differentiation. Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown. We show here that PPARdelta is directly implicated in fatty acid-induced cell proliferation. Ectopic expression of PPARdelta renders 3T3C2 cells capable of responding to treatment with long-chain fatty acids by a resumption of mitosis, and this effect is limited to a few days after confluence. This response is restricted to PPARdelta activators and, for fatty acids, takes place within the range of concentrations found to trigger differentiation of preadipocytes both in vitro and in vivo. Furthermore, the use of a mutated inactive PPARdelta demonstrated that transcriptional activity of the nuclear receptor is required to mediate fatty acid-induced proliferation. These data demonstrate that PPARdelta, as a transcription factor, is directly implicated in fatty acid-induced proliferation, and this could explain the hyperplastic development of adipose tissue that occurs in high-fat-fed animals.

Laforin is a dual specificity protein phosphatase involved in Lafora disease (LD), a fatal form of progressive myoclonus epilepsy characterized by neurodegeneration and the presence of intracellular polyglucosan inclusions (Lafora bodies) in different tissues. In this work, we describe that mice lacking laforin (epm2a-/-) have enhanced insulin response leading to altered whole-body energy balance. This enhanced insulin response overactivates the Akt pathway which increases glucose uptake in the heart, resulting in increased glycogen levels and the formation of polyglucosan inclusions. In addition, enhanced insulin response resulted in increased liver lipid biosynthesis, resulting in hepatic steatosis. On the contrary, overexpression in rat hepatoma FTO2B cells of native laforin but not of a form lacking phosphatase activity (C266S) resulted in attenuation of insulin signaling. These results define laforin as a new regulator of insulin sensitivity, which provides novel insights into LD pathogenesis and identifies this phosphatase as a potential novel component of the insulin signaling cascade.

In both developed and emerging countries, sedentary life style and over exposition to high energy dense foods has led to a thermodynamic imbalance and consequently obesity. Obesity often involves a behavioural component in which, similar to drugs abuse, compulsive consumption of palatable food rich in lipids and sugar drives energy intake far beyond metabolic demands. The hypothalamus is one of the primary integration sites of circulating energy-related signals like leptin or ghrelin and is therefore considered as one of the main central regulators of energy balance. However, food intake is also modulated by sensory inputs, such as tastes and odours, as well as by affective or emotional states. The mesolimbic pathway is well established as a key actor of the rewarding aspect of feeding. Particularly, the hedonic and motivational aspects of food are closely tied to the release of the neurotransmitter dopamine (DA) in striatal structure such as the Nucleus Accumbens (Nacc). In both rodent and humans several studies shows an attenuated activity of dopaminergic signal associated with obesity and there is evidence that consumption of palatable food per se leads to DA signalling alterations. Furthermore impaired cognition in obese mice is improved by selectively lowering triglycerides (TG) and intracerebroventricular administration of TG induces by itself acquisition impairment in several cognitive paradigms in normal body weight mice. Together, these observations raise the possibility that nutritional lipids, particularly TG, directly affect cognitive and reward processes by modulating the mesolimbic pathway and might contribute to the downward spiral of compulsive consumption of palatable food and obesity. This review is an attempt to capture recent evolution in the field that might point toward a direct action of nutritional lipid in the reward circuitry.

Sterol O-acyltransferase 2 (Soat2) encodes acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2), which synthesizes cholesteryl esters in hepatocytes and enterocytes fated either to storage or to secretion into nascent triglyceride-rich lipoproteins.We aimed to unravel the molecular mechanisms leading to reduced hepatic steatosis when Soat2 is depleted in mice.Soat2-/- and wild-type mice were fed a high-fat, a high-carbohydrate, or a chow diet, and parameters of lipid and glucose metabolism were assessed.Glucose, insulin, homeostatic model assessment for insulin resistance (HOMA-IR), oral glucose tolerance (OGTT), and insulin tolerance tests significantly improved in Soat2-/- mice, irrespective of the dietary regimes (2-way ANOVA). The significant positive correlations between area under the curve (AUC) OGTT (r = 0.66, p < 0.05), serum fasting insulin (r = 0.86, p < 0.05), HOMA-IR (r = 0.86, p < 0.05), Adipo-IR (0.87, p < 0.05), hepatic triglycerides (TGs) (r = 0.89, p < 0.05), very-low-density lipoprotein (VLDL)-TG (r = 0.87, p < 0.05) and the hepatic cholesteryl esters in wild-type mice disappeared in Soat2-/- mice. Genetic depletion of Soat2 also increased whole-body oxidation by 30% (p < 0.05) compared to wild-type mice.Our data demonstrate that ACAT2-generated cholesteryl esters negatively affect the metabolic control by retaining TG in the liver and that genetic inhibition of Soat2 improves liver steatosis via partitioning of lipids into secretory (VLDL-TG) and oxidative (fatty acids) pathways.

The reinforcing and motivational aspects of food are tied to the release of the dopamine in the mesolimbic system (ML). Free fatty acids from triglyceride (TG)-rich particles are released upon action of TG-lipases found at high levels in peripheral oxidative tissue (muscle, heart), but also in the ML. This suggests that local TG-hydrolysis in the ML might regulate food seeking and reward. Indeed, evidence now suggests that dietary TG directly target the ML to regulate amphetamine-induced locomotion and reward seeking behavior. Though the cellular mechanisms of TG action are unresolved, TG act in part through ML lipoprotein lipase, upstream of dopamine 2 receptor (D2R), and show desensitization in conditions of chronically elevated plasma TG as occur in obesity. TG sensing in the ML therefore represents a new mechanism by which chronic consumption of dietary fat might lead to adaptations in the ML and dysregulated feeding behaviors.

Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism.We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc-/- (YFP-Ptc-/-) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake.Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc-/- mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc-/- mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks.Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.

Nutritional long-chain fatty acids control adipose tissue mass by regulating the number and the size of adipocytes.It is now established that peroxisome-proliferator-activated receptors (PPARs) play crucial functions in the control of gene expression and the level of cell differentiation.PPARγ, which is activated by specific prostanoids, is a key factor in activating terminal differentiation and adipogenesis.We have recently demonstrated that PPARδ, once activated by fatty acids, drives the expression of a limited set of genes, including that encoding PPARγ, thereby inducing adipose differentiation.Thus far, the mechanism of action of fatty acids in the control of preadipocyte proliferation has remained unknown.We show here that PPARδ is directly implicated in fatty acid-induced cell proliferation.Ectopic expression of PPARδ renders 3T3C2 cells capable of responding to Abbreviations used : AF-2, activation function-2 ; ALBP, adipocyte lipid binding protein ; BrdU, 5-bromo-2h-deoxyuridine ; FAT, fatty acid translocase ; PPAR, peroxisome-proliferator-activated receptor ; PPRE, PPAR-responsive element ; RXR, retinoid X receptor.1 To whom correspondence should be addressed (e-mail grimaldi!taloa.unice.fr

Impaired skeletal muscle mitochondrial fatty acid oxidation (mFAO) has been implicated in the etiology of insulin resistance. Carnitine palmitoyltransferase-1 (CPT1) is a key regulatory enzyme of mFAO whose activity is inhibited by malonyl-CoA, a lipogenic intermediate. Whereas increasing CPT1 activity in vitro has been shown to exert a protective effect against lipid-induced insulin resistance in skeletal muscle cells, only a few studies have addressed this issue in vivo. We thus examined whether a direct modulation of muscle CPT1/malonyl-CoA partnership is detrimental or beneficial for insulin sensitivity in the context of diet-induced obesity. By using a Cre- LoxP recombination approach, we generated mice with skeletal muscle-specific and inducible expression of a mutated CPT1 form (CPT1mt) that is active but insensitive to malonyl-CoA inhibition. When fed control chow, homozygous CPT1mt transgenic (dbTg) mice exhibited decreased CPT1 sensitivity to malonyl-CoA inhibition in isolated muscle mitochondria, which was sufficient to substantially increase ex vivo muscle mFAO capacity and whole body fatty acid utilization in vivo. Moreover, dbTg mice were less prone to high-fat/high-sucrose (HFHS) diet-induced insulin resistance and muscle lipotoxicity despite similar body weight gain, adiposity, and muscle malonyl-CoA content. Interestingly, these CPT1mt-protective effects in dbTg-HFHS mice were associated with preserved muscle insulin signaling, increased muscle glycogen content, and upregulation of key genes involved in muscle glucose metabolism. These beneficial effects of muscle CPT1mt expression suggest that a direct modulation of the malonyl-CoA/CPT1 partnership in skeletal muscle could represent a potential strategy to prevent obesity-induced insulin resistance.

<h2>Abstract</h2><h3>Background</h3> Bariatric surgery is an effective treatment for type 2 diabetes. Early post-surgical enhancement of insulin secretion is key for diabetes remission. The full complement of mechanisms responsible for improved pancreatic beta cell functionality after bariatric surgery is still unclear. Our aim was to identify pathways, evident in the islet transcriptome, that characterize the adaptive response to bariatric surgery independently of body weight changes. <h3>Methods</h3> We performed entero-gastro-anastomosis (EGA) with pyloric ligature in leptin-deficient ob/ob mice as a surrogate of Roux-en-Y gastric bypass (RYGB) in humans. Multiple approaches such as determination of glucose tolerance, GLP-1 and insulin secretion, whole body insulin sensitivity, ex vivo glucose-stimulated insulin secretion (GSIS) and functional multicellular Ca²⁺-imaging, profiling of mRNA and of miRNA expression were utilized to identify significant biological processes involved in pancreatic islet recovery. <h3>Findings</h3> EGA resolved diabetes, increased pancreatic insulin content and GSIS despite a persistent increase in fat mass, systemic and intra-islet inflammation, and lipotoxicity. Surgery differentially regulated 193 genes in the islet, most of which were involved in the regulation of glucose metabolism, insulin secretion, calcium signaling or beta cell viability, and these were normalized alongside changes in glucose metabolism, intracellular Ca²⁺ dynamics and the threshold for GSIS. Furthermore, 27 islet miRNAs were differentially regulated, four of them hubs in a miRNA-gene interaction network and four others part of a blood signature of diabetes resolution in <i>ob/ob</i> mice and in humans. <h3>Interpretation</h3> Taken together, our data highlight novel miRNA-gene interactions in the pancreatic islet during the resolution of diabetes after bariatric surgery that form part of a blood signature of diabetes reversal. <h3>Funding</h3> European Union's Horizon 2020 research and innovation programme via the Innovative Medicines Initiative 2 Joint Undertaking (RHAPSODY), INSERM, Société Francophone du Diabète, Institut Benjamin Delessert, Wellcome Trust Investigator Award (212625/Z/18/Z), MRC Programme grants (MR/R022259/1, MR/J0003042/1, MR/L020149/1), Diabetes UK (BDA/11/0004210, BDA/15/0005275, BDA 16/0005485) project grants, National Science Foundation (310030–188447), Fondation de l'Avenir.

Hypothalamic AgRP/NPY neurons are key players in the control of feeding behaviour. Ghrelin, a major orexigenic hormone, activates AgRP/NPY neurons to stimulate food intake and adiposity. However, cell-autonomous ghrelin-dependent signalling mechanisms in AgRP/NPY neurons remain poorly defined. Here we show that calcium/calmodulin-dependent protein kinase ID (CaMK1D), a genetic hot spot in type 2 diabetes, is activated upon ghrelin stimulation and acts in AgRP/NPY neurons to mediate ghrelin-dependent food intake. Global Camk1d-knockout male mice are resistant to ghrelin, gain less body weight and are protected against high-fat-diet-induced obesity. Deletion of Camk1d in AgRP/NPY, but not in POMC, neurons is sufficient to recapitulate above phenotypes. In response to ghrelin, lack of CaMK1D attenuates phosphorylation of CREB and CREB-dependent expression of the orexigenic neuropeptides AgRP/NPY in fibre projections to the paraventricular nucleus (PVN). Hence, CaMK1D links ghrelin action to transcriptional control of orexigenic neuropeptide availability in AgRP neurons.

Background Daily variations in lipid concentrations in both gut lumen and blood are detected by specific sensors located in the gastrointestinal tract and in specialized central areas. Deregulation of the lipid sensors could be partly involved in the dysfunction of glucose homeostasis. The study aimed at comparing the effect of Medialipid (ML) overload on insulin secretion and sensitivity when administered either through the intestine or the carotid artery in mice. Methodology/Principal Findings An indwelling intragastric or intracarotid catheter was installed in mice and ML or an isocaloric solution was infused over 24 hours. Glucose and insulin tolerance and vagus nerve activity were assessed. Some mice were treated daily for one week with the anti-lipid peroxidation agent aminoguanidine prior to the infusions and tests. The intestinal but not the intracarotid infusion of ML led to glucose and insulin intolerance when compared with controls. The intestinal ML overload induced lipid accumulation and increased lipid peroxidation as assessed by increased malondialdehyde production within both jejunum and duodenum. These effects were associated with the concomitant deregulation of vagus nerve. Administration of aminoguanidine protected against the effects of lipid overload and normalized glucose homeostasis and vagus nerve activity. Conclusions/Significance Lipid overload within the intestine led to deregulation of gastrointestinal lipid sensing that in turn impaired glucose homeostasis through changes in autonomic nervous system activity.

Signaling through adrenergic receptors (ARs) by norepinephrine (NE) and epinephrine (Epi) regulates weight gain when mice are fed a high-fat diet (HFD) by controlling diet-induced thermogenesis. Thus, one would predict that mice unable to make NE/Epi because of inactivation of the dopamine beta-hydroxylase gene (Dbh-null mice) would have a propensity to become obese. We characterized the response of Dbh-null and control mice to a HFD.Dbh-null and control mice were fed an HFD or a regular diet (RD) for 2 months. Body weight, adiposity, muscle triglyceride levels, and adipocyte size were measured, as were circulating leptin, adiponectin, triglyceride, glucose, and insulin levels. A glucose tolerance test was also preformed.Dbh-null mice gain weight normally on an HFD and have the same adiposity. Their serum triglyceride and leptin levels are normal, but adipocytes are approximately 30% smaller than controls. Dbh-null mice maintain low blood glucose levels and glucose tolerance when exposed to the HFD in contrast to controls.Complete lack of NE/Epi does not predispose to obesity. Because mice lacking all three betaARs become obese on an HFD, an imbalance of signaling through alpha- and betaARs seems to be responsible for obesity. Surprisingly, Dbh-null mice maintain glucose tolerance.

Background The nuclear receptor chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) is an important coordinator of glucose homeostasis. We report, for the first time, a unique differential regulation of its expression by the nutritional status in the mouse hypothalamus compared to peripheral tissues. Methodology/Principal Findings Using hyperinsulinemic-euglycemic clamps and insulinopenic mice, we show that insulin upregulates its expression in the hypothalamus. Immunofluorescence studies demonstrate that COUP-TFII gene expression is restricted to a subpopulation of ventromedial hypothalamic neurons expressing the melanocortin receptor. In GT1-7 hypothalamic cells, the MC4-R agonist MTII leads to a dose dependant increase of COUP-TFII gene expression secondarily to a local increase in cAMP concentrations. Transfection experiments, using a COUP-TFII promoter containing a functional cAMP responsive element, suggest a direct transcriptional activation by cAMP. Finally, we show that the fed state or intracerebroventricular injections of MTII in mice induce an increased hypothalamic COUP-TFII expression associated with a decreased hepatic and pancreatic COUP-TFII expression. Conclusions/Significance These observations strongly suggest that hypothalamic COUP-TFII gene expression could be a central integrator of insulin and melanocortin signaling pathway within the ventromedial hypothalamus. COUP-TFII could play a crucial role in brain integration of circulating signal of hunger and satiety involved in energy balance regulation.

During the last several decades, the world has witnessed a pandemic expansion of pathologies related to high-fat and high-carbohydrate diets including obesity, diabetes, dyslipidemia, and cardiovascular diseases—collectively referred to as metabolic syndrome. Obesity is now considered by the World Health Organization (WHO) to be a worldwide epidemic, having more than doubled since 1980. In 2008, there were 1.5 billion overweight adults in both developed and developing countries (http://www.who.int/mediacentre/factsheets/fs311/en/). The WHO believes the fundamental cause of obesity and being overweight is an energy imbalance between calories consumed and calories expended. Appropriate energy balance is reached when energy intake and energy expenditure are adapted to meet energy demands and nutrient availability. In the central nervous system (CNS), the hypothalamus was identified as a key integrator of nutrients-induced signals of hunger and satiety directing information regarding energy stores and food availability. The arcuate nucleus (ARC) subdivision of the hypothalamus lies at the bottom of the third ventricle close to a circumventricular organ called median eminence (ME). In this region, the blood–brain barrier is fenestrated and allows for facilitated blood–brain exchange. Neurons that reside there are referred to as “first order neurons” because they would be the first to respond to the circulating signals of hunger and satiety such as leptin, insulin or ghrelin. The ARC contains at least two crucial populations of neurons that continuously monitor signals reflecting energy status and promote the appropriate behavioral and metabolic responses to changes in nutritional availability and demand [1]. Neurons in the ARC that make both pro-opiomelanocortin (POMC) and cocaine- and amphetamine-related transcript secrete the melanocortin peptides adrenocorticotropic hormone (ACTH) and α, β and γ-melanocyte-stimulating hormone (MSH), which are derived from posttranslational processing of POMC. POMC neurons decrease food intake and increase energy expenditure through activation of melanocortin receptors (MCR) via the release of α-MSH (Fig. 1). Conversely, activity of neighboring neurons expressing the orexigenic neuropeptides, agouti-related protein (AgRP) and neuropeptide Y (NPY) (AgRP neurons), increase feeding by opposing the anorexigenic actions of the neighboring POMC neurons, in part through the release of AgRP, a competitive inhibitor of MCRs. The two populations project to several nuclei within and outside of the hypothalamus [2] and are an integral part of the so called “melanocortin system” which represent a canonical neurocircuitry involved in the regulation of energy balance. Any mutation in the melanocortin signaling pathway results in hyperphagia, hypometabolism, hyperinsulinemia, and hyperglycemia in both rodents and humans. Figure 1 MiRNAs processing in POMC neurons is critical for central regulation of energy balance. Arcuate(ARC) POMC and AgRP neurons define the two antagonistic branch of the melanocortin system that lies at the bottom of the 3rd ventricules. POMC and AgRP neurons ... In recent years, several processes were described to link high fat feeding and obesity to aberrant development and function of POMC neurocircuitry, such as defective leptin signaling [3,4], inflammation [5] or autopaghy processes [6]. Moreover, high fat feeding was found to alter not only POMC neuron activity but also the ability of this network to renew and rewire during adulthood [7,8]. In this issue of Molecular Metabolism, a study from Schneeberger et al. provides an important molecular clue to understand the molecular mechanisms that could relay peripheral nutrient excess and/or obesity to the function and maintenance of hunger-related neurocircuitry. The authors applied a variety of elegant techniques combined with integrated approaches to tackle the role of microRNA (miRNA) processing enzyme Dicer to the development, activity and survival of POMC neurons [9]. miRNAs are small non-coding RNAs that are processed through primary transcript (pri-miRNA) maturation into pre-miRNA and subsequently exported from the nucleus to be transformed from double stranded RNA into small 20–25 bp long interfering single strand RNA (siRNA) through the specific action of the endoribonuclease Dicer [10]. In turn, Dicer promotes the formation of the RNA-induced silencing complex (RISC) in which siRNA association with target mRNA leads to either translational repression or degradation of the transcript (Fig. 1). miRNAs biogenesis recently emerged as a fundamental regulatory process involved in a wide variety of biological functions including metabolism, cancer and neurological disorders. The authors demonstrate first the expression of Dicer in both AgRP and POMC neurons, they went on showing that hypothalamic but not hindbrain Dicer mRNA expression is regulated by nutritional manipulation and genetic obesity. Fasting induces Dicer mRNA while in animals fed high fat diets or lack leptin (ob/ob) showed reduced Dicer mRNA levels, consistent with an integral role of Dicer in energy sensing. Using sophisticated approaches in which deletion of Dicer1 was restricted to POMC-expressing cells (Pomc-Cre; Dicer1flox/flox), the authors described increased body weight gain, adiposity, hyperglycemia, hyperinsulinemia and glucose intolerance. This phenotype was observed at 12 weeks of age while 6-week old mice remained asymptomatic. Lack of Dicer expression is associated with a sharp reduction of POMC and CART peptide abundance in the hypothalamus at 3 weeks and became undetectable at 6 weeks. This defect paralleled a progressive loss of arcuate POMC neurons. Importantly, the authors provide evidence that POMC survival and maintenance in adulthood critically requires the integrity of miRNA biogenesis while POMC neurons neurogenesis remained unaffected. Since POMC is expressed in the pituitary, the authors also explored thoroughly the consequence of Dicer1 inactivation in these cells. They found that POMC-specific deletion of Dicer1 leads to the absence of pituitary intermediary lobe, a reduction of adrenocorticotropic hormone (ACTH) together with specific deletion of melanotroph and corticotroph genes and altered adrenal morphology. Finally, the authors also provided DNA array data supporting that defective MAPK signaling, proteasome, and ribosomal function are likely involved in the neurodegenerative processes that results from Dicer1 inactivation. This elegant work firmly establishes that DICER-dependent processing of miRNA biogenesis in POMC expressing cells is critically required to sustain survival and activity of POMC cells in both hypothalamus and the pituitary. Importantly, the metabolic syndrome described here is presumably attributable to hypothalamic cell loss since reduced corticosterone level and adrenal corticotropic function is often associated with reduced obesity. In conclusion, this works provides fundamental clues to understand how energy surplus may impact on feeding-related neurocircuitry. The progressive degeneration of POMC neurons observed in POMCDicerKO mice somewhat hampers the conclusion that can be drawn regarding the role of DICER in POMC neurons activity in adult animals. Therefore additional genetic approaches allowing for temporal deletion of Dicer1 will be needed in the future to explore several aspect miRNA-mediated post-transcriptional regulation of POMC neuron electrophysiological activity, neuropeptide processing, neurotransmitter release, synaptic remodeling, or receptor expression. There is no doubt that the breakthrough achieved by such discovery will complement the arsenal of strategies that could be mobilized to curtail the obesity epidemic.

EDITORIAL article Front. Endocrinol., 19 June 2019Sec. Neuroendocrine Science Volume 10 - 2019 | https://doi.org/10.3389/fendo.2019.00399