Pharmacological chaperone action in humanized mouse models of MC4R-linked obesity.

MC4R mutations represent the largest monogenic cause of obesity, resulting mainly from receptor misfolding and intracellular retention by the cellular quality control system. The present study aimed at determining whether pharmacological chaperones (PCs) that restore folding and plasma membrane trafficking by stabilizing near native protein conformation may represent valid therapeutic avenues for the treatment of melanocortin type 4 receptor-linked (MC4R-linked) obesity. To test the therapeutic PC potential, we engineered humanized MC4R (hMC4R) mouse models expressing either the WT human MC4R or a prevalent obesity-causing mutant (R165W). Administration of a PC able to rescue cell surface expression and functional activity of R165W-hMC4R in cells restored the anorexigenic response of the R165W-hMC4R obese mice to melanocortin agonist, providing a proof of principle for the therapeutic potential of MC4R-targeting PCs in vivo. Interestingly, the expression of the WT-hMC4R in mice revealed lower sensitivity of the human receptor to α-melanocyte-stimulating hormone (α-MSH) but not ß-MSH or melanotan II, resulting in a lower penetrance obese phenotype in the WT-hMC4R versus R165W-hMC4R mice. In conclusion, we created 2 new obesity models, a hypomorphic highlighting species differences and an amorphic providing a preclinical model to test the therapeutic potential of PCs to treat MC4R-linked obesity.

Glial Endozepines Reverse High-Fat Diet-Induced Obesity by Enhancing Hypothalamic Response to Peripheral Leptin.

Research on energy homeostasis has focused on neuronal signaling; however, the role of glial cells has remained little explored. Glial endozepines exert anorexigenic actions by mechanisms which remain poorly understood. In this context, the present study was designed to decipher the mechanisms underlying the anorexigenic action of endozepines and to investigate their potential curative effect on high-fat diet-induced obesity. We carried out a combination of physiological, pharmacological, and molecular analyses together to dissect the underlying mechanisms of endozepine-induced hypophagia. To evaluate the potential anti-obesity effect of endozepines, different model of obesity were used, i.e., ob/ob and diet-induced obese mice. We show that the intracerebral administration of endozepines enhances satiety by targeting anorexigenic brain circuitry and induces STAT3 phosphorylation, a hallmark of leptin signaling. Strikingly, endozepines are entirely ineffective at reducing food intake in the presence of a circulating leptin antagonist and in leptin-deficient mice (ob/ob) but potentiate the reduced food intake and weight loss induced by exogenous leptin administration in these animals. Endozepines reversed high fat diet-induced obesity by reducing food intake and restored leptin-induced STAT3 phosphorylation in the hypothalamus. Interestingly, we observed that glucose and insulin synergistically enhance tanycytic endozepine expression and release. Finally, endozepines, which induce ERK activation necessary for leptin transport into the brain in cultured tanycytes, require tanycytic leptin receptor expression to promote STAT3 phosphorylation in the hypothalamus. Our data identify endozepines as potential anti-obesity compounds in part through the modulation of the LepR-ERK-dependent tanycytic leptin shuttle.

Endozepines and their receptors: Structure, functions and pathophysiological significance.

The existence of specific binding sites for benzodiazepines (BZs) in the brain has prompted the search for endogenous BZ receptor ligands designated by the generic term « endozepines ¼. This has led to the identification of an 86-amino acid polypeptide capable of displacing [3H]diazepam binding to brain membranes, thus called diazepam-binding inhibitor (DBI). It was subsequently found that the sequence of DBI is identical to that of a lipid carrier protein termed acyl-CoA-binding protein (ACBP). The primary structure of DBI/ACBP has been well preserved, suggesting that endozepines exert vital functions. The DBI/ACBP gene is expressed by astroglial cells in the central nervous system, and by various cell types in peripheral organs. Endoproteolytic cleavage of DBI/ACBP generates several bioactive peptides including a triakontatetraneuropeptide that acts as a selective ligand of peripheral BZ receptors/translocator protein, and an octadecaneuropeptide that activates a G protein-coupled receptor and behaves as an allosteric modulator of the GABAAR. Although DBI/ACBP is devoid of a signal peptide, endozepines are released by astrocytes in a regulated manner. Consistent with the diversity and wide distribution of BZ-binding sites, endozepines appear to exert a large array of biological functions and pharmacological effects. Thus, intracerebroventricular administration of DBI or derived peptides induces proconflict and anxiety-like behaviors, and reduces food intake. Reciprocally, the expression of DBI/ACBP mRNA is regulated by stress and metabolic signals. In vitro, endozepines stimulate astrocyte proliferation and protect neurons and astrocytes from apoptotic cell death. Endozepines also regulate neurosteroid biosynthesis and neuropeptide expression, and promote neurogenesis. In peripheral organs, endozepines activate steroid hormone production, stimulate acyl chain ceramide synthesis and trigger pro-inflammatory cytokine secretion. The expression of the DBI/ACBP gene is enhanced in addiction/withdrawal animal models, in patients with neurodegenerative disorders and in various types of tumors. We review herein the current knowledge concerning the various actions of endozepines and discuss the physiopathological implications of these regulatory gliopeptides.

Emerging Signaling Pathway in Arcuate Feeding-Related Neurons: Role of the Acbd7.

The understanding of the mechanisms whereby energy balance is regulated is essential to the unraveling of the pathophysiology of obesity. In the last three decades, focus was put on the metabolic role played by the hypothalamic neurons expressing proopiomelanocortin (POMC) and cocaine and amphetamine regulated transcript (CART) and the neurons co-localizing agouti-related peptide (AgRP), neuropeptide Y (NPY), and gamma-aminobutyric acid (GABA). These neurons are part of the leptin-melanocortin pathway, whose role is key in energy balance regulation. More recently, the metabolic involvement of further hypothalamic uncharacterized neuron populations has been suggested. In this review, we discuss the potential homeostatic implication of hypothalamic GABAergic neurons that produce Acyl-Coa-binding domain containing protein 7 (ACBD7), precursor of the nonadecaneuropeptide (NDN), which has recently been characterized as a potent anorexigenic neuropeptide capable of relaying the leptin anorectic/thermogenic effect via the melanocortin system.

DEPTOR in POMC neurons affects liver metabolism but is dispensable for the regulation of energy balance.

We have recently demonstrated that specific overexpression of DEP-domain containing mTOR-interacting protein (DEPTOR) in the mediobasal hypothalamus (MBH) protects mice against high-fat diet-induced obesity, revealing DEPTOR as a significant contributor to energy balance regulation. On the basis of evidence that DEPTOR is expressed in the proopiomelanocortin (POMC) neurons of the MBH, the present study aimed to investigate whether these neurons mediate the metabolic effects of DEPTOR. Here, we report that specific DEPTOR overexpression in POMC neurons does not recapitulate any of the phenotypes observed when the protein was overexpressed in the MBH. Unlike the previous model, mice overexpressing DEPTOR only in POMC neurons 1) did not show differences in feeding behavior, 2) did not exhibit changes in locomotion activity and oxygen consumption, 3) did not show an improvement in systemic glucose metabolism, and 4) were not resistant to high-fat diet-induced obesity. These results support the idea that other neuronal populations are responsible for these phenotypes. Nonetheless, we observed a mild elevation in fasting blood glucose, insulin resistance, and alterations in liver glucose and lipid homeostasis in mice overexpressing DEPTOR in POMC neurons. Taken together, these results show that DEPTOR overexpression in POMC neurons does not affect energy balance regulation but could modulate metabolism through a brain-liver connection.

Mediobasal hypothalamic overexpression of DEPTOR protects against high-fat diet-induced obesity.

BACKGROUND/OBJECTIVE: The mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions into distinct protein complexes (mTORC1 and mTORC2) that regulate energy homeostasis. DEP-domain containing mTOR-interacting protein (DEPTOR) is part of these complexes and is known to dampen mTORC1 function, consequently reducing mTORC1 negative feedbacks and promoting insulin signaling and Akt/PKB activation in several models. Recently, we observed that DEPTOR is expressed in several structures of the brain including the mediobasal hypothalamus (MBH), a region that regulates energy balance. Whether DEPTOR in the MBH plays a functional role in regulating energy balance and hypothalamic insulin signaling has never been tested. METHODS: We have generated a novel conditional transgenic mouse model based on the Cre-LoxP system allowing targeted overexpression of DEPTOR. Mice overexpressing DEPTOR in the MBH were subjected to a metabolic phenotyping and MBH insulin signaling was evaluated. RESULTS: We first report that systemic (brain and periphery) overexpression of DEPTOR prevents high-fat diet-induced obesity, improves glucose metabolism and protects against hepatic steatosis. These phenotypes were associated with a reduction in food intake and feed efficiency and an elevation in oxygen consumption. Strikingly, specific overexpression of DEPTOR in the MBH completely recapitulated these phenotypes. DEPTOR overexpression was associated with an increase in hypothalamic insulin signaling, as illustrated by elevated Akt/PKB activation. CONCLUSION: Altogether, these results support a role for MBH DEPTOR in the regulation of energy balance and metabolism.

Involvement of the Acyl-CoA binding domain containing 7 in the control of food intake and energy expenditure in mice.

Acyl-CoA binding domain-containing 7 (Acbd7) is a paralog gene of the diazepam-binding inhibitor/Acyl-CoA binding protein in which single nucleotide polymorphism has recently been associated with obesity in humans. In this report, we provide converging evidence indicating that a splice variant isoform of the Acbd7 mRNA is expressed and translated by some POMC and GABAergic-neurons in the hypothalamic arcuate nucleus (ARC). We have demonstrated that the ARC ACBD7 isoform was produced and processed into a bioactive peptide referred to as nonadecaneuropeptide (NDN) in response to catabolic signals. We have characterized NDN as a potent anorexigenic signal acting through an uncharacterized endozepine G protein-coupled receptor and subsequently via the melanocortin system. Our results suggest that ACBD7-producing neurons participate in the hypothalamic leptin signalling pathway. Taken together, these data suggest that ACBD7-producing neurons are involved in the hypothalamic control exerted on food intake and energy expenditure by the leptin-melanocortin pathway.

Increased Hypothalamic Levels of Endozepines, Endogenous Ligands of Benzodiazepine Receptors, in a Rat Model of Sepsis.

BACKGROUND: The mechanisms involved in septic anorexia are mainly related to the secretion of inflammatory cytokines. The term endozepines designates a family of neuropeptides, including the octadecaneuropeptide (ODN), originally isolated as endogenous ligands of benzodiazepine receptors. Previous data showed that ODN, produced and released by astrocytes, is a potent anorexigenic peptide. We have studied the effect of sepsis by means of a model of cecal ligation and puncture (CLP) on the hypothalamic expression of endozepines (DBI mRNA and protein levels), as well as on the level of neuropeptides controlling energy homeostasis mRNAs: pro-opiomelanocortin, neuropeptide Y, and corticotropin-releasing hormone. In addition, we have investigated the effects of two inflammatory cytokines, TNF-α and IL-1ß, on DBI mRNA levels in cultured rat astrocytes. METHODS: Studies were performed on Sprague-Dawley male rats and on cultures of rat cortical astrocytes. Sepsis was induced using the CLP method. Sham-operated control animals underwent the same procedure, but the cecum was neither ligated nor incised. RESULTS: Sepsis caused by CLP evoked an increase of DBI mRNA levels in ependymal cells bordering the third ventricle and in tanycytes of the median eminence. CLP-induced sepsis was also associated with stimulated ODN-like immunoreactivity (ODN-LI) in the hypothalamus. In addition, TNF-α, but not IL-1ß, induced a dose-dependent increase in DBI mRNA in cultured rat astrocytes. An increase in the mRNA encoding the precursor of the anorexigenic peptide α-melanocyte stimulating hormone, the pro-opiomelanocortin, and the corticotropin-releasing hormone was observed in the hypothalamus. CONCLUSION: These results suggest that during sepsis, hypothalamic mRNA encoding endozepines, anorexigenic peptide as well as stress hormone could play a role in the anorexia/cachexia associated with inflammation due to sepsis and we suggest that this hypothalamic mRNA expression could involve TNF-α.

Hypothalamic control of brown adipose tissue thermogenesis.

It has long been known, in large part from animal studies, that the control of brown adipose tissue (BAT) thermogenesis is insured by the central nervous system (CNS), which integrates several stimuli in order to control BAT activation through the sympathetic nervous system (SNS). SNS-mediated BAT activity is governed by diverse neurons found in brain structures involved in homeostatic regulations and whose activity is modulated by various factors including oscillations of energy fluxes. The characterization of these neurons has always represented a challenging issue. The available literature suggests that the neuronal circuits controlling BAT thermogenesis are largely part of an autonomic circuitry involving the hypothalamus, brainstem and the SNS efferent neurons. In the present review, we recapitulate the latest progresses in regards to the hypothalamic regulation of BAT metabolism. We briefly addressed the role of the thermoregulatory pathway and its interactions with the energy balance systems in the control of thermogenesis. We also reviewed the involvement of the brain melanocortin and endocannabinoid systems as well as the emerging role of steroidogenic factor 1 (SF1) neurons in BAT thermogenesis. Finally, we examined the link existing between these systems and the homeostatic factors that modulate their activities.

The PVH as a site of CB1-mediated stimulation of thermogenesis by MC4R agonism in male rats.

The present study was designed to investigate the involvement of the cannabinoid receptor 1 (CB1) in the stimulating effects of the melanocortin-4 receptor (MC4R) agonism on whole-body and brown adipose tissue (BAT) thermogenesis. In a first series of experiments, whole-body and BAT thermogenesis were investigated in rats infused in the third ventricle of the brain with the MC4R agonist melanotan II (MTII) and the CB1 agonist δ9-tetrahydrocannabinol (δ(9)-THC) or the CB1 antagonist AM251. Whole-body thermogenesis was measured by indirect calorimetry and BAT thermogenesis assessed from interscapular BAT (iBAT) temperature. δ(9)-THC blunted the effects of MTII on energy expenditure and iBAT temperature, whereas AM251 tended to potentiate the MTII effects. δ(9)-THC also blocked the stimulating effect of MTII on (14)C-bromopalmitate and (3)H-deoxyglucose uptakes in iBAT. Additionally, δ(9)-THC attenuated the stimulating effect of MTII on the expression of peroxisome proliferator-activated receptor-γ coactivator 1-α (Pgc1α), type II iodothyronine deiodinase (Dio2), carnitine palmitoyltransferase 1B (Cpt1b), and uncoupling protein 1 (Ucp1). In a second series of experiments, we addressed the involvement of the paraventricular hypothalamic nucleus (PVH) in the CB1-mediated effects of MTII on iBAT thermogenesis, which were assessed following the infusion of MTII in the PVH and δ(9)-THC or AM251 in the fourth ventricle of the brain. We demonstrated the ability of δ(9)-THC to blunt MTII-induced iBAT temperature elevation. δ(9)-THC also blocked the PVH effect of MTII on (14)C-bromopalmitate uptake as well as on Pgc1α and Dio2 expression in iBAT. Altogether the results of this study demonstrate the involvement of the PVH in the CB1-mediated stimulating effects of the MC4R agonist MTII on whole-body and BAT thermogenesis.

The medial preoptic nucleus as a site of the thermogenic and metabolic actions of melanotan II in male rats.

The present study was designed to investigate the role of the medial preoptic nucleus (MPO) as a site of the thermogenic and metabolic effects of the α-melanocyte-stimulating hormone analog melanotan II (MTII). We also assessed the involvement of the dorsomedial hypothalamic nucleus (DMH) by investigating the effects of the MPO infusion of MTII in rats with DMH lesions produced by kainic acid. Infusion of MTII in the MPO led to increases in interscapular brown adipose tissue (iBAT) temperature and iBAT uptake of 14C-bromopalmitate. Both increases were blocked by DMH lesions. iBAT temperature increase (area under curve) and 14C-bromopalmitate uptake emerged as two correlated variables (r = 0.63, P < 0.001). DMH lesions also blocked MTII-induced expression of mRNAs coding for proteins involved in 1) thermogenesis [type II iodothyronine deiodinase (Dio2) and peroxisome proliferator-activated receptor gamma coactivator 1-α (Pgc1α)], 2) lipolysis [hormone-sensitive lipase (Hsl)], and 3) lipogenesis [diacylglycerol-O-acyltransferase 2 (Dgat2), fatty acid synthase (Fas)], in iBAT of rats killed 1 h after MPO infusion of MTII. MTII also stimulated expression of genes in iWAT but only in rats with DMH lesions. These genes included glucose transporter member 4 (Glut4), glycerol-3-phosphate acyltransferase 3 (Gpat3), Dgat1, Dgat2, triglyceride lipase (Atgl), Hsl, and carnitine palmitoyltransferase 1ß (Cpt1ß). Altogether, the present results reveal the MPO as a site of the thermogenic and metabolic actions of MTII. They also contribute to establish the MPO-DMH duet as a significant target for melanocortins to modulate energy homeostasis.

Gliotransmission and brain glucose sensing: critical role of endozepines.

Hypothalamic glucose sensing is involved in the control of feeding behavior and peripheral glucose homeostasis, and glial cells are suggested to play an important role in this process. Diazepam-binding inhibitor (DBI) and its processing product the octadecaneuropeptide (ODN), collectively named endozepines, are secreted by astroglia, and ODN is a potent anorexigenic factor. Therefore, we investigated the involvement of endozepines in brain glucose sensing. First, we showed that intracerebroventricular administration of glucose in rats increases DBI expression in hypothalamic glial-like tanycytes. We then demonstrated that glucose stimulates endozepine secretion from hypothalamic explants. Feeding experiments indicate that the anorexigenic effect of central administration of glucose was blunted by coinjection of an ODN antagonist. Conversely, the hyperphagic response elicited by central glucoprivation was suppressed by an ODN agonist. The anorexigenic effects of centrally injected glucose or ODN agonist were suppressed by blockade of the melanocortin-3/4 receptors, suggesting that glucose sensing involves endozepinergic control of the melanocortin pathway. Finally, we found that brain endozepines modulate blood glucose levels, suggesting their involvement in a feedback loop controlling whole-body glucose homeostasis. Collectively, these data indicate that endozepines are a critical relay in brain glucose sensing and potentially new targets in treatment of metabolic disorders.