Rôle des endozépines dans la régulation hypothalamique de l’homéostasie énergétique

The term endozepine refers to a family of peptides including the diazepam-binding inhibitor (DBI) and its processing products, including the octadecaneuropeptide ODN (DBI33-50). Endozepins are true gliotransmitters since (i) DBI is expressed by CNS astroglial cells, (ii) their release is regulated by several neuropeptides, (iii) ODN decreases the expression of POMC and increases that of NPY at the arcuate nucleus, (iv) ODN exerts a strong anorectic effect in rodents. This effect is relayed by a metabotropic receptor distinct from the conventional benzodiazepine receptors. Some studies suggest that hypothalamic astroglial cells perceive certain circulating signals reflecting the energy status of the body. However, the nature of the signals involved in "glia-neuron" coupling remains unknown. All of these data suggest that the ODN could be a relay between the peripheral factors and the neuronal populations of the arcuate nucleus.We show for the first time that an acute 18-hour fast reduces the level of mRNAs encoding DBI in the astroglial cells of the medobasal hypothalamus. The insulin and leptin receptors are present on cells expressing DBI, however, the intraperitoneal injection of insulin or leptin is unable to reverse the effect of fasting.In a second step, we investigated the links that may exist between blood glucose and endozepines produced by astroglial cells. Central glucose injection increases the level of DBI-encoding transcripts at the periventricular zone of the hypothalamus of previously hungry rats, and the addition of glucose to the extracellular medium also stimulates the release of endozepines from hypothalamic explants. The anorexigenic effect of central or peripheral glucose injection in rats previously deprived of food is blocked by the icv injection of cyclo1-8 [DLeu5] OP, a metabotropic ODN antagonist analogue, while that the orexigenic effect of a central injection of 2-DG in normally fed rats is abolished by the co-injection of OP, an agonist analogue of this receptor. Finally, the anorexigenic effect of a central injection of glucose or OP is blocked by the co-injection of an MCR3 / 4 receptor antagonist, SHU-9119, demonstrating for the first time that endozepines, produced by glial cells, constitute an essential relay in the response of the melanocortic system to changes in blood glucose. Central injection of cyclo1-8 [dLeu5] OP results in increased blood glucose levels in normally fed rats while icv injection of ODN in hungry animals increases glucose tolerance, suggesting that the central endozepinergic system exerts an hypoglycemic tonic effect.Using the C6 cell line and the promoter / reporter strategy, we show the existence of a glucose response element in the proximal promoter of the DBI gene. Preliminary work conducted in vitro indicates that the activation of the DBI promoter by glucose is relayed via the hexosamine pathway, an ancillary pathway for glycolysis controlling the O-glycosylation of proteins. The immunohistochemical localization of O-glycosylated residues in the rat hypothalamus reveals the existence of intense labeling in glial cells expressing DBI, indicating that this pathway is functional in vivo.All of our work suggests that endozepines produced by hypothalamic glia play a key role in central glucose perception and participate in a feedback mechanism that controls glucose homeostasis in the body.On désigne sous le terme endozépine une famille de peptides incluant le diazepam-binding inhibitor (DBI) et ses produits de maturation, dont l’octadecaneuropeptide ODN (DBI33-50). Les endozépines sont de véritables gliotransmetteurs puisque (i) le DBI est exprimé par les cellules astrogliales du SNC, (ii) leur libération est régulée par plusieurs neuropeptides, (iii) l’ODN diminue l’expression de la POMC et augmente celle du NPY au niveau du noyau arqué, (iv) l’ODN exerce un puissant effet anorexigène chez le rongeur. Cet effet est relayé par un récepteur métabotropique distinct des récepteurs classiques des benzodiazépines. Quelques travaux suggèrent que les cellules astrogliales hypothalamiques perçoivent certains signaux circulants reflétant le statut énergétique de l’organisme. Toutefois, la nature des signaux impliqués dans le couplage « glie-neurone » reste inconnue. L’ensemble de ces données suggère que l’ODN pourrait constituer un relais entre les facteurs périphériques et les populations neuronales du noyau arqué.Nous montrons pour la première fois qu’un jeûne aigu de 18 h réduit le taux des ARNm codant le DBI dans les cellules astrogliales de l’hypothalamus médiobasal. Les récepteurs de l’insuline et de la leptine sont présents sur des cellules exprimant le DBI, toutefois, l’injection intrapéritonéale d’insuline ou de leptine est incapable de reverser l’effet du jeûne. Dans un second temps, nous avons recherché les liens pouvant exister entre le glucose sanguin et les endozépines produites par les cellules astrogliales. L’injection centrale de glucose augmente le niveau des transcrits codant le DBI au niveau de la zone périventriculaire de l’hypothalamus de rats préalablement affamés, et l’ajout de glucose dans le milieu extracellulaire stimule également la libération d’endozépines à partir d’explants hypothalamiques. L’effet anorexigène d’une injection centrale ou périphérique de glucose, chez des rats préalablement privés de nourriture, est bloqué par l’injection icv de cyclo1-8[DLeu5]OP, un analogue antagoniste du récepteur métabotropique de l’ODN, tandis que l’effet orexigène d’une injection centrale de 2-DG chez des rats normalement nourris est aboli par la co-injection d’OP, un analogue agoniste de ce récepteur. Enfin, l’effet anorexigène d’une injection centrale de glucose ou d’OP est bloqué par la co-injection d’un antagoniste des récepteurs MCR3/4, le SHU-9119, démontrant pour la première fois que les endozépines, produites par les cellules gliales, constituent un relais essentiel dans la réponse du système mélanocortinique aux variations de la glycémie. L’injection centrale de cyclo1-8[dLeu5]OP entraîne une augmentation de la glycémie chez des rats normalement nourris tandis que l’injection icv d’ODN chez des animaux affamés augmente la tolérance au glucose, suggérant que le système endozépinergique central exerce un effet tonique hypoglycémiant.A l’aide de la lignée cellulaire C6 et de la stratégie promoteur/rapporteur, nous montrons l’existence d’un élément de réponse au glucose dans le promoteur proximal du gène DBI. Des travaux préliminaires menés in vitro indiquent que l’activation du promoteur DBI par le glucose est relayée par la voie des hexosamines, une voie annexe de la glycolyse contrôlant la O-glycosylation des protéines. La localisation immunohistochimique des résidus O-glycosylés dans l’hypothalamus de rat révèle l’existence d’un marquage intense dans les cellules gliales exprimant le DBI, indiquant que cette voie est fonctionnelle in vivo.L’ensemble de nos travaux suggère que les endozépines produites par les cellules gliales hypothalamiques jouent un rôle essentiel dans la perception centrale du glucose et participent à un mécanisme de rétroaction contrôlant l’homéostasie glucidique de l’organisme

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

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 β-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

Hypothalamic Glucose-sensing: Role of Glia-to-Neuron Signaling

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Implication des neurones produisant l’Acyl-Coa Binding Domain Containing 7 (ACBD7) dans la régulation hypothalamique de l’homéostasie énergétique

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Implication des neurones produisant l’Acyl-Coa Binding Domain Containing 7 (ACBD7) dans la régulation hypothalamique de l’homéostasie énergétique

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

International audienceBackground: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-1β, 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-1β, 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

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. Keywords: mTOR, DEPTOR, Hypothalamus, Energy balance, Glucose metabolis

Endozepines and their receptors: Structure, functions and pathophysiological significance

International audienceThe 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 ». 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 (PBR/TSPO), 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 discusses the physiopathological implications of these regulatory gliopeptides