Martine Begeot

Leptin Infusion and Obesity in Mouse Cause Alterations in the Hypothalamic Melanocortin System

The objectives of this study were to identify potential alterations in gene expression of melanocortin‐4 receptor (MC4‐R), proopiomelanocortin (POMC), and Agouti‐related protein (AgRP) in mouse hypothalamus under a chronic peripheral infusion of leptin or at early (8 weeks) and advanced (16 weeks) phases of diet‐induced obesity. Control or diet‐induced obesity mice (8 or 16 weeks of high‐fat diet) were either treated or not treated with leptin. Metabolic features were analyzed and expression of the genes of interest was measured by quantitative reverse transcriptase‐PCR (RT‐qPCR) and western blot. We reported that in control mice, but not in obese mice, leptin infusion induced an increase in POMC mRNA level as well as in MC4‐R mRNA level suggesting that leptin could act directly and/or through α‐melanocyte‐stimulating hormone (α‐MSH). This hypothesis was reinforced after in vitro studies, using the mouse hypothalamic GT1‐7 cell line, since both leptin and Norleucine4, D‐Phenylalanine7‐α‐MSH (NDP‐α‐MSH) treatments increased MC4‐R expression. After 8 weeks of high‐fat diet, nondiabetic obese mice became resistant to the central action of leptin and their hypothalamic content of POMC and AgRP mRNA were decreased without modification of MC4‐R mRNA level. After 16 weeks of high‐fat diet, mice exhibited more severe metabolic disorders with type 2 diabetes. Moreover, hypothalamic expression of MC4‐R was highly increased. In conclusion, several alterations of the melanocortin system were found in obese mice that are probably consecutive to their central resistance to leptin. Moreover, when the metabolic status is highly degraded (with all characteristics of a type 2 diabetes), other regulatory mechanisms (independent of leptin) can also take place.

Metabolic and melanocortin gene expression alterations in male offspring of obese mice.

To study the consequences of maternal obesity during gestation and suckling periods on metabolic features and expression of genes belonging to the melanocortinergic system, we developed Diet-Induced-Obesity (DIO) in mice fed high-fat-diet (HFD). After weaning, F1-descendants were fed the same diet than dams up to 16 weeks or received a 2-week standard chow at several time points. From birth, F1-DIO displayed higher body weight than F1-control. Hyperinsulinemia, hypertriglyceridemia, hyperleptinemia were detected from P10 and fasting hyperglycaemia from 2 week-post-weaning. From late gestation to 16-week-post-weaning the expression of MC4-R gene and/or the POMC/AgRP ratio was increased, suggesting an activation of this pathway to compensate the deleterious effects of HFD. Standard chow replacement at weaning normalized metabolic status but a partial recovery was obtained for later changes. Concomitant variations in the expression of the melanocortinergic genes were observed. Therefore, early nutritional intervention could override the impact of maternal and postnatal over-nutrition.

Regulation of CYP11A gene expression in bovine ovarian granulosa cells in primary culture by cAMP and phorbol esters is conferred by a common cis-acting element.

Production and secretion of steroid hormones throughout the ovarian cycle occurs in a highly episodic and coordinated fashion that requires precise and finely tuned regulatory mechanisms. The regulation of ovarian steroidogenesis by the gonadotropin follicle stimulating hormone (FSH) and luteinizing hormone (LH) as well as by other factors occurs, at least in part, at the level of expression of the genes encoding steroidogenic enzymes. The present study is aimed at the elucidation of regulatory mechanisms by which cyclic adenosine monophosphate (cAMP) and protein kinase C regulate cytochrome P450scc (CYP11A) gene expression in bovine granulosa cells in primary culture. As a first step we characterized the bovine granulosa cell cultures with regard to regulation of P450scc activity and mRNA levels upon treatment with forskolin and/or the phorbol ester TPA. Forskolin, a potent stimulator of cAMP generation, increased both progesterone secretion and P450scc mRNA levels. In contrast, treatment with TPA alone decreased both basal progesterone production and P450scc mRNA accumulation. Co-treatment with forskolin and TPA decreased progesterone and P450scc mRNA levels as compared to forskolin treatment alone. The possibility that TPA interfered with the forskolin-stimulated cAMP production could be excluded because simultaneous treatment of granulosa cells with TPA and forskolin potentiated the formation of cAMP. In order to identify regulatory sequences within the 5 flanking region of the bovine CYP11A gene, chimeric DNA constructs comprizing regions of the CYP11A gene fused to a beta-globin-derived reporter gene were transfected into granulosa cells in primary culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Agouti-related protein in adrenal steroidogenesis.

The levels of Agouti-related protein (AgRP) mRNA in the adrenal are second only to those in the hypothalamus, raising questions regarding its target binding sites and its specific role in adrenal steroidogenesis. We and others demonstrated the presence of a population of melanocortin receptor-4 (MC4R) positively coupled to steroidogenesis in adrenal cells. Moreover, AgRP inhibited both the acute and long-term steroidogenic responses of these cells to NDP-alphaMSH through its antagonistic properties towards MC4R. Although AgRP had no antagonistic properties towards the MC2R and did not modify the acute steroidogenic response to ACTH, it exerted a biphasic sustained inhibitory effect on the long-term response to ACTH through an undefined alternate mechanism. Since adrenal cells release a relatively large amount of AgRP, this protein likely exerts a local paracrine/autocrine control on adrenal steroidogenesis.