Low-dose food contaminants trigger sex-specific, hepatic metabolic changes in the progeny of obese mice.

Environmental contaminants are suspected to be involved in the epidemic incidence of metabolic disorders, food ingestion being a primarily route of exposure. We hypothesized that life-long consumption of a high-fat diet that contains low doses of pollutants will aggravate metabolic disorders induced by obesity itself. Mice were challenged from preconception throughout life with a high-fat diet containing pollutants commonly present in food (2,3,7,8-tetrachlorodibenzo-p-dioxin, polychlorinated biphenyl 153, diethylhexyl phthalate, and bisphenol A), added at low doses in the tolerable daily intake range. We measured several blood parameters, glucose and insulin tolerance, hepatic lipid accumulation, and gene expression in adult mice. Pollutant-exposed mice exhibited significant sex-dependent metabolic disorders in the absence of toxicity and weight gain. In males, pollutants increased the expression of hepatic genes (from 36 to 88%) encoding proteins related to cholesterol biosynthesis and decreased (40%) hepatic total cholesterol levels. In females, there was a marked deterioration of glucose tolerance, which may be related to the 2-fold induction of estrogen sulfotransferase and reduced expression of estrogen receptor α (25%) and estrogen target genes (>34%). Because of the very low doses of pollutants used in the mixture, these findings may have strong implications in terms of understanding the potential role of environmental contaminants in food in the development of metabolic diseases.

Link between intestinal CD36 ligand binding and satiety induced by a high protein diet in mice.

CD36 is a ubiquitous membrane glycoprotein that binds long-chain fatty acids. The presence of a functional CD36 is required for the induction of satiety by a lipid load and its role as a lipid receptor driving cellular signal has recently been demonstrated. Our project aimed to further explore the role of intestinal CD36 in the regulation of food intake. Duodenal infusions of vehicle or sulfo-N-succinimidyl-oleate (SSO) was performed prior to acute infusions of saline or Intralipid (IL) in mice. Infusion of minute quantities of IL induced a decrease in food intake (FI) compared to saline. Infusion of SSO had the same effect but no additive inhibitory effect was observed in presence of IL. No IL- or SSO-mediated satiety occurred in CD36-null mice. To determine whether the CD36-mediated hypophagic effect of lipids was maintained in animals fed a satietogen diet, mice were subjected to a High-Protein diet (HPD). Concomitantly with the satiety effect, a rise in intestinal CD36 gene expression was observed. No satiety effect occurred in CD36-null mice. HPD-fed WT mice showed a diminished FI compared to control mice, after saline duodenal infusion. But there was no further decrease after lipid infusion. The lipid-induced decrease in FI observed on control mice was accompanied by a rise in jejunal oleylethanolamide (OEA). Its level was higher in HPD-fed mice than in controls after saline infusion and was not changed by lipids. Overall, we demonstrate that lipid binding to intestinal CD36 is sufficient to produce a satiety effect. Moreover, it could participate in the satiety effect induced by HPD. Intestine can modulate FI by several mechanisms including an increase in OEA production and CD36 gene expression. Furthermore, intestine of mice adapted to HPD have a diminished capacity to modulate their food intake in response to dietary lipids.

Direct and indirect impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on adult mouse Leydig cells: an in vitro study.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related substances are ubiquitous environmental pollutants that exert adverse effects on reproductive processes. In testis, Leydig cells which produce testosterone are under hormonal and local control exerted by cytokines including TNFα. Using mouse Leydig primary cell cultures as a model, we studied the effects of TCDD on the steroidogenic outcome of Leydig cells and the gene expression levels of Ccl5 and Cxcl4, previously shown to be target genes of TCDD in testis. We found that TCDD did not alter the steroidogenic outcome of Leydig cells but that it up-regulated Cxcl4 gene expression levels. TCDD also impacted Ccl5 gene expression when cells had been co-treated with TNFα. TCDD action probably initiated with binding to the aryl hydrocarbon receptor (AhR) present on Leydig cells. TCDD regulated the gene expression levels of AhR (transient down-regulation) and its repressor AhRR and Cyp1b1 (up-regulation). The trophic human chorionic gonadotropin (hCG) hormone did not impact AhR, its repressor AhRR or Cyp1b1 but it opposed the TCDD-enhanced AhRR mRNA levels. Conversely, TNFα stimulated AhR gene expression levels. Collectively, it is suggested that the impact of TCDD on expression of target genes in Leydig cells may operate under the complex network of hormones and cytokines.

Role of hypothalamic melanocortin system in adaptation of food intake to food protein increase in mice.

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.

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.

Nonclassic lipoid congenital adrenal hyperplasia masquerading as familial glucocorticoid deficiency.

CONTEXT: Familial glucocorticoid deficiency (FGD) is an autosomal recessive disorder resulting from resistance to the action of ACTH on the adrenal cortex. Affected individuals are deficient in cortisol and, if untreated, are likely to succumb to hypoglycemia and/or overwhelming infection. Mutations of the ACTH receptor (MC2R) and the melanocortin 2 receptor accessory protein (MRAP), FGD types 1 and 2 respectively, account for approximately 45% of cases. OBJECTIVE: A locus on chromosome 8 has previously been linked to the disease in three families, but no underlying gene defect has to date been identified. DESIGN: The study design comprised single-nucleotide polymorphism genotyping and mutation detection. SETTING: The study was conducted at secondary and tertiary referral centers. PATIENTS: Eighty probands from families referred for investigation of the genetic cause of FGD participated in the study. INTERVENTIONS: There were no interventions. RESULTS: Analysis by single-nucleotide polymorphism array of the genotype of one individual with FGD previously linked to chromosome 8 revealed a large region of homozygosity encompassing the steroidogenic acute regulatory protein gene, STAR. We identified homozygous STAR mutations in this patient and his affected siblings. Screening of our total FGD patient cohort revealed homozygous STAR mutations in a further nine individuals from four other families. CONCLUSIONS: Mutations in STAR usually cause lipoid congenital adrenal hyperplasia, a disorder characterized by both gonadal and adrenal steroid deficiency. Our results demonstrate that certain mutations in STAR (R192C and the previously reported R188C) can present with a phenotype indistinguishable from that seen in FGD.

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 alpha-melanocyte-stimulating hormone (alpha-MSH). This hypothesis was reinforced after in vitro studies, using the mouse hypothalamic GT1-7 cell line, since both leptin and Norleucine(4), D-Phenylalanine(7)-alpha-MSH (NDP-alpha-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.

A novel inhibitory protein in adipose tissue, the aldo-keto reductase AKR1B7: its role in adipogenesis.

The aldo-keto reductase 1B7 (AKR1B7) encodes an aldose-reductase that has been reported as a detoxification enzyme until now. We have demonstrated that AKR1B7 is differently expressed in various mouse white adipose tissues depending on their location. Its expression is associated with a higher ratio of preadipocytes vs. adipocytes. The cells that express AKR1B7 did not contain lipid droplets, and the expression level of akr1b7 was very low in mature adipocytes. We have defined the role of AKR1B7 in adipogenesis using either primary cultures of adipose stromal cells (containing adipocyte precursors) or the 3T3-L1 cell line. Under the same differentiation conditions, adipose stromal cells from tissues that expressed AKR1B7 had a decreased capacity to accumulate lipids compared with those that did not express it. Moreover, the overexpression of sense or antisense AKR1B7 in 3T3-L1 preadipocytes inhibited or accelerated, respectively, their rate of differentiation into adipocytes. In vivo experiments demonstrated that AKR1B7-encoding mRNA expression decreased in adipose tissues from mice where obesity was induced by a high-fat diet. All these results attributed for the first time a novel role to AKR1B7, which is the inhibition of adipogenesis in some adipose tissues.

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.

Compound heterozygosity of a frameshift mutation in the coding region and a single base substitution in the promoter of the ACTH receptor gene in a family with isolated glucocorticoid deficiency.

Isolated glucocorticoid deficiency (IGD) is an autosomal recessive syndrome characterized by glucocorticoid insufficiency without mineralocorticoid deficiency. Mutations in the coding region of the ACTH receptor (MC2R) have been reported in several families with IGD. We amplified and sequenced the entire MC2R coding region in a new family with IGD. The proband was found to be heterozygous (paternal allele) for the mutation Gly217fs, which changes the open reading frame of the MC2R protein resulting in a truncated receptor. No other abnormality was found in the MC2R coding region. However, sequencing of the promoter region of the MC2R gene (-1017/44 bp) of the proband revealed a heterozygous T-->C substitution in the maternal allele at -2 bp position from initiation of the transcription start site. This substitution was found in only 6.5% in a healthy unrelated population. Constructs containing this polymorphism consistently showed a significant 15% decrease in promoter activity compared to wild type. In conclusion, we provide evidence that the IGD in this previously unreported family with ACTH resistance appears to be secondary to compound heterozygosity of a coding region and a promoter mutation in the MC2R gene.

CCAAT/enhancer-binding proteins (C/EBPs) regulate the basal and cAMP-induced transcription of the human 11beta-hydroxysteroid dehydrogenase encoding gene in adipose cells.

Android obesity is often associated with a metabolic syndrome characterized, in particular, by a type 2 diabetes and cardiovascular problems. This could be induced by an excess of local production of glucocorticoids (GC) by adipose tissue (or other tissues). This production of GC by its target tissues depends on the 11beta-hydroxysteroid dehydrogenase type 1 (11betaHSD1) enzyme. Our aim was to characterize some mechanisms which control the expression of the human 11betaHSD1 gene (hHSD11B1) in preadipocytes. By using different luciferase constructs containing fragments of the hHSD11B1 promoter, we demonstrate that two members of the CCAAT/enhancer-binding protein family, C/EBPalpha and C/EBPbeta, are required for the basal transcriptional activity of HSD11B1 in 3T3-L1 preadipocyte cells. This effect depends on the binding of each isoform to specific binding sites. Mutation of either one of these sites induced a 40-50% decrease of the constitutive activity of the hHSD11B1 promoter. A forskolin treatment of 3T3-L1 preadipocyte cells induced an increased endogenous expression of HSD11B1. By transfection studies using the hHSD11B1 luciferase constructs, it appears that C/EBPbeta was strongly involved in this induction, as the forskolin stimulation was suppressed after mutation of the C/EBPbeta binding site. Part of the mechanism involved the increase of nuclear C/EBPbeta protein levels induced by forskolin and a phosphorylation step associated with an enhanced binding of the transcription factor to its site. These data indicate that members of the C/EBP family control intracellular levels of GC in preadipocytes via the regulation of the constitutive and cAMP-dependent expressions of HSD11B1.

Expression of the human melanocortin-2 receptor in different eukaryotic cells.

The human melanocortin-2 receptor (hMC2R) is mainly present in the adrenal cortex and has been difficult to express in heterologous cells. The hMC2R fused to the EGFP at its C-terminus has been stably transfected in the murine M3 melanoma and HEK293 cells. In the M3 cells, the hMC2R-EGFP was well-addressed to the cell membrane and functional whereas in the HEK293 cells, the hMC2R-EGFP was retained intracellularly. These results suggest that some specific factors, missing in cells, which do not express any melanocortin receptor, are involved in the correct addressing of the hMC2R to the cell membrane.

Leptin modulates both resorption and formation while preventing disuse-induced bone loss in tail-suspended female rats.

In vitro studies have demonstrated leptin-positive effects on the osteoblast lineage and negative effects on osteoclastogenesis. Therefore, we tested the hypothesis that leptin may prevent tail-suspension-induced bone loss characterized by an uncoupling pattern of bone remodeling, through both mechanisms. Female rats were randomly tail-suspended or not and treated either with ip administration of leptin or vehicle for 3, 7, and 14 d. As measured by dual energy x-ray absorptiometry, tail-suspension induced a progressive decrease in tibia-metaphysis bone mineral density, which was prevented by leptin. Histomorphometry showed that this was related to the prevention of the transient increase in osteoclast number observed with suspension at d 7. These effects could be mediated by the receptor activator of nuclear factor kappaB-ligand (RANKL)/osteoprotegerin (OPG) pathway since we observed using direct RT-PCR, a suspension-induced increase in RANKL gene expression in proximal tibia at d 3, which was counterbalanced by leptin administration with a similar 3-fold increase in OPG expression and a RANKL to OPG ratio close to nonsuspended conditions. In addition, leptin prevented the decrease in bone formation rate induced by tail-suspension at d 14. The latter could be related to the role of leptin in mediating the reciprocal differentiation between adipocytes and osteoblasts, because leptin concurrently blunted the disuse-induced increase in bone marrow adipogenesis. In summary, these data suggest that peripheral administration of leptin could prevent disuse-induced bone loss through, first, a major inhibitory effect on bone resorption and, second, a delayed effect preventing the decrease in bone formation.

Mutations in MRAP, encoding a new interacting partner of the ACTH receptor, cause familial glucocorticoid deficiency type 2.

Familial glucocorticoid deficiency (FGD), or hereditary unresponsiveness to adrenocorticotropin (ACTH; OMIM 202200), is an autosomal recessive disorder resulting from resistance to the action of ACTH on the adrenal cortex, which stimulates glucocorticoid production. Affected individuals are deficient in cortisol and, if untreated, are likely to succumb to hypoglycemia or overwhelming infection in infancy or childhood. Mutations of the ACTH receptor (melanocortin 2 receptor, MC2R) account for approximately 25% of cases of FGD. FGD without mutations of MC2R is called FGD type 2. Using SNP array genotyping, we mapped a locus involved in FGD type 2 to chromosome 21q22.1. We identified mutations in a gene encoding a 19-kDa single-transmembrane domain protein, now known as melanocortin 2 receptor accessory protein (MRAP). We show that MRAP interacts with MC2R and may have a role in the trafficking of MC2R from the endoplasmic reticulum to the cell surface.

Sustained inhibitory effect of Agouti Related Protein on the ACTH-induced cortisol production by bovine cultured adrenal cells.

The adrenal gland is the second tissue after hypothalamus exhibiting high expression level of Agouti Related Protein (Agrp) mRNA, which suggests that this peptide may control adrenal cell functions. However, its role in this tissue remained to be determined. In this report, we studied the effects of a long-term treatment (24 h) of cultured bovine adrenal cells by Agrp on the (Nle4, d-Phe7)-alphaMSH (NDP-alphaMSH)- or ACTH-induced cortisol release. We showed that Agrp inhibited, in a dose-dependent manner, the 10(-9) M NDP-alphaMSH-induced cortisol production through its antagonistic properties towards MSH at the level of MC4-R. Surprisingly, we found that Agrp in the same conditions of cell treatment also induced a strong inhibition of the ACTH-induced cortisol release. These effects were stronger using low concentrations of Agrp and disappeared for higher concentrations resulting in U-shaped curve data. There was no effect of SHU9119 in the same conditions of stimulation of the cells. Our data confirmed that Agrp is not an antagonist of ACTH at the level of MC2-R and that its sustained effect on ACTH-induced steroidogenesis did not involve its antagonistic properties at the level of MC4-R. The hypothesis would be that Agrp is acting on adrenal steroidogenesis through an alternate mechanism.

Inactivation and intracellular retention of the human I183N mutated melanocortin 3 receptor associated with obesity.

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G protein-coupled receptors (GPCRs) by stimulating adenylate cyclase. The melanocortin 3 receptor (MC3R) in the melanocortin receptor (MCR) family has been identified as a neural receptor subtype mainly expressed in the brain in mammals. Until now, only one heterozygous mutation (I183N) has been identified in the coding region of this receptor in two obese patients of the same family. In this study, we reported the functional characterization of the I183N mutated MC3R compared with that of the wild-type MC3R after transfection in HEK293 cells. Our results showed that the I183N mutation totally abolished the activity of the mutated receptor to generate intracellular cAMP. Furthermore, confocal microscopy observation revealed that the mutation induced an intracellular retention of the mutated receptor. Moreover, we demonstrated for the first time by co-transfection studies that the mutated receptor could reduce the wild-type receptor activity through a dominant negative effect.

Characterization of cell lines stably expressing human normal or mutated EGFP-tagged MC4R.

The melanocortin receptor type 4 (MC4-R) is involved in food intake and represents a potential target for the treatment of some forms of obesity. The fluorescent protein EGFP was fused to the wild-type or mutated coding sequence of the human MC4-R. After transfection in HEK 293, clones stably expressing hMC4-R-EGFP were selected. Wild-type chimeric hMC4-R was well addressed to the cell membrane as demonstrated using confocal microscopy and displayed the same pharmacological characteristics as native hMC4R. NDP-alpha MSH induced a time-dependent internalization of MC4-R that was partially prevented by AgRP. The two mutated chimeric receptors studied here (CTCT-deleted and C271A) showed a high alteration of their response to ligand and were retained inside the cells. In conclusion, we have developed a model of clones stably expressing EGFP-tagged-hMC4-R. This is the only such model available to date and it provides a useful tool to follow the trafficking of MC4-R inside living cells.

Agouti-related protein antagonizes glucocorticoid production induced through melanocortin 4 receptor activation in bovine adrenal cells: a possible autocrine control.

Agouti-related protein (Agrp), primarily expressed in the hypothalamus, is an endogenous antagonist of alphaMSH at the level of melanocortin 3 receptor (MC3-R) and MC4-R, but the adrenal gland represents the second major Agrp-expressing tissue. In adrenal fasciculata cells, the glucocorticoid secretion is under the control of ACTH, which binds specifically MC2-R, the only functional melanocortin receptor described in these cells to date. Nevertheless, using cultured bovine fasciculata adrenal cells, we report that Agrp has no antagonistic properties against ACTH at the level of MC2-R. In our studies, (Nle4, d-Phe7)-alphaMSH (NDP-alphaMSH) stimulated the production of cortisol in a dose-dependent manner, and these effects were abolished by Agrp or SHU9119, a synthetic antagonist of MC3-R and MC4-R. Using a more specific antagonist (JKC-363) and RT-PCR analysis, we can postulate that the effects of NDP-alphaMSH were mediated via MC4-R. These results are suggestive that adrenal glucocorticoid production could be regulated through MC4-R that may have some relevance in the physiology of adrenal cells. Moreover, Agrp might exert an autocrine control on adrenal cells because a protein with biological Agrp-like activity is secreted by these cells. This peptide could then modulate locally the functions of some peripheral tissues such as adrenals.

Stable expression of human melanocortin 3 receptor fused to EGFP in the HEK293 cells.

Among the melanocortins alpha-MSH is known to be involved in feeding behavior. These hormones mediate their effects through G protein-coupled receptors by stimulating adenylate cyclase. In this study, we have developed an in vitro expression model for human melanocortin 3 receptor (hMC3R) tagged at its C terminus with EGFP. The corresponding chimeric cDNA was stably expressed in HEK293 cells. The selected clones expressing the hMC3R-EGFP exhibited cell surface fluorescence and responded to NDP-MSH stimulation by producing cAMP in a dose-dependent manner (EC(50): 0.3 nM). Binding studies revealed a single class of binding sites with a K(D) of 2.24 nM. Moreover, Agouti-related protein was also demonstrated to be an antagonist of the hMC3R-EGFP. Thus, the hMC3R tagged with EGFP stably expressed in HEK293 cells, exhibiting the same characteristics than the wild-type hMC3R, is the only model of expression of this receptor allowing its direct localization inside living cells.