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.

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.

beta-Nerve growth factor participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.

NGF appears to be involved in spermatogenesis. However, mice lacking NGF or TrkA genes do not survive more than a few days whereas p75(NTR) knockout mice are viable and fertile. Therefore, we addressed the effect of betaNGF on spermatogenesis by using the systems of rat germ cell culture we established previously. betaNGF did not modify the number of Sertoli cells, pachytene spermatocytes, secondary spermatocytes nor the half-life of round spermatids, but increased the number of secondary meiotic metaphases and decreased the number of round spermatids formed in vitro. These effects of betaNGF were reversible and maximal at about 4 x 10(-11) M. Conversely, K252a, a Trk-specific kinase inhibitor, enhanced the number of round spermatids above that of control cultures. The presence of betaNGF and its receptors TrkA and p75(NTR) was investigated in testis sections, in Sertoli cell and germ cell fractions, and in germ cell and Sertoli cell co-cultures. betaNGF was detected only in germ cells from pachytene spermatocytes of stages VII up to spermatids of stages IX-X. TrkA and p75(NTR) were detected in Sertoli cells and in these germ cells. Taken together, these results indicate that betaNGF should participate in an auto/paracrine pathway of regulation of the second meiotic division of rat spermatocytes in vivo.

Transforming growth factor beta-1 decreases the yield of the second meiotic division of rat pachytene spermatocytes in vitro.

BACKGROUND: TGF beta and its receptors are present in both germ cells and somatic cells of the male gonad. However, knock-out strategies for studying spermatogenesis regulation by TGF beta have been disappointing since TGF beta-or TGF beta receptor-null mice do not survive longer than a few weeks. METHODS: In the present study, we addressed the role of TGF beta-1 on the completion of meiosis by rat pachytene spermatocytes (PS) cocultured with Sertoli cells. Identification and counting of meiotic cells were performed by cytology and cytometry. RESULTS: Under our culture conditions, some PS differentiated into round spermatids (RS). When TGF beta-1 was added to the culture medium, neither the number of PS or of secondary spermatocytes nor the half-life of RS was modified by the factor. By contrast, the number of RS and the amount of TP1 mRNA were lower in TGF beta-1-treated cultures than in control cultures. Very few metaphase I cells were ever observed both in control and TGF beta-1-treated wells. Higher numbers of metaphase II were present and their number was enhanced by TGF beta-1 treatment. A TGF beta-like bioactivity was detected in control culture media, the concentration of which increased with the time of culture. CONCLUSION: These results indicate that TGF beta-1 did not change greatly, if any, the yield of the first meiotic division but likely enhanced a bottleneck at the level of metaphase II. Taken together, our results suggest strongly that TGF beta participates in an auto/paracrine pathway of regulation of the meiotic differentiation of rat spermatocytes.