Danielle Naville

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 ∼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.

Mutant WD-repeat protein in triple-A syndrome

Triple-A syndrome (MIM 231550; also known as Allgrove syndrome) is an autosomal recessive disorder characterized by adrenocorticotropin hormone (ACTH)-resistant adrenal insufficiency, achalasia of the oesophageal cardia and alacrima. Whereas several lines of evidence indicate that triple-A syndrome results from the abnormal development of the autonomic nervous system, late-onset progressive neurological symptoms (including cerebellar ataxia, peripheral neuropathy and mild dementia) suggest that the central nervous system may be involved in the disease as well. Using fine-mapping based on linkage disequilibrium in North African inbred families, we identified a short ancestral haplotype on chromosome 12q13 (<1 cM), sequenced a BAC contig encompassing the triple-A minimal region and identified a novel gene (AAAS) encoding a protein of 547 amino acids that is mutant in affected individuals. We found five homozygous truncating mutations in unrelated patients and ascribed the founder effect in North African families to a single splice-donor site mutation that occurred more than 2,400 years ago. The predicted product of AAAS, ALADIN (for alacrima-achalasia-adrenal insufficiency neurologic disorder), belongs to the WD-repeat family of regulatory proteins, indicating a new disease mechanism involved in triple-A syndrome. The expression of the gene in both neuroendocrine and cerebral structures points to a role in the normal development of the peripheral and central nervous systems.

Regulation of corticotropin receptor number and messenger RNA in cultured human adrenocortical cells by corticotropin and angiotensin II.

The regulation of ACTH receptor binding sites and mRNA by ACTH and angiotensin II (A-II) was studied using cultured human adrenal fasciculata reticularis cells (HAC). These cells expressed two major ACTH receptor transcripts of 1.8 and 3.4 kb and three minor ones of 4, 7, and 11 kb. ACTH increased the levels of all these transcripts in a time- and dose-dependent manner. At a maximal concentration of 10(-8) M, ACTH enhanced 21- and 4-fold the level of ACTH receptor mRNA and the number of receptors per cell, respectively. Pretreatment of HAC with A-II produced a dose-dependent enhancement of ACTH receptor mRNA that was associated with an increase of both ACTH receptor number and responsiveness to this hormone. The effects of A-II were completely blocked by an AT1 receptor subtype antagonist but not by an AT2 antagonist. The effects of ACTH together with A-II on ACTH receptor mRNA were greater than those induced by each hormone alone. These results show that ACTH receptor number and mRNA are positively regulated by the two main hormones (ACTH and A-II) which, in vivo, regulate adrenocortical functions. In addition, they also show that HAC are a target for A-II. Thus, regulation of ACTH receptors may be one mechanism by which ACTH and A-II regulate adrenocortical functions under both normal and pathological conditions.

Chronic Consumption of Farmed Salmon Containing Persistent Organic Pollutants Causes Insulin Resistance and Obesity in Mice

Background Dietary interventions are critical in the prevention of metabolic diseases. Yet, the effects of fatty fish consumption on type 2 diabetes remain unclear. The aim of this study was to investigate whether a diet containing farmed salmon prevents or contributes to insulin resistance in mice. Methodology/Principal Findings Adult male C57BL/6J mice were fed control diet (C), a very high-fat diet without or with farmed Atlantic salmon fillet (VHF and VHF/S, respectively), and Western diet without or with farmed Atlantic salmon fillet (WD and WD/S, respectively). Other mice were fed VHF containing farmed salmon fillet with reduced concentrations of persistent organic pollutants (VHF/S-POPs). We assessed body weight gain, fat mass, insulin sensitivity, glucose tolerance, ex vivo muscle glucose uptake, performed histology and immunohistochemistry analysis, and investigated gene and protein expression. In comparison with animals fed VHF and WD, consumption of both VHF/S and WD/S exaggerated insulin resistance, visceral obesity, and glucose intolerance. In addition, the ability of insulin to stimulate Akt phosphorylation and muscle glucose uptake was impaired in mice fed farmed salmon. Relative to VHF/S-fed mice, animals fed VHF/S-POPs had less body burdens of POPs, accumulated less visceral fat, and had reduced mRNA levels of TNFα as well as macrophage infiltration in adipose tissue. VHF/S-POPs-fed mice further exhibited better insulin sensitivity and glucose tolerance than mice fed VHF/S. Conclusions/Significance Our data indicate that intake of farmed salmon fillet contributes to several metabolic disorders linked to type 2 diabetes and obesity, and suggest a role of POPs in these deleterious effects. Overall, these findings may participate to improve nutritional strategies for the prevention and therapy of insulin resistance.

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.

Somatomedin-C/insulin-like growth factor 1-like material secreted by porcine sertoli cells in vitro: Characterization and regulation

Conditioned medium from pig Sertoli cells cultured in a chemically defined medium containing 3H-leucine contains a peptide with properties similar to that of human purified plasma and recombinant DNA somatomedin-C/insulin-like growth factor 1 (Sm-C/IGF-1). Purification of this peptide was achieved by affinity chromatography using mouse monoclonal anti-Sm-C/IGF-1 antibodies and reverse-phase high pressure liquid chromatography on a Bondapak C18 column. Polyacrylamide gel electrophoresis of the purified material gives a single spot after staining or in the autoradiogram, with identical molecular weight to that of pure human Sm-C/IGF-1. The purified peptide behaves like both the pure and recombinant DNA Sm-C/IGF-1 in the specific RIA and/or radioreceptor assays. Under basal conditions the amount of Sm-C/IGF-1 secreted by Sertoli cells was about 4 ng/10(6) cells/24 h, but it decreased during culture. Neither growth hormone nor follitropin were able to stimulate Sm-C/IGF-1 secretion, but both fibroblast growth factor and epidermal growth factor enhanced two- to three-fold its secretion. In addition, cells pretreated for 24 h with these growth factors became sensitive to the stimulatory effect of FSH. Since previous in vitro studies have shown that Sm-C/IGF-1 is a mitogenic and differentiating factor for both Sertoli and Leydig cells, it is possible that Sm-C/IGF-1 secreted by Sertoli cells might play a paracrine and/or autocrine role in the regulation of testicular function.

Control of production of insulin-like growth factor I by pig Leydig and Sertoli cells cultured alone or together. Cell-cell interactions

The production of insulin-like growth factor I (IGF-I) by pig Leydig cells and pig Sertoli cells cultured alone or together was investigated. Human chorionic gonadotropin (hCG) and basic fibroblast growth factor (FGF) stimulate in a dose-dependent manner IGF-I production by Leydig cells. At maximal concentrations the effects of both factors were almost additive. Insulin at micromolar concentrations enhanced IGF-I production and potentiated the effects of hCG and FGF. The secretion of IGF-I by Sertoli cells was stimulated by FSH and FGF. Under basal conditions, the production of IGF-I by the coculture was similar to the addition of the production by each cell cultured alone. In contrast, in the presence of hCG, FSH or FGF, the production of IGF-I by the coculture largely exceeded that expected from the monocultures. Moreover, stimulation of the coculture with both hCG and FSH resulted in a further increase in IGF-I production. These results indicate that Leydig as well as Sertoli cells secrete IGF-I and that the secretion of both cell types is stimulated by the corresponding gonadotropin. In addition, they indicate that Leydig-Sertoli interactions play a role in the control of IGF-I production, supporting the contention that this growth factor plays an important role in the paracrine and autocrine control of testicular functions.

Regulation of corticotropin and steroidogenic enzyme mRNAs in human fetal adrenal cells by corticotropin, angiotensin-II and transforming growth factor beta 1.

Using cultured human fetal adrenal cells, we have investigated the basal secretion of cortisol and dehydroepiandrosterone sulfate (DHAS) and the effect of corticotropin (ACTH), angiotensin-II (A-II) and transforming growth factor beta 1 (TGF beta 1) on the secretion of these steroids and on the mRNA levels of ACTH receptor (ACTHR), cytochrome P-450scc (cholesterol side-chain cleavage), P450 17 alpha (17 alpha-hydroxylase/17-20 lyase) and 3 beta-HSD (3 beta-hydroxysteroid dehydrogenase). The basal DHAS/cortisol ratio declined progressively between 12.5 and 21 weeks. ACTH treatment enhanced the secretion of cortisol and to a lesser extent that of DHAS, and increased the steroidogenic response to an acute stimulation with ACTH. These changes were associated with increased mRNA levels of ACTHR and of the steroidogenic enzymes. A-II treatment also increased the secretion of both DHAS and cortisol, but less than ACTH, enhanced the responsiveness to ACTH and increased ACTHR, P450scc and P450 17 alpha mRNA levels. In contrast, TGF beta 1 alone or together with ACTH decreased DHAS secretion, but not cortisol secretion. Moreover, TGF beta 1 had no effect on ACTHR and P450scc mRNA levels, decreased by about 50% the mRNA levels of P450 17 alpha both in the absence or presence of ACTH, but enhanced the stimulatory effects of ACTH on 3 beta-HSD mRNA. These results, along with those previously reported, suggest that both A-II and TGF beta may play a role in fetal adrenal function. In addition, they show that the effects of both peptides are qualitatively different from, even sometimes opposite to, those previously reported in bovine and ovine adrenal cells.

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.

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.—Naville, D., Pinteur, C., Vega, N., Menade, Y., Vigier, M., Le Bourdais, A., Labaronne, E., Debard, C., Luquain‐Costaz, C., Bégeot, M., Vidal, H., Le Magueresse‐Battistoni, B., Low‐dose food contaminants trigger sex‐specific, hepatic metabolic changes in the progeny of obese mice. FASEB J. 27, 3860–3870 (2013). www.fasebj.org