Marc Lombès

Immunohistochemical and biochemical evidence for a cardiovascular mineralocorticoid receptor.

The presence of mineralocorticoid receptors (MRs) and their physicochemical characteristics were investigated in the heart and blood vessels of rabbits. Immunohistochemical methods using the monoclonal anti-idiotypic antibody H10E, which interacts with the steroid binding domain of MRs, revealed the presence of immunoreactive material in the heart and large blood vessels. In the heart, a positive staining was observed in myocytes and endothelial cells of atria and ventricles. In vessels, MRs were detected in the aorta and pulmonary artery. They were localized in endothelial and vascular smooth muscle cells. No staining was present in the small vascular bed, arterioles, and capillaries. In all these studies, the mineralocorticoid specificity of the staining was assessed by in situ competition experiments with aldosterone and RU486, a glucocorticoid antagonist. The presence of MRs in the heart and vessels was further demonstrated by specific aldosterone binding to one class of high affinity binding sites in the cytosol of the adrenalectomized rabbit heart (Kd, 0.25 nM; maximum MR concentration, 15-20 fmol/mg protein), whose mineralocorticoid specificity has been clearly established by competition studies. Sedimentation gradient analyses revealed that the cardiovascular MR is an 8.5S hetero-oligomer that includes the heat shock protein 90. The physicochemical characteristics of the cardiovascular MRs are virtually identical to those of the renal MRs. Altogether, our results clearly demonstrate the presence of MRs in the cardiovascular system. This supports the possibility of direct aldosterone actions in the heart and blood vessels.

Prerequisite for Cardiac Aldosterone Action Mineralocorticoid Receptor and 11β-Hydroxysteroid Dehydrogenase in the Human Heart

BACKGROUND It has been proposed that aldosterone exerts direct effects on heart function, most notably on the development of myocardial fibrosis during ventricular hypertrophy in rat. Initial events in aldosterone action entail its binding to mineralocorticoid receptor (MR). Because MR displays similar affinities for aldosterone and glucocorticoids, the in vivo aldosterone selectivity of MR requires the presence of an enzyme, 11 beta-hydroxysteroid dehydrogenase (11-HSD), which metabolizes glucocorticoids into inactive derivatives. Although evidence exists for the presence of MR in rodent heart, no data are available for humans; moreover, the existence of cardiac 11-HSD is controversial. METHODS AND RESULTS The heart samples used originated from tissue removed during cardiac surgery in nontransplant patients or from endocavitary biopsies done for the follow-up of heart transplantation. The expression of MR was examined at the mRNA and protein level by in situ hybridization with cRNA probes specific for human MR mRNA and by immunodetection with two specific anti-MR antibodies. 11-HSD catalytic activity was determined by measurement of the metabolic rate of tritiated corticosteroids by cardiac samples. In nontransplanted hearts, an in situ hybridization signal equivalent to that found in the whole kidney was present on cardiomyocytes. Specific immunolabeling of cardiomyocytes with anti-MR antibodies demonstrated the presence of the MR protein. Cardiac 11-HSD activity was detected (243 +/- 26 fmol.30 min-1.mg protein-1) and was dependent on the cofactor NAD, not NADP, suggesting that it corresponds to the form of the enzyme specifically responsible for MR protection. In transplanted hearts that presented severe alterations, MR immunodetection was weaker and irregular, with no specific hybridization signal. CONCLUSIONS Our results demonstrate that MR is coexpressed with 11-HSD in human heart, which thus possesses the cellular machinery required for direct aldosterone action.

The G0/G1 Switch Gene 2 Regulates Adipose Lipolysis through Association with Adipose Triglyceride Lipase

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme for triacylglycerol (TAG) hydrolysis in adipocytes. The precise mechanisms whereby ATGL is regulated remain uncertain. Here, we demonstrate that a protein encoded by G(0)/G(1) switch gene 2 (G0S2) is a selective regulator of ATGL. G0S2 is highly expressed in adipose tissue and differentiated adipocytes. When overexpressed in HeLa cells, G0S2 localizes to lipid droplets and prevents their degradation mediated by ATGL. Moreover, G0S2 specifically interacts with ATGL through the hydrophobic domain of G0S2 and the patatin-like domain of ATGL. More importantly, interaction with G0S2 inhibits ATGL TAG hydrolase activity. Knockdown of endogenous G0S2 accelerates basal and stimulated lipolysis in adipocytes, whereas overexpression of G0S2 diminishes the rate of lipolysis in both adipocytes and adipose tissue explants. Thus, G0S2 functions to attenuate ATGL action both in vitro and in vivo and by this mechanism regulates TAG hydrolysis.

Pivotal role of the mineralocorticoid receptor in corticosteroid-induced adipogenesis

In addition to their role in controlling water and salt homeostasis, recent work suggests that aldosterone and mineralocorticoid receptors (MR) may be involved in adipocyte biology. This is of particular relevance given the role of MR as a high‐affinity receptor for both mineralocorticoids and glucocorticoids. We have thus examined the effect of aldosterone and MR on white adipose cell differentiation. When cells are cultured in a steroid‐free medium, aldosterone promotes acquisition of the adipose phenotype of 3T3‐L1 and 3T3‐F442A cells in a time‐, dose‐, and MR‐dependent manner. In contrast, late and long‐term exposure to dexamethasone inhibits adipocyte terminal maturation. The aldosterone effect on adipose maturation was accompanied by induction of PPARγ mRNA expression, which was blocked by the MR antagonist spironolactone. Under permissive culture conditions, specific MR down‐regulation by siRNAs markedly inhibited 3T3‐L1 differentiation by interfering with the transcriptional control of adipogenesis, an effect not mimicked by specific inactivation of the glucocorticoid receptor. These results demonstrate that MR represents an important proadipogenic transcription factor that may mediate both aldosterone and glucocorticoid effects on adipose tissue development. MR thus may be of pathophysiological relevance to the development of obesity and the metabolic syndrome.–Caprio, M., Fève, B., Claës, A., Viengchareun, S., Lombès, M., Zennaro, M‐C. Pivotal role of the mineralocorticoid receptor in corticosteroid‐induced adipogenesis. FASEB J. 21, 2185–2194 (2007)

Isolated Familial Hypogonadotropic Hypogonadism and a GNRH1 Mutation

We investigated whether mutations in the gene encoding gonadotropin-releasing hormone 1 (GNRH1) might be responsible for idiopathic hypogonadotropic hypogonadism (IHH) in humans. We identified a homozygous GNRH1 frameshift mutation, an insertion of an adenine at nucleotide position 18 (c.18-19insA), in the sequence encoding the N-terminal region of the signal peptide-containing protein precursor of gonadotropin-releasing hormone (prepro-GnRH) in a teenage brother and sister, who had normosmic IHH. Their unaffected parents and a sibling who was tested were heterozygous. This mutation results in an aberrant peptide lacking the conserved GnRH decapeptide sequence, as shown by the absence of immunoreactive GnRH when expressed in vitro. This isolated autosomal recessive GnRH deficiency, reversed by pulsatile GnRH administration, shows the pivotal role of GnRH in human reproduction.

The mineralocorticoid receptor: insights into its molecular and (patho)physiological biology

The last decade has witnessed tremendous progress in the understanding of the mineralocorticoid receptor (MR), its molecular mechanism of action, and its implications for physiology and pathophysiology. After the initial cloning of MR, and identification of its gene structure and promoters, it now appears as a major actor in protein-protein interaction networks. The role of transcriptional coregulators and the determinants of mineralocorticoid selectivity have been elucidated. Targeted oncogenesis and transgenic mouse models have identified unexpected sites of MR expression and novel roles for MR in non-epithelial tissues. These experimental approaches have contributed to the generation of new cell lines for the characterization of aldosterone signaling pathways, and have also facilitated a better understanding of MR physiology in the heart, vasculature, brain and adipose tissues. This review describes the structure, molecular mechanism of action and transcriptional regulation mediated by MR, emphasizing the most recent developments at the cellular and molecular level. Finally, through insights obtained from mouse models and human disease, its role in physiology and pathophysiology will be reviewed. Future investigations of MR biology should lead to new therapeutic strategies, modulating cell-specific actions in the management of cardiovascular disease, neuroprotection, mineralocorticoid resistance, and metabolic disorders.

Forkhead Transcription Factor FoxO1 in Adipose Tissue Regulates Energy Storage and Expenditure

OBJECTIVE—Adipose tissue serves as an integrator of various physiological pathways, energy balance, and glucose homeostasis. Forkhead box–containing protein O subfamily (FoxO) 1 mediates insulin action at the transcriptional level. However, physiological roles of FoxO1 in adipose tissue remain unclear. RESEARCH DESIGN AND METHODS—In the present study, we generated adipose tissue–specific FoxO1 transgenic mice (adipocyte protein 2 [aP2]-FLAG-Δ256) using an aP2 promoter/enhancer and a mutant FoxO1 (FLAGΔ256) in which the carboxyl terminal transactivation domain was deleted. Using these mice, we analyzed the effects of the overexpression of FLAGΔ256 on glucose metabolism and energy homeostasis. RESULTS—The aP2-FLAG-Δ256 mice showed improved glucose tolerance and insulin sensitivity accompanied with smaller-sized adipocytes and increased adiponectin (adipoq) and Glut 4 (Slc2a4) and decreased tumor necrosis factor α (Tnf) and chemokine (C-C motif) receptor 2 (Ccr2) gene expression levels in white adipose tissue (WAT) under a high-fat diet. Furthermore, the aP2-FLAG-Δ256 mice had increased oxygen consumption accompanied with increased expression of peroxisome proliferator–activated receptor γ coactivator (PGC)-1α protein and uncoupling protein (UCP)-1 (Ucp1), UCP-2 (Ucp2), and β3-AR (Adrb3) in brown adipose tissue (BAT). Overexpression of FLAGΔ256 in T37i cells, which are derived from the hibernoma of SV40 large T antigen transgenic mice, increased expression of PGC-1α protein and Ucp1. Furthermore, knockdown of endogenous FoxO1 in T37i cells increased Pgc1α (Ppargc1a), Pgc1β (Ppargc1b), Ucp1, and Adrb3 gene expression. CONCLUSIONS—These data suggest that FoxO1 modulates energy homeostasis in WAT and BAT through regulation of adipocyte size and adipose tissue–specific gene expression in response to excessive calorie intake.

Hibernoma development in transgenic mice identifies brown adipose tissue as a novel target of aldosterone action.

Aldosterone is a major regulator of salt balance and blood pressure, exerting its effects via the mineralocorticoid receptor (MR). To analyze the regulatory mechanisms controlling tissue-specific expression of the human MR (hMR) in vivo, we have developed transgenic mouse models expressing the SV40 large T antigen (TAg) under the control of each of the two promoters of the hMR gene (P1 or P2). Unexpectedly, all five P1-TAg founder animals died prematurely from voluminous malignant liposarcomas originating from brown adipose tissue, as evidenced by the expression of the mitochondrial uncoupling protein ucp1, indicating that the proximal P1 promoter was transcriptionally active in brown adipocytes. No such hibernoma occurred in P2-TAg transgenic mice. Appropriate tissue-specific usage of P1 promoter sequences was confirmed by demonstrating the presence of endogenous MR in both neoplastic and normal brown adipose tissue. Several cell lines were derived from hibernomas; among them, the T37i cells can undergo terminal differentiation into brown adipocytes, which remain capable of expressing ucp1 upon adrenergic or retinoic acid stimulation. These cells possess endogenous functional MR, thus providing a new model to explore molecular mechanisms of mineralocorticoid action. Our data broaden the known functions of aldosterone and suggest a potential role for MR in adipocyte differentiation and regulation of thermogenesis.

Brown adipocytes are novel sites of expression and regulation of adiponectin and resistin

Adiponectin and resistin, two recently identified adipocyte‐specific secretory factors, are able to modulate insulin actions in target tissues. To investigate their expression and hormonal regulation in brown adipocytes, we used the brown adipocyte cell line T37i, which, beside uncoupling protein expression, secretes leptin. Adiponectin and resistin mRNA were detected as a function of cell differentiation. Both transcripts were expressed at relatively high levels in differentiated T37i cells, reaching maximal levels on day 7, while resistin expression drastically fell afterwards. These stable transcripts (t 1/2>8 h) were differentially regulated by factors involved in insulin responsiveness. Insulin and thiazolidinedione, a peroxisome proliferator‐activated receptor γ agonist, stimulated resistin expression two‐ to four‐fold in differentiated T37i cells, whereas adiponectin mRNA levels increased 1.5–2‐fold. In contrast, dexamethasone and isoproterenol reduced by two‐fold the level of adiponectin and resistin transcripts in differentiated T37i cells. This study provides the first direct evidence that differentiated brown adipocytes are endocrine cells capable of expressing adiponectin and resistin. The complex hormonal regulation of their expression in brown adipocytes clearly differs from that reported in white adipose tissue, pointing to differential physiological and pathophysiological implications of brown fat in energy homeostasis.

Alteration of Cardiac and Renal Functions in Transgenic Mice Overexpressing Human Mineralocorticoid Receptor

The mineralocorticoid receptor (MR), a ligand-dependent transcription factor, mediates aldosterone actions in a large variety of tissues. To explore the functional implication of MR in pathophysiology, transgenic mouse models were generated using the proximal human MR (hMR) promoter to drive expression of hMR in aldosterone target tissues. Tissue-specific analysis of transgene expression in two independent transgenic animal (TG) lines by ribonuclease protection assays revealed that hMR is expressed in all mineralocorticoid-sensitive tissues, most notably in the kidney and the heart. TG exhibit both renal and cardiac abnormalities. Enlarged kidneys were histologically associated with renal tubular dilation and cellular vacuolization whose prevalence increased with aging. Renal clearance studies also disclosed a significant decrease in urinary potassium excretion rate in TG. hMR-expressing animals had normal blood pressure but developed mild dilated cardiomyopathy (increased left ventricle diameters and decreased shortening fraction), which was accompanied by a significant increase in heart rate. Differential gene expression analysis revealed a 2- to 5-fold increase in cardiac expression of atrial natriuretic peptide, serum- and glucocorticoid-induced kinase, and early growth response gene 1 as detected by microarrays; renal serum- and glucocorticoid-induced kinase was also induced significantly. Altogether, TG exhibited specific alteration of renal and cardiac functions, thus providing useful pathophysiological models to gain new insights into the tissue-specific mineralocorticoid signaling pathways.