The mineralocorticoid receptor (MR) controls adipocyte function, but its role in the conversion of white adipose tissue (WAT) into thermogenic fat has not been elucidated. We investigated responses to the MR antagonists spironolactone (spiro; 20 mg/kg/d) and drospirenone (DRSP; 6 mg/kg/d) in C57BL/6 mice fed a high-fat (HF) diet for 90 d. DRSP and spiro curbed HF diet-induced impairment in glucose tolerance, and prevented body weight gain and white fat expansion. Notably, either MR antagonist induced up-regulation of brown adipocyte-specific transcripts and markedly increased protein levels of uncoupling protein 1 (UCP1) in visceral and inguinal fat depots when compared with the HF diet group. Positron emission tomography and magnetic resonance spectroscopy confirmed acquisition of brown fat features in WAT. Interestingly, MR antagonists markedly reduced the autophagic rate both in murine preadipocytes in vitro (10(-5) M) and in WAT depots in vivo, with a concomitant increase in UCP1 protein expression. Moreover, the autophagy repressor bafilomycin A1 (10(-8) M) mimicked the effect of MR antagonists, increasing UCP1 protein expression in primary preadipocytes. Hence, we showed that adipocyte MR regulates brown remodeling of WAT through a modulation of autophagy. These results provide a rationale for the use of MR antagonists to prevent the adverse metabolic consequences of adipocyte dysfunction.

White adipose tissue (WAT) is no longer considered a depot for energy storage in the form of triglycerides, but is a secretory organ that releases factors, known as adipokines, capable of regulating several physiological processes. Alteration of WAT function with subsequent dysfunctional expression and secretion of adipokines plays a key role in the pathogenesis of obesity, diabetes, and other metabolic diseases. For this reason, a deeper understanding of the molecular mechanisms regulating adipocyte function is deemed necessary for planning strategies to treat and prevent obesity and its metabolic complications. This review examines cell culture models currently available for studying adipocyte biology. We focus on advantages, disadvantages and main differences between established preadipocyte cell lines and primary preadipocyte cultures. We revise protocols used to promote adipocyte differentiation and mature adipocytes dedifferentiation into preadipocytes. Finally, we briefly describe co-cultures of adipocytes with other cell types and three-dimensional adipocyte culture systems. These models allow investigation of cell-cell interactions with the cross-talk physiologically occurring between adipocytes and other cell types residing within or outside adipose tissue.

The mineralocorticoid receptor (MR) mediates aldosterone- and glucocorticoid-induced adipocyte differentiation. Drospirenone (DRSP) is a potent synthetic antimineralocorticoid with progestogenic and antiandrogenic properties, which is widely used for contraception and hormone replacement therapy. We investigated its potential role on adipocyte differentiation. The effects of DRSP were studied in murine preadipocyte cell lines and primary cultures of human preadipocytes. Differentiation markers and mechanisms underlying phenotypic variations in response to DRSP were explored. Early exposure to DRSP during differentiation led to a marked dose-dependent inhibition of adipose differentiation and triglyceride accumulation in 3T3-L1 and 3T3-F442A cells. DRSP also markedly inhibited adipose conversion of human primary preadipocytes derived from visceral (mesenteric and epicardial) and subcutaneous fat. This effect was MR-dependent and did not involve the glucocorticoid, androgen, or progesterone receptors. DRSP inhibited clonal expansion of preadipocytes and decreased expression of PPARγ, a key transcriptional mediator of adipogenesis, but had no effect on lipolysis, glucose uptake, and PPARγ binding to its ligands. DRSP exerts a potent antiadipogenic effect that is related to an alteration of the transcriptional control of adipogenesis via an antagonistic effect on the MR. Selective MR blockade therefore has promise as a novel therapeutic option for the control of excessive adipose tissue deposition and its related metabolic complications.

Clinical evidence shows that in males obesity is frequently associated with hypogonadism and vice versa; also, low testosterone levels have been considered a “hallmark” of metabolic syndrome in men. These observations indicate that there is a strict connection between anatomically and functionally distinct cell types such as white adipocytes and Leydig cells, that synthesize testosterone. Adipose tissue is able to control several functions of the testis through its products secreted in the bloodstream. On the other hand, circulating levels of testosterone and estradiol deeply affect adipocyte proliferation, differentiation, and fat mass distribution, hereby controlling critical metabolic functions, such as food intake, insulin sensitivity, vascular reactivity, and immunity. This paper highlights the existing clinical and experimental evidence linking androgens and adipose tissue and illustrates the consequences occurring when the balance between fat mass distribution and eugonadism is lost.

Objective Elevated aldosterone is associated with increased risk of atherosclerosis complications, whereas treatment with mineralocorticoid receptor (MR) antagonists decreases the rate of cardiovascular events. Here we test the hypothesis that aldosterone promotes early atherosclerosis by modulating intercellular adhesion molecule-1 (ICAM-1) expression and investigate the molecular mechanisms by which aldosterone regulates ICAM-1 expression. Methods and Results Apolipoprotein-E (ApoE)−/− mice fed an atherogenic diet and treated with aldosterone for 4 weeks showed increased vascular expression of ICAM-1, paralleled by enhanced atherosclerotic plaque size in the aortic root. Moreover, aldosterone treatment resulted in increased plaque lipid and inflammatory cell content, consistent with an unstable plaque phenotype. ApoE/ICAM-1 double knockout (ApoE−/−/ICAM-1−/−) littermates were protected from the aldosterone-induced increase in plaque size, lipid content and macrophage infiltration. Since aldosterone is known to regulate ICAM-1 transcription via MR in human endothelial cells, we explored MR regulation of the ICAM-1 promoter. Luciferase reporter assays performed in HUVECs using deletion constructs of the human ICAM-1 gene promoter showed that a region containing a predicted MR-responsive element (MRE) is required for MR-dependent transcriptional regulation of ICAM-1. Conclusions Pro-atherogenic effects of aldosterone are mediated by increased ICAM-1 expression, through transcriptional regulation by endothelial MR. These data enhance our understanding of the molecular mechanism by which MR activation promotes atherosclerosis complications.

Introduction Prolonged tadalafil administration in men with erectile dysfunction is associated with increased testosterone (T): estradiol (E2) ratio mainly related to reduction of E2 levels. Aim To investigate the presence of phosphodiesterase type 5 (PDE5) isoenzyme in primary human visceral adipocytes and whether different PDE5 inhibitors (PDE5i) could directly modulate aromatase (ARO) expression in differentiated human visceral adipocytes in culture. Main Outcome Measures PDE5 mRNA and protein expression in primary human visceral adipocytes as well as mRNA and protein expression of ARO, with functional activity after selective PDE5 blockade by tadalafil and sildenafil. Methods Purified primary human visceral pre-adipocytes were differentiated ex vivo and were exposed to tadalafil or sildenafil (1 µM) for different intervals of time (6-12-24-96 hours). ARO mRNA content and expression were measured by Western Blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. T and E2 in supernatants were measured by ELISA also in the presence of letrozole. Results Differentiated adipocytes were found to express detectable levels of PDE5 transcripts. Acute exposure (6 hours) to both PDE5i tadalafil and sildenafil increased ARO mRNA expression by 4.7- and 2.8-fold, respectively (P < 0.001). ARO mRNA and protein levels were increased by the treatment with PDE5i in a time- and dose-dependent manner. Such effect was mimicked by 8-bromo-cGMP but was lost after 24 and 96 hours; differently, the PDE3B specific inhibitor milrinone (1 µM), displayed no effect. Accordingly, long-term exposure (24 and 96 hours) to PDE5i caused a significant increase in E2 concentrations in the supernatant (1.7 and 2 fold, respectively; P < 0.001), with a parallel reduction of T (15% and 30%, respectively; P < 0.001). Such effect was reversed by the co-incubation with the specific ARO-inhibitor letrozole. Conclusions Our results demonstrate that PDE5 is expressed in human visceral adipocytes and that acute exposure to PDE5i selectively stimulates ARO expression, which is related to a specific PDE5 blockade. We speculate that modulation of ARO activity by PDE5i could be one of the mechanisms responsible, at least in part, for the beneficial effects of PDE5i on endothelial and metabolic functions.

Combined GR/MR pharmacological antagonism markedly reduced HFD-driven weight gain and fat mass expansion in mice through the increase in adipose PGC-1α, suggesting that both receptors represent strategic therapeutic targets to fight obesity. The effects of CORT118335 in adipocytes seem predominantly mediated by MR antagonism.

fatty acids and minimal obesity, absent GH activation leads to steatosis because phosphorylated STAT5 prevents hepatic steatosis. These results raise the possibility of low-dose GH treatment for nonalcoholic fatty liver disease. Endocrinology, published November 17, 2010, 10.1210/en. 2010 The Somatotrope as a Metabolic Sensor: Deletion of Leptin Receptors Causes Obesity Gwen V. Childs, Noor Akhter, Anessa Haney, Mohsin Syed, Angela Odle, Michael Cozart, Zachary Brodrick, Dana Gaddy, Larry J. Suva, Nisreen Akel, Christopher Crane, Helen Benes, Amanda Charlesworth, Raul Luque, Streamson Chua, and Rhonda D. Kineman Leptin, the product of the Lep gene, reports levels of adiposity to the hypothalamus and other regulatory cells, including pituitary somatotropes, which secrete GH. Leptin deficiency is associated with a decline in somatotrope numbers and function, suggesting that leptin may be important in their maintenance. This hypothesis was tested in a new animal model in which exon 17 of the leptin receptor (Lepr) was selectively deleted in somatotropes by Cre-loxP technology. Organ genotyping confirmed the recombination of the floxed LepR allele only in the pituitary. Deletion mutant mice showed a 72% reduction in pituitary cells bearing leptin receptor (LEPR)-b, a 43% reduction in LEPR proteins and a 60% reduction in percentages of immunopositive GH cells, which correlated with reduced serum GH. In mutants, LEPR expression by other pituitary cells was like that of normal animals. Leptin stimulated phosphorylated Signal transducer and activator of transcription 3 expression in somatotropes from normal animals but not from mutants. Pituitary weights, cell numbers, IGF-I, and the timing of puberty were not different from control values. Growth curves were normal during the first 3 months. Deletion mutant mice became approximately 30-46% heavier than controls with age, which was attributed to an increase in fat mass, with no significant effects on lean mass. Serum leptin levels were either normal in younger animals or reflected the level of obesity in older animals. The specific ablation of the Lepr exon 17 gene in somatotropes resulted in GH deficiency with a consequential reduction in lipolytic activity normally maintained by GH and increased adiposity. Endocrinology, published November 17, 2010, 10.1210/en. 2010 During pregnancy, trophoblasts grow to adapt the feto-maternal unit to fetal requirements. Aldosterone and cortisol levels increase, the latter being inactivated by a healthy placenta. By contrast, preeclamptic placental growth is reduced while aldosterone levels are low and placental cortisol tissue levels are high due to improper deactivation. Aldosterone acts as a growth factor in many tissues, whereas cortisol inhibits growth. We hypothesized that in preeclampsia low aldosterone and enhanced cortisol availability might mutually affect placental growth and function. Proliferation of cultured human trophoblasts was time-and dose-dependently increased with aldosterone (P Ͻ 0.04 to P Ͻ 0.0001) and inhibit...