Essential role of ICAM-1 in aldosterone-induced atherosclerosis.

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 4weeks 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.

Mineralocorticoid receptor antagonism induces browning of white adipose tissue through impairment of autophagy and prevents adipocyte dysfunction in high-fat-diet-fed mice.

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.

Androgens and adipose tissue in males: a complex and reciprocal interplay.

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.

Exercise training reduces serum capacity to induce endothelial cell death in patients with chronic heart failure.

AIMS: Physical training improves endothelial function and exercise capacity in patients with heart failure (HF). Serum from patients with cardiovascular diseases increases apoptosis of human endothelial cells suggesting the importance of humoral factors in the progression of the disease. We evaluated whether exercise training influences the apoptotic capacity of serum from patients with chronic HF (CHF). METHODS AND RESULTS: The study included 39 patients with HF (NYHA II) and 10 age-matched healthy controls. Patients were allocated to either a structured programme of exercise training (24 patients) or standard care (15 patients). Human umbilical vein endothelial cells (HUVECs) were incubated with a medium containing 20% serum obtained before and after either a 3-week exercise training programme or standard care. At baseline, serum from patients with CHF induced a higher degree of lactate dehydrogenase (LDH) release and apoptosis in HUVECs compared with healthy controls (43 ± 1.5 vs. 16 ± 1.1%, P< 0.001 and 67 ± 5.4 vs. 23 ± 5.8%, P< 0.001, respectively). Exercise training significantly increased performance in the 6 min walking test (+34.7%) and reduced the ability of serum to induce LDH release and apoptosis of HUVECs. The reduction of apoptosis after exercise training correlated with the improvement in functional capacity. The expression of the apoptosis markers Bax and Caspase-3 was significantly reduced in HUVECs exposed to serum collected after exercise training. Circulating tumour necrosis factor-alpha, matrix metalloproteinase-1 (MMP-1), and tissue inhibitor of metalloproteinase-1 (TIMP-1) levels were significantly reduced by exercise training and the MMP-9/TIMP-1 ratio increased. CONCLUSION: A short term in-hospital structured cardiovascular training programme reduces the ability of serum-derived factors to induce endothelial cell death in patients with CHF.

Antiadipogenic effects of the mineralocorticoid receptor antagonist drospirenone: potential implications for the treatment of metabolic syndrome.

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.

Exposure to phosphodiesterase type 5 inhibitors stimulates aromatase expression in human adipocytes in vitro.

INTRODUCTION: Prolonged tadalafil administration in men with erectile dysfunction is associated with increased testosterone (T): estradiol (E(2)) ratio mainly related to reduction of E(2) 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 E(2) 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 E(2) 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.

Cellular models for understanding adipogenesis, adipose dysfunction, and obesity.

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.