Dihydrotestosterone (DHT) rapidly increase after maximal aerobic exercise in healthy males: the lowering effect of phosphodiesterase's type 5 inhibitors on DHT response to exercise-related stress.

PURPOSE: Few data exist on dihydrotestosterone (DHT) adaptation to exercise-related stress. The aim of the study was to investigate on serum DHT and other androgens' responses to acute aerobic exercises, and to verify if a long-acting phosphodiesterase's type 5 inhibitors could influence these responses, as previously observed for salivary testosterone. METHODS: In a double-blind cross over study, 12 healthy trained male volunteers were submitted to both an acute sub-maximal and maximal exercise tests on cycle ergometer, after randomly receiving a two days placebo or tadalafil administration (20 mg, Cialis®, Ely-Lilly, Indianapolis, IN, USA). Blood sample collections were performed at different time points before and after exercise. Serum DHT, total testosterone (TT), dehydroepiandrosterone sulfate (DHEAS) and luteinizing hormone (LH), were assayed. RESULTS: Serum DHT increase in placebo treatment immediately post maximal aerobic exercise and return to basal values at 60 min of recovery whereas tadalafil administration significantly reduced the DHT increase after exercise. The values of areas under curves showed the increase of TT after acute sub-maximal and maximal exercise and of DHEAS only after acute maximal aerobic exercise independently from treatment. CONCLUSIONS: In addition to testosterone, also DHT plays an exercise-related adaptive role during high intensity aerobic exercise, but its rapid useful effects during exercise have to be determined. We hypothesized that the increased androgens secretion during exercise could be mainly related to steroidogenic enzymes modifications in peripheral tissues (i.e., muscles). Moreover, the blunting effect of tadalafil on DHT increase support a possible role of peripheral nitric oxide/GMPc related pathways in influencing physical-stress related DHT metabolism.

Vitamin D, sport and health: a still unresolved clinical issue.

Vitamin D metabolites have a pleiotropic role in human physiology, both in static and dynamic conditions, and a lot of vitamin D-related biological effects could influence physical and sport performances in athletes. Probably due to different factors (e.g., drugs, doping, nutrition, ultraviolet B radiation exposure), in athletes a very high prevalence of vitamin D inadequacy (i.e., deficiency or insufficiency) has been observed. Vitamin D inadequacy in athletes could be associated with specific health risks and to alterations of functional capacities, potentially influencing the fine adjustment of physical performances during training and sport competitions. When risk factors for vitamin D inadequacy exist, a preventive vitamin D supplementation is indicated, and if a vitamin D inadequacy is diagnosed, its supplementation is recommended. Unfortunately, on these issues many concerns remain unresolved. Indeed, it is not clear if athletes should be classified as a special population at increased risk for vitamin D inadequacy; moreover, in comparison to the non-athletic population, it is still not clear if athletes should have different reference ranges and different optimal target levels for serum vitamin D, if they have additional health risks, and if they need different type of supplementations (doses) for prevention and/or replacement therapy. Moreover, in athletes also the abuse of vitamin D supplements for ergogenic purposes raise different ethical and safety concerns. In this review, the main physio-pathological, functional and clinical issues that relate vitamin D to the world of athletes are described.

Sulodexide counteracts endothelial dysfunction induced by metabolic or non-metabolic stresses through activation of the autophagic program.

OBJECTIVE: Endothelial dysfunction (ED) predisposes to venous thrombosis (VT) and post-thrombotic syndrome (PTS), a long-term VT-related complication. Sulodexide (SDX) is a highly purified glycosaminoglycan with antithrombotic, pro-fibrinolytic and anti-inflammatory activity used in the treatment of chronic venous disease (CVD), including patients with PTS. SDX has recently obtained clinical evidence in the "extension therapy" after initial-standard anticoagulant treatment for the secondary prevention of recurrent deep vein thrombosis (DVT). Herein, we investigated how SDX counteracts ED. MATERIALS AND METHODS: Human umbilical vein endothelial cells (HUVEC) were used. Metabolic and non metabolic-induced ED was induced by treating with methylglyoxal (MGO) or irradiation (IR), respectively. Bafilomycin A1 was used to inhibit autophagy. The production of reactive oxygen species (ROS), tetrazolium bromide (MTT) assay for cell viability, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay for cell apoptosis, Real-time PCR and Western blot analysis for gene and protein expression were used. RESULTS: SDX protected HUVEC from MGO- or IR-induced apoptosis by counteracting the activation of the intrinsic and extrinsic caspase cascades. The cytoprotective effects of SDX resulted from a reduction in a) ROS production, b) neo-synthesis and release of pro-inflammatory cytokines (TNFα, IL1, IL6, IL8), c) DNA damage induced by MGO or IR. These effects were reduced when autophagy was inhibited. CONCLUSIONS: Data herein collected indicate the ability of SDX to counteract ED induced by metabolic or non-metabolic stresses by involving the intracellular autophagy pathway. Our experience significantly increases the knowledge of the mechanisms of action of SDX against ED and supports the use of SDX in the treatment of CVD, PTS and in the secondary prevention of recurrent DVT.

Comparative study of testosterone and vitamin D analogue, elocalcitol, on insulin-controlled signal transduction pathway regulation in human skeletal muscle cells.

PURPOSE: Skeletal muscle (Skm) plays a key role in regulating energetic metabolism through glucose homeostasis. Several hormones such as Testosterone (T) and Vitamin D (VD) have been shown to affect energy-dependent cell trafficking by determining Insulin (I)-like effects. AIM: To elucidate possible hormone-related differences on muscular metabolic control, we analyzed and compared the effects of T and elocalcitol (elo), a VD analogue, on the activation of energy-dependent cell trafficking, metabolism-related-signal transduction pathways and transcription of gene downstream targets. METHODS: Human fetal skeletal muscle cells (Hfsmc) treated with T or elo were analyzed for GLUT4 localization, phosphorylation/activation status of AKT, ERK1/2, IRS-1 signaling and c-MYC protein expression. RESULTS: T, similar to elo, induced GLUT4 protein translocation likely in lipid raft microdomains. While both T and elo induced a rapid IRS-1 phosphorylation, the following dynamic in phosphorylation/activation of AKT and ERK1/2 signaling was different. Moreover, T but not elo increased c-MYC protein expression. CONCLUSIONS: All together, our evidence indicates that whether both T and elo are able to affect upstream I-like pathway, they differently determine downstream effects in I-dependent cascade, suggesting diverse physiological roles in mediating I-like response in human skeletal muscle.

Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

The phosphodiesterase 5 inhibitor sildenafil decreases the proinflammatory chemokine IL-8 in diabetic cardiomyopathy: in vivo and in vitro evidence.

PURPOSE: Interleukin (IL)-8 is a proinflammatory C-X-C chemokine involved in inflammation underling cardiac diseases, primary or in comorbid condition, such diabetic cardiomyopathy (DCM). The phosphodiesterase type 5 inhibitor sildenafil can ameliorate cardiac conditions by counteracting inflammation. The study aim is to evaluate the effect of sildenafil on serum IL-8 in DCM subjects vs. placebo, and on IL-8 release in human endothelial cells (Hfaec) and peripheral blood mononuclear cells (PBMC) under inflammatory stimuli. METHODS: IL-8 was quantified: in sera of (30) DCM subjects before (baseline) and after sildenafil (100 mg/day, 3-months) vs. (16) placebo and (15) healthy subjects, by multiplatform array; in supernatants from inflammation-challenged cells after sildenafil (1 µM), by ELISA. RESULTS: Baseline IL-8 was higher in DCM vs. healthy subjects (149.14 ± 46.89 vs. 16.17 ± 5.38 pg/ml, p < 0.01). Sildenafil, not placebo, significantly reduced serum IL-8 (23.7 ± 5.9 pg/ml, p < 0.05 vs. baseline). Receiver operating characteristic (ROC) curve for IL-8 was 0.945 (95% confidence interval of 0.772 to 1.0, p < 0.01), showing good capacity of discriminating the response in terms of drug-induced IL-8 decrease (sensitivity of 0.93, specificity of 0.90). Sildenafil significantly decreased IL-8 protein release by inflammation-induced Hfaec and PBMC and downregulated IL-8 mRNA in PBMC, without affecting cell number or PDE5 expression. CONCLUSION: Sildenafil might be suggested as potential novel pharmacological tool to control DCM progression through IL-8 targeting at systemic and cellular level.

The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism.

PURPOSE: Tadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells. METHODS: RTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used. RESULTS: Tadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels. CONCLUSION: In human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.

Testosterone insulin-like effects: an in vitro study on the short-term metabolic effects of testosterone in human skeletal muscle cells.

PURPOSE: Testosterone by promoting different metabolic pathways contributes to short-term homeostasis of skeletal muscle, the largest insulin-sensitive tissue and the primary site for insulin-stimulated glucose utilization. Despite evidences indicate a close relationship between testosterone and glucose metabolism, the molecular mechanisms responsible for a possible testosterone-mediated insulin-like effects on skeletal muscle are still unknown. METHODS: Here we used undifferentiated proliferating or differentiated human fetal skeletal muscle cells (Hfsmc) to investigate the short-term effects of testosterone on the insulin-mediated biomolecular metabolic machinery. GLUT4 cell expression, localization and the phosphorylation/activation of AKT, ERK, mTOR and GSK3ß insulin-related pathways at different time points after treatment with testosterone were analyzed. RESULTS: Independently from cells differentiation status, testosterone, with an insulin-like effect, induced Glut4-mRNA expression, GLUT4 protein translocation to the cytoplasmic membrane, while no effect was observed on GLUT4 protein expression levels. Furthermore, testosterone treatment modulated the insulin-dependent signal transduction pathways inducing a rapid and persistent activation of AKT, ERK and mTOR, and a transient inhibition of GSK3ß. T-related effects were shown to be androgen receptor dependent. CONCLUSION: All together our data indicate that testosterone through the activation of non-genomic pathways, participates in skeletal muscle glucose metabolism by inducing insulin-related effects.