The regulatory effect of sex steroids on the RhoA/ROCK pathway in the rat distal vagina.

BACKGROUND: Sex steroids have been demonstrated as important modulators of vaginal function. The RhoA/ROCK calcium-sensitizing pathway plays a role in genital smooth muscle contractile mechanism, but its regulation has never been elucidated. AIM: This study investigated the sex steroid regulation of the vaginal smooth muscle RhoA/ROCK pathway using a validated animal model. METHODS: Ovariectomized (OVX) Sprague-Dawley rats were treated with 17ß-estradiol (E2), testosterone (T), and T with letrozole (T + L) and compared with intact animals. Contractility studies were performed to test the effect of the ROCK inhibitor Y-27632 and the nitric oxide (NO) synthase inhibitor L-NAME. In vaginal tissues, ROCK1 immunolocalization was investigated; mRNA expression was analyzed by semiquantitative reverse transcriptase-polymerase chain reaction; and RhoA membrane translocation was evaluated by Western blot. Finally, rat vaginal smooth muscle cells (rvSMCs) were isolated from the distal vagina of intact and OVX animals, and quantification of the RhoA inhibitory protein RhoGDI was performed after stimulation with NO donor sodium nitroprusside, with or without administration of the soluble guanylate cyclase inhibitor ODQ or PRKG1 inhibitor KT5823. OUTCOMES: Androgens are critical in inhibiting the RhoA/ROCK pathway of the smooth muscle compartment in the distal vagina. RESULTS: ROCK1 was immunolocalized in the smooth muscle bundles and blood vessel wall of the vagina, with weak positivity detected in the epithelium. Y-27632 induced a dose-dependent relaxation of noradrenaline precontracted vaginal strips, decreased by OVX and restored by E2, while T and T + L decreased it below the OVX level. In Western blot analysis, when compared with control, OVX significantly induced RhoA activation, as revealed by its membrane translocation, with T reverting it at a level significantly lower than in controls. This effect was not exerted by E2. Abolishing NO formation via L-NAME increased Y-27632 responsiveness in the OVX + T group; L-NAME had partial effects in controls while not modulating Y-27632 responsiveness in the OVX and OVX + E2 groups. Finally, stimulation of rvSMCs from control animals with sodium nitroprusside significantly increased RhoGDI protein expression, counteracted by ODQ and partially by KT5823 incubation; no effect was observed in rvSMCs from OVX rats. CLINICAL IMPLICATIONS: Androgens, by inhibiting the RhoA/ROCK pathway, could positively contribute to vaginal smooth muscle relaxation, favoring sexual intercourse. STRENGTHS AND LIMITATIONS: This study describes the role of androgens in maintaining vaginal well-being. The absence of a sham-operated animal group and the use of the only intact animal as control represented a limitation to the study.

Neuroprotective Effects of Testosterone in the Hypothalamus of an Animal Model of Metabolic Syndrome.

Metabolic syndrome (MetS) is known to be associated to inflammation and alteration in the hypothalamus, a brain region implicated in the control of several physiological functions, including energy homeostasis and reproduction. Previous studies demonstrated the beneficial effects of testosterone treatment (TTh) in counteracting some MetS symptoms in both animal models and clinical studies. This study investigated the effect of TTh (30 mg/kg/week for 12 weeks) on the hypothalamus in a high-fat diet (HFD)-induced animal model of MetS, utilizing quantitative RT-PCR and immunohistochemical analyses. The animal model recapitulates the human MetS features, including low testosterone/gonadotropin plasma levels. TTh significantly improved MetS-induced hypertension, visceral adipose tissue accumulation, and glucose homeostasis derangements. Within hypothalamus, TTh significantly counteracted HFD-induced inflammation, as detected in terms of expression of inflammatory markers and microglial activation. Moreover, TTh remarkably reverted the HFD-associated alterations in the expression of important regulators of energy status and reproduction, such as the melanocortin and the GnRH-controlling network. Our results suggest that TTh may exert neuroprotective effects on the HFD-related hypothalamic alterations, with positive outcomes on the circuits implicated in the control of energy metabolism and reproductive tasks, thus supporting a possible role of TTh in the clinical management of MetS.

Insight on the Intracrinology of Menopause: Androgen Production within the Human Vagina.

In this study, we investigated steroidogenic gene mRNA expression in human vaginas and verified the ability of human vagina smooth muscle cells (hvSMCs) to synthesize androgens from upstream precursor dehydroepiandrosterone (DHEA). As a readout for androgen receptor (AR) activation, we evaluated the mRNA expression of various androgen-dependent markers. hvSMCs were isolated from vagina tissues of women undergoing surgery for benign gynecological diseases. In these cells, we evaluated mRNA expression of several steroidogenic enzymes and sex steroid receptors using real time reverse transcription-polymerase chain reaction. Androgen production was quantified with liquid chromatography tandem-mass spectrometry (LC-MS/MS). In vaginal tissues, AR mRNA was significantly less expressed than estrogen receptor α, whereas in hvSMCs, its mRNA expression was higher than progestin and both estrogen receptors. In hvSMCs and in vaginal tissue, when compared to ovaries, the mRNA expression of proandrogenic steroidogenic enzymes (HSD3ß1/ß2, HSD17ß3/ß5), along with 5α-reductase isoforms and sulfotransferase, resulted as being more abundant. In addition, enzymes involved in androgen inactivation were less expressed than in the ovaries. The LC-MS/MS analysis revealed that, in hvSMCs, short-term DHEA supplementation increased Δ4-androstenedione levels in spent medium, while increasing testosterone and DHT secretion after longer incubation. Finally, androgenic signaling activation was evaluated through AR-dependent marker mRNA expression, after DHEA and T stimulation. This study confirmed that the human vagina is an androgen-target organ with the ability to synthesize androgens, thus providing support for the use of androgens for local symptoms of genitourinary syndrome in menopause.

Anti-inflammatory effects of androgens in the human vagina.

Chronic inflammation is involved in the genitourinary syndrome of menopause (GSM) and beneficial effects of androgens in the vagina have been described. We investigated the potential involvement of human vagina smooth muscle cells (hvSMCs) in the inflammatory response and the immunomodulatory effect of androgen receptor (AR) agonist dihydrotestosterone (DHT). HvSMCs isolated from menopausal women were evaluated for sex steroids receptors and toll-like receptors mRNA expression, and left untreated or treated in vitro with lipopolysaccharide (LPS) or IFNγ, in the presence or absence of DHT. We evaluated mRNA expression (by RT-PCR) and secretion in cell culture supernatants (by a bead-based immunoassay) of pro-inflammatory markers. Nuclear translocation of NF-κB (by immunofluorescence) and cell surface HLA-DR expression (by flow cytometry) were also evaluated. Similar experiments were repeated in rat vSMCs (rvSMCs). In hvSMCs and rvSMCs, AR was highly expressed. DHT pre-treatment inhibited LPS-induced mRNA expression of several pro-inflammatory mediators (i.e. COX2, IL-6, IL-12A and IFNγ), effect significantly blunted by AR antagonist bicalutamide. DHT significantly counteracted the secretion of IL-1RA, IL-2, IL-5, IL-15, FGF, VEGF and TNFα. LPS-induced NF-κB nuclear translocation was significantly inhibited by DHT, an effect counteracted by bicalutamide. DHT pre-treatment significantly decreased IFNγ-induced expression of HLA-DR, mRNA expression of iNOS, COX2 and MCP1, and secretion of IL-1, IL-2, IL-5, IL-6, MCP1 and GCSF. Similar effects were observed in rvSMCs. The activation of AR suppresses the inflammatory response in hvSMCs, reducing their potential to be involved in the initiation and maintaining of inflammation, thus representing a therapeutic strategy in conditions, such as the GSM.

Testosterone improves muscle fiber asset and exercise performance in a metabolic syndrome model.

Lifestyle modifications, including physical exercise (PhyEx), are well-known treatments for metabolic syndrome (MetS), a cluster of metabolic and cardiovascular risk factors often associated to hypogonadism. Given the trophic role of testosterone on skeletal muscle (SkM), this study was aimed at evaluating the effects of testosterone treatment on SkM metabolism and exercise performance in male rabbits with high-fat diet (HFD)-induced MetS. HFD rabbits, treated or not with testosterone (30 mg/kg/week) for 12 weeks, were compared to regular diet animals (RD). A subset of each group was exercise-trained for 12 weeks. HFD increased type-II (fast, glycolytic) and decreased type-I (slow, oxidative) muscle fibers compared to RD as evaluated by RT-PCR and histochemistry. Testosterone reverted these effects, also inducing the expression of mitochondrial respiration enzymes and normalizing HFD-induced mitochondrial cristae reduction. Moreover, testosterone significantly increased the expression of myogenic/differentiation markers and genes related to glucidic/lipid metabolism. At the end of the PhyEx protocol, when compared to RD, HFD rabbits showed a significant reduction of running distance and running time, while testosterone counteracted this effect, also decreasing lactate production. In the trained groups, muscle histology showed a significant reduction of oxidative fibers in HFD compared to RD and the positive effect of testosterone in maintaining oxidative metabolism, as also demonstrated by analyzing mitochondrial ultrastructure, succinate dehydrogenase activity and ATP production. Our results indicate that testosterone could be useful to promote oxidative muscle metabolism altered by MetS, thus improving exercise performance. Conversely, testosterone administration to otherwise eugonadal rabbits (RD) only increased muscle fiber diameter but not endurance performance.

Physical activity counteracts metabolic syndrome-induced hypogonadotropic hypogonadism and erectile dysfunction in the rabbit.

Metabolic syndrome (MetS) clusters cardiovascular and metabolic risk factors along with hypogonadism and erectile dysfunction. Lifestyle modifications including physical exercise (PhyEx) are well-known treatments for this condition. In this study, we analyzed the effect of PhyEx on hypothalamic-pituitary-testis axis and erectile function by use of an animal MetS model, previously established in rabbits fed a high-fat diet (HFD). Rabbits fed a regular diet (RD) were used as controls. A subset of both groups was trained on a treadmill. HFD rabbits showed typical MetS features, including HG (reduced T and LH) and impairment of erectile function. PhyEx in HFD rabbits completely restored plasma T and LH and the penile alterations. At testicular and hypothalamic levels, an HFD-induced inflammatory status was accompanied by reduced T synthesis and gonadotropin-releasing hormone (GnRH) immunopositivity, respectively. In the testis, PhyEx normalized HFD-related macrophage infiltration and increased the expression of steroidogenic enzymes and T synthesis. In the hypothalamus, PhyEx normalized HFD-induced gene expression changes related to inflammation and glucose metabolism, restored GnRH expression, particularly doubling mRNA levels, and regulated expression of molecules related to GnRH release (kisspeptin, dynorphin). Concerning MetS components, PhyEx significantly reduced circulating cholesterol and visceral fat. In multivariate analyses, cholesterol levels resulted as the main factor associated with MetS-related alterations in penile, testicular, and hypothalamic districts. In conclusion, our results show that PhyEx may rescue erectile function, exert anti-inflammatory effects on hypothalamus and testis, and increase LH levels and T production, thus supporting a primary role for lifestyle modification to combat MetS-associated hypogonadism and erectile dysfunction.

INT-767 prevents NASH and promotes visceral fat brown adipogenesis and mitochondrial function.

The bile acid receptors, farnesoid X receptor (FXR) and Takeda G-protein-coupled receptor 5 (TGR5), regulate multiple pathways, including glucose and lipid metabolism. In a rabbit model of high-fat diet (HFD)-induced metabolic syndrome, long-term treatment with the dual FXR/TGR5 agonist INT-767 reduces visceral adipose tissue accumulation, hypercholesterolemia and nonalcoholic steatohepatitis. INT-767 significantly improves the hallmarks of insulin resistance in visceral adipose tissue (VAT) and induces mitochondrial and brown fat-specific markers. VAT preadipocytes isolated from INT-767-treated rabbits, compared to preadipocytes from HFD, show increased mRNA expression of brown adipogenesis markers. In addition, INT-767 induces improved mitochondrial ultrastructure and dynamic, reduced superoxide production and improved insulin signaling and lipid handling in preadipocytes. Both in vivo and in vitro treatments with INT-767 counteract, in preadipocytes, the HFD-induced alterations by upregulating genes related to mitochondrial biogenesis and function. In preadipocytes, INT-767 behaves mainly as a TGR5 agonist, directly activating dose dependently the cAMP/PKA pathway. However, in vitro experiments also suggest that FXR activation by INT-767 contributes to the insulin signaling improvement. INT-767 treatment counteracts HFD-induced liver histological alterations and normalizes the increased pro-inflammatory genes. INT-767 also induces a significant reduction of fatty acid synthesis and fibrosis markers, while increasing lipid handling, insulin signaling and mitochondrial markers. In conclusion, INT-767 significantly counteracts HFD-induced liver and fat alterations, restoring insulin sensitivity and prompting preadipocytes differentiation toward a metabolically healthy phenotype.

Anti-fibrotic effects of chronic treatment with the selective FXR agonist obeticholic acid in the bleomycin-induced rat model of pulmonary fibrosis.

Farnesoid X receptor (FXR) activation by obeticholic acid (OCA) has been demonstrated to inhibit inflammation and fibrosis development in liver, kidney and intestine in multiple disease models. FXR activation has also been demonstrated to suppress the inflammatory response and to promote lung repair after lung injury. This study investigated the protective effects of OCA treatment (3 or 10mg/kg/day) on inflammation, tissue remodeling and fibrosis in the bleomycin-induced pulmonary fibrosis rat model. Effects of OCA treatment on morphological and molecular alterations of the lung, as well as remodeling of the alveoli and the right ventricle were also evaluated. Lung function was assessed by measuring airway resistance to inflation. In the acute phase (7days), bleomycin promoted an initial thickening and fibrosis of the lung interstitium, with upregulation of genes related to epithelial proliferation, tissue remodeling and hypoxia. At 28days, an evident increase in the deposition of collagen in the lungs was observed. This excessive deposition was accompanied by an upregulation of transcripts related to the extracellular matrix (TGFß1, SNAI1 and SNAI2), indicating lung fibrosis. Administration of OCA protected against bleomycin-induced lung damage by suppressing molecular mechanisms related to epithelial-to-mesenchymal transition (EMT), inflammation and collagen deposition, with a dose-dependent reduction of proinflammatory cytokines such as IL-1ß and IL-6, as well as TGF-ß1 and SNAI1 expression. Pirfenidone, a recently approved treatment for idiopathic pulmonary fibrosis (IPF), significantly counteracted bleomycin-induced pro-fibrotic genes expression, but did not exert significant effects on IL-1ß and IL-6. OCA treatment in bleomycin-challenged rats also improved pulmonary function, by effectively normalizing airway resistance to inflation and lung stiffness in vivo. Results with OCA were similar, or even superior, to those obtained with pirfenidone. In conclusion, our results suggest an important protective effect of OCA against bleomycin-induced lung fibrosis by blunting critical mediators in the pathogenesis of IPF.

Cardiopulmonary protective effects of the selective FXR agonist obeticholic acid in the rat model of monocrotaline-induced pulmonary hypertension.

Farnesoid X receptor (FXR) activation by obeticholic acid (OCA) has been demonstrated to inhibit inflammation and fibrosis development and even induce fibrosis regression in liver, kidney and intestine in multiple disease models. OCA also inhibits liver fibrosis in nonalcoholic steatohepatitis patients. FXR activation has also been demonstrated to suppress the inflammatory response and to promote lung repair after lung injury. This study investigated the effects of OCA treatment (3, 10 or 30mg/kg, daily for 5days a week, for 7 and/or 28 days) on inflammation, tissue remodeling and fibrosis in the monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) rat model. Treatment with OCA attenuated MCT-induced increased pulmonary arterial wall thickness and right ventricular hypertrophy, by i) blunting pathogenic inflammatory mechanisms (downregulation of interleukin 6, IL-6, and monocyte chemoattractant protein-1, MCP-1) and ii) enhancing protective mechanisms counteracting fibrosis and endothelial/mesenchymal transition. MCT-injected rats also showed a marked decrease of pulmonary artery responsiveness to both endothelium-dependent and independent relaxant stimuli, such as acetylcholine and a nitric oxide donor, sodium nitroprusside. Administration of OCA (30mg/kg) normalized this decreased responsiveness. Accordingly, OCA treatment induced profound beneficial effects on lung histology. In particular, both OCA doses markedly reduced the MCT-induced medial wall thickness increase in small pulmonary arteries. To evaluate the objective functional improvement by OCA treatment of MCT-induced PAH, we performed a treadmill test and measured duration of exercise. MCT significantly reduced, and OCA normalized treadmill endurance. Results with OCA were similar, or even superior, to those obtained with tadalafil, a well-established treatment of PAH. In conclusion, OCA treatment demonstrates cardiopulmonary protective effects, modulating lung vascular remodeling, reducing right ventricular hypertrophy and significantly improving exercise capacity. Thus, OCA can restore the balance between relaxant and contractile pathways in the lung, promoting cardiopulmonary protective actions.

Differential Effects of Testosterone and Estradiol on Clitoral Function: An Experimental Study in Rats.

INTRODUCTION: Female sexual response is a complex phenomenon in which psychological, neurologic, and vascular mechanisms and hormonal factors interact. During the arousal phase, they cooperate to increase genital blood flow, thus inducing engorgement of the clitoris and lubrication of the vagina. Regulation of vascular and non-vascular smooth muscle tone is the crucial event in the erectile process. Preclinical studies have suggested that nitric oxide (NO) is the main vasodilator neurotransmitter modulating, through the second messenger cyclic guanosine monophosphate (cGMP), clitoral flow vessels. AIM: To investigate the effects of sexual steroid hormones on pro-erectile and relaxant (mediated by NO and cGMP) and anti-erectile and contractile (mediated by ras homolog gene family member A [RhoA] and Rho-associated protein kinase [ROCK]) mechanisms in the clitoris using a validated animal model of female ovariectomized Sprague-Dawley rats. METHODS: Subgroups of ovariectomized rats were treated with 17ß-estradiol, progesterone, testosterone, or testosterone and letrozole for 6 weeks. The experimental groups were compared with a control group of intact rats. MAIN OUTCOME MEASURES: Sex steroids plasma levels were assessed and in vitro contractility studies were carried out in order to investigate the effect of ovariectomy and in vivo treatments on clitoris smooth muscle activity. Smooth muscle cells (SMCs) from rat clitoral biopsies were isolated and characterized. RhoA activity was determined in SMCs cell cultures. RNA from tissues and cells was analyzed by quantitative real-time RT-PCR. RESULTS: Using real-time polymerase chain reaction, testosterone treatment upregulated the expression of NO-mediated pathway genes (endothelial and neuronal NO synthase, guanylate cyclase soluble subunit-α3, guanylate cyclase soluble subunit-ß3, cGMP-dependent protein kinase 1, and phosphodiesterase type 5). Conversely, estrogen replacement upregulated the expression of calcium-sensitizing RhoA-ROCK pathway genes. In vitro contractility studies were performed on phenylephrine pre-contracted clitoris strips. Ovariectomy resulted in a decreased responsiveness to Y-27632, a ROCK inhibitor, which was fully restored by 17ß-estradiol supplementation. To further examine the effect of 17ß-estradiol on the RhoA-ROCK pathway, smooth muscle cells were isolated from rat clitoris and their migration capacity was evaluated. CONCLUSION: Collectively, these data demonstrate that testosterone improves the relaxation of vascular smooth muscle cells through the NO-cGMP pathway, and that testosterone and 17ß-estradiol are necessary to maintain a functional contractile and relaxant machinery in the clitoris. This new concept might provide support for the concomitant use of estrogen and testosterone during the treatment of sexual arousal disorders related to hormonal imbalance or insufficiency.

Tadalafil reduces visceral adipose tissue accumulation by promoting preadipocytes differentiation towards a metabolically healthy phenotype: Studies in rabbits.

Development of metabolically healthy adipocytes within dysfunctional adipose tissue may represent an attractive way to counteract metabolic syndrome (MetS). In an experimental animal model of high fat diet (HFD)-induced MetS, in vivo, long- and short-term tadalafil treatments were able to reduce visceral adipose tissue (VAT) accumulation and hypertriglyceridemia, and to induce the expression in VAT of the brown fat-specific marker, uncoupling protein 1 (UCP1). VAT preadipocytes (PAD), isolated from the tadalafil-treated HFD rabbits, showed: i) a multilocular morphology; ii) an increased expression of brown fat-specific genes (such as UCP1 and CIDEA); iii) improved mitochondrial structure and dynamic and reduced superoxide production; iv) improved insulin sensitivity. Similar effects were obtained after in vitro tadalafil treatment in HFD rPAD. In conclusion, tadalafil counteracted HFD-associated VAT alterations, by restoring insulin-sensitivity and prompting preadipocytes differentiation towards a metabolically healthy phenotype.

Estrogen mediates metabolic syndrome-induced erectile dysfunction: a study in the rabbit.

INTRODUCTION: Estrogen receptor (ER) α is critical in mediating the harmful effects of hyperestrogenism in fetal or neonatal life on the developing penis. In contrast, little is known on the impact of an excess of estrogens on penile function in adulthood. AIM: To investigate the effect of estrogens on metabolic syndrome (MetS)-associated erectile dysfunction (ED). METHODS: We employed a recently established animal model of high fat diet (HFD)-induced MetS. Subgroups of MetS rabbits were dosed with either testosterone (T) or tamoxifen. We evaluated penile responsiveness to acetylcholine (Ach) as well as the expression of genes related to penile smooth muscle relaxation and contractility. MAIN OUTCOME MEASURE: Associations between MetS-induced penile alterations and sex steroids were investigated in an animal model of HFD-induced MetS. To understand the role of either androgen deficiency or estrogen excess on ED, we treated subgroups of MetS rabbits with either T or tamoxifen, a classical ER antagonist. RESULTS: Feeding an HFD-induced MetS was associated to elevated estradiol (E2) and low T levels. E2, but not T, was independently and negatively associated with genes able to affect penile erection. Smooth muscle-related markers decreased as a function of E2 and were positively associated with all the variables investigated. Increasing concentrations of circulating E2 were negatively associated with Ach-induced relaxation. In HFD rabbits, in vivo T dosing significantly improved MetS and completely normalized circulating E2. Conversely, in vivo tamoxifen dosing reduced visceral adiposity and partially restored T level. Ach-induced relaxation was severely impaired by HFD and significantly restored, up to the control level, by both tamoxifen and T dosing. In rabbit smooth muscle cells cultures 17ß-E2 (1 nM) significantly reduced the expression of α-smooth muscle actin, transgelin, and phosphodiesterase type 5. The effects of 17ß-E2 were completely reverted by tamoxifen (100 nM). CONCLUSIONS: This study demonstrates, for the first time, that HFD-induced ED is more associated with a high E2, rather than to a low T, milieu. HFD-induced ED is partially restored by in vivo treatment not only with T but also with the nonsteroidal ER antagonist, tamoxifen.

Metformin in vitro and in vivo increases adenosine signaling in rabbit corpora cavernosa.

INTRODUCTION: In subjects with erectile dysfunction responding poorly to sildenafil, metformin was reported to improve erections. AIMS: The aim of this study is to investigate metformin's mechanism of action on erectile function, particularly focusing on adenosine (ADO) and nitric oxide (NO) signaling in an animal model of high-fat diet (HFD)-induced metabolic syndrome. METHODS: In vitro contractility studies of penile strips. Penile expression of genes related to ADO or NO signaling was also evaluated. MAIN OUTCOME MEASURE: In vitro contractility studies were used to investigate the effect of in vivo and ex vivo metformin administration on ADO- or acetylcholine (Ach)-induced relaxation of penile strips from HFD as compared with animals fed a regular diet (RD). RESULTS: Expression of ADO receptor type 3 (A3 R), ADO deaminase (ADA), AMP deaminase type 1 (AMPD1), and 2 (AMPD2) was decreased in HFD as compared with RD. Accordingly, in HFD the ADO relaxant effect was potentiated as compared with RD (P < 0.02). In vivo metformin treatment in both RD and HFD significantly increased the ADO relaxing effect (P < 0.0001 and P < 0.01, respectively, vs. relative untreated groups) although to a different extent. In fact, the half-maximal inhibitory concentration (IC50 )/IC50 ratio in RD increased fourfold vs. HFD (RD IC50 ratio = 13.75 ± 2.96; HFD IC50 ratio = 2.85 ± 0.52). In corpora cavernosa (CC) from HFD, in vivo metformin (i) normalized A3 R, ADA, and AMPD1; (ii) further decreased AMPD2; (iii) increased dimethylarginine dimethylamino-hydrolase; and (iv) partially restored impaired Ach-induced relaxation. Ex vivo metformin time and dose dependently increased the relaxant effect of ADO in RD. The potentiating effect of metformin on ADO-induced relaxation was significantly reduced by preincubation with NO synthase inhibitor N(ω) -Nitro-L-arginine methyl ester hydrochloride (L-NAME). Interestingly, in vivo testosterone supplementation in HFD rabbits (i) increased penile expression of endothelial NO synthase and AMPD2 and (ii) restored metformin's potentiating effect on ADO-induced relaxation up to RD level. CONCLUSION: Metformin in vivo and ex vivo increases ADO signaling in CC, most probably interfering with NO formation and ADO breakdown.

Tadalafil effect on metabolic syndrome-associated bladder alterations: an experimental study in a rabbit model.

INTRODUCTION: Metabolic syndrome (MetS) and lower urinary tract symptoms (LUTS) are often associated. Bladder detrusor hyper-contractility-a major LUTS determinant-is characterized by increased Ras homolog gene family, member A/Rho-associated protein kinase (RhoA/ROCK) signaling, which is often upregulated in MetS. AIM: This study investigated the effects of tadalafil dosing on RhoA/ROCK signaling in bladder, in a rabbit model of high-fat diet (HFD)-induced MetS. METHODS: Adult male rabbits feeding a HFD for 12 weeks. A subset of HFD animals was treated with tadalafil (2 mg/kg/day, 1 week: the last of the 12 weeks) and compared with HFD and control (feeding a regular diet) rabbits. MAIN OUTCOME MEASURES: In vitro contractility studies to evaluate the relaxant effect of the selective ROCK inhibitor, Y-27632, in carbachol precontracted bladder strips. Evaluation of RhoA activation by its membrane translocation. Immunohistochemistry for ROCK expression has been performed to evaluate ROCK expression in bladder from the different experimental groups. mRNA expression of inflammation, pro-fibrotic markers by quantitative RT-PCR has been performed to evaluate the effect of tadalafil on MetS-induced inflammation and fibrosis within the bladder. The in vitro effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cells was evaluated by using chemotaxis assay. RESULTS: Bladder strips from HFD rabbits showed hyper-responsiveness to Y-27632, indicating RhoA/ROCK overactivity in HFD bladder compared with matched controls. Accordingly, the fraction of activated (translocated to the membrane) RhoA as well as ROCK expression are increased in HFD bladder. Tadalafil dosing normalized HFD-induced bladder hypersensitivity to Y-27632, by reducing RhoA membrane translocation and ROCK overexpression. Tadalafil dosing reduced mRNA expression of inflammatory, pro-fibrotic, and hypoxia markers. A direct inhibitory effect of tadalafil on RhoA/ROCK signaling in bladder smooth muscle cell was demonstrated by using chemotaxis assay. Pre-treatment with tadalafil inhibited both basal and PDGF-induced migration of bladder smooth muscle cells. CONCLUSIONS: Tadalafil dosing reduced RhoA/ROCK signaling and smooth muscle overactivity in an animal model of MetS-associated bladder alterations. Our findings suggest a novel mechanism of action of tadalafil in alleviating LUTS in MetS patients.

Nonalcoholic steatohepatitis as a novel player in metabolic syndrome-induced erectile dysfunction: an experimental study in the rabbit.

A pathogenic link between erectile dysfunction (ED) and metabolic syndrome (MetS) is now well established. Nonalcoholic steatohepatitis (NASH), the hepatic hallmark of MetS, is regarded as an active player in the pathogenesis of MetS-associated cardiovascular disease (CVD). This study was aimed at evaluating the relationship between MetS-induced NASH and penile dysfunction. We used a non-genomic, high fat diet (HFD)-induced, rabbit model of MetS, and treated HFD rabbits with testosterone (T), with the selective farnesoid X receptor (FXR) agonist obeticholic acid (OCA), or with the anti-TNFα mAb infliximab. Rabbits fed a regular diet were used as controls. Liver histomorphological and gene expression analysis demonstrated NASH in HFD rabbits. Several genes related to inflammation (including TNFα), activation of stellate cells, fibrosis, and lipid metabolism parameters were negatively associated to maximal acetylcholine (Ach)-induced relaxation in penis. When all these putative liver determinants of penile Ach responsiveness were tested as covariates in a multivariate model, only the association between hepatic TNFα expression and Ach response was confirmed. Accordingly, circulating levels of TNFα were increased 15-fold in HFD rabbits. T and OCA dosing in HFD rabbits both reduced TNFα liver expression and plasma levels, with a parallel increase of penile eNOS expression and responsiveness to Ach. Also neutralization of TNFα with infliximab treatment fully normalized HFD-induced hypo-responsiveness to Ach, as well as responsiveness to vardenafil, a phosphodiesterase type 5 inhibitor. Thus, MetS-induced NASH in HFD rabbits plays an active role in the pathogenesis of ED, likely through TNFα, as indicated by treatments reducing liver and circulating TNFα levels (T or OCA), or neutralizing TNFα action (infliximab), which significantly improve penile responsiveness to Ach in HFD rabbits.

Metabolic syndrome induces inflammation and impairs gonadotropin-releasing hormone neurons in the preoptic area of the hypothalamus in rabbits.

Rabbits with high fat diet (HFD)-induced metabolic syndrome (MetS) developed hypogonadotropic hypogonadism (HH) and showed a reduced gonadotropin-releasing hormone (GnRH) immunopositivity in the hypothalamus. This study investigated the relationship between MetS and hypothalamic alterations in HFD-rabbits. Gonadotropin levels decreased as a function of MetS severity, hypothalamic gene expression of glucose transporter 4 (GLUT4) and interleukin-6 (IL-6). HFD determined a low-grade inflammation in the hypothalamus, significantly inducing microglial activation, expression and immunopositivity of IL-6, as well as GLUT4 and reduced immunopositivity for KISS1 receptor, whose mRNA expression was negatively correlated to glucose intolerance. Correcting glucose metabolism with obetcholic acid improved hypothalamic alterations, reducing GLUT4 and IL-6 immunopositivity and significantly increasing GnRH mRNA, without, however, preventing HFD-related HH. No significant effects at the hypothalamic level were observed after systemic anti-inflammatory treatment (infliximab). Our results suggest that HFD-induced metabolic derangements negatively affect GnRH neuron function through an inflammatory injury at the hypothalamic level.

Exendin-4 induces cell adhesion and differentiation and counteracts the invasive potential of human neuroblastoma cells.

Exendin-4 is a molecule currently used, in its synthetic form exenatide, for the treatment of type 2 diabetes mellitus. Exendin-4 binds and activates the Glucagon-Like Peptide-1 Receptor (GLP-1R), thus inducing insulin release. More recently, additional biological properties have been associated to molecules that belong to the GLP-1 family. For instance, Peptide YY and Vasoactive Intestinal Peptide have been found to affect cell adhesion and migration and our previous data have shown a considerable actin cytoskeleton rearrangement after exendin-4 treatment. However, no data are currently available on the effects of exendin-4 on tumor cell motility. The aim of this study was to investigate the effects of this molecule on cell adhesion, differentiation and migration in two neuroblastoma cell lines, SH-SY5Y and SK-N-AS. We first demonstrated, by Extra Cellular Matrix cell adhesion arrays, that exendin-4 increased cell adhesion, in particular on a vitronectin substrate. Subsequently, we found that this molecule induced a more differentiated phenotype, as assessed by i) the evaluation of neurite-like protrusions in 3D cell cultures, ii) the analysis of the expression of neuronal markers and iii) electrophysiological studies. Furthermore, we demonstrated that exendin-4 reduced cell migration and counteracted anchorage-independent growth in neuroblastoma cells. Overall, these data indicate for the first time that exendin-4 may have anti-tumoral properties.

PDE5 inhibitors blunt inflammation in human BPH: a potential mechanism of action for PDE5 inhibitors in LUTS.

BACKGROUND: Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH)/low urinary tract symptoms (LUTS) are often comorbid. Chronic inflammation is one of the putative links between these diseases. Phosphodiesterase type 5 inhibitors (PDE5i) are recognized as an effective treatment of BPH-related LUTS. One proposed mechanism of action of PDE5 is the inhibition of intraprostatic inflammation. In this study we investigate whether PDE5i could blunt inflammation in the human prostate. METHODS: Evaluation of the effect of tadalafil and vardenafil on secretion of interleukin 8 (IL-8, a surrogate marker of prostate inflammation) by human myofibroblast prostatic cells (hBPH) exposed to different inflammatory stimuli. We preliminary evaluate histological features of prostatic inflammatory infiltrates in BPH patients enrolled in a randomized, double bind, placebo controlled study aimed at investigating the efficacy of vardenafil (10 mg/day, for 12 weeks) on BPH/LUTS. RESULTS: In vitro treatment with tadalafil or vardenafil on hBPH reduced IL-8 secretion induced by either TNFα or metabolic factors, including oxidized low-density lipoprotein, oxLDL, to the same extent as a PDE5-insensitive PKG agonist Sp-8-Br-PET-cGMP. These effects were reverted by the PKG inhibitor KT5823, suggesting a cGMP/PKG-dependency. Treatment with tadalafil or vardenafil significantly suppressed oxLDL receptor (LOX-1) expression. Histological evaluation of anti-CD45 staining (CD45 score) in prostatectomy specimens of BPH patients showed a positive association with MetS severity. Reduced HDL-cholesterol and elevated triglycerides were the only MetS factors significantly associated with CD45 score. In the MetS cohort there was a significant lower CD45 score in the vardenafil-arm versus the placebo-one.

FXR activation normalizes insulin sensitivity in visceral preadipocytes of a rabbit model of MetS.

Insulin resistance is the putative key underlying mechanism linking adipose tissue (AT) dysfunction with liver inflammation and steatosis in metabolic syndrome (MetS). We have recently demonstrated that the selective farnesoid X receptor (FXR) agonist obeticholic acid (OCA) ameliorates insulin resistance and the metabolic profile with a marked reduction in the amount of visceral AT (VAT) in a high-fat diet (HFD)-induced rabbit model of MetS. These effects were mediated by the activation of FXR, since treatment with the selective TGR5 agonist INT-777 was not able to ameliorate the metabolic parameters evaluated. Herein, we report the effects of in vivo OCA dosing on the liver, the VAT, and the adipogenic capacity of VAT preadipocytes (rPADs) isolated from rabbits on a HFD compared with those on a control diet. VAT and liver were studied by immunohistochemistry, Western blot analysis, and RT-PCR. rPADs were exposed to a differentiating mixture to evaluate adipogenesis. Adipocyte size, hypoxia, and the expression of perilipin and cytosolic insulin-regulated glucose transporter GLUT4 (SLC2A4) were significantly increased in VAT isolated from the HFD rabbits, and normalized by OCA. The expression of steatosis and inflammation markers was increased in the liver of the HFD rabbits and normalized by OCA. rPADs isolated from the HFD rabbits were less sensitive to insulin, as demonstrated by the decreased insulin-induced glucose uptake, triglyceride synthesis, and adipogenic capacity, as well as by the impaired fusion of lipid droplets. OCA treatment preserved all the aforementioned metabolic functions. In conclusion, OCA dosing in a MetS rabbit model ameliorates liver and VAT functions. This could reflect the ability of OCA to restore insulin sensitivity in AT unable to finalize its storage function, counteracting MetS-induced metabolic alterations and pathological AT deposition.

Fat boosts, while androgen receptor activation counteracts, BPH-associated prostate inflammation.

BACKGROUND: Metabolic syndrome (MetS) and benign prostate hyperplasia (BPH) are often comorbid. Chronic inflammation, a determinant pathogenic factor for BPH, is a putative link between the two conditions. METHODS: In a multi-center cohort of BPH patients (n = 244) who underwent prostatectomy, we evaluated whether MetS is associated with prostatic inflammation in BPH specimens. In addition, we investigated the in vitro inflammatory effects of metabolic insults on human prostatic myofibroblastic cells (hBPH). RESULTS: Inflammatory infiltrates score (IS) in prostatectomy specimens showed a step-wise association with the number of MetS factors present (P = 0.001). After adjusting for age, reduced HDL cholesterol, and elevated triglycerides were the only factors significantly associated with IS. Increased IS was also significantly associated with hypogonadism. In an age- and testosterone (T)-adjusted model, dyslipidemia was still associated with IS. To investigate whether metabolic factors could directly trigger prostate inflammation, we performed preliminary studies in myofibroblastic hBPH. Among the different factors, oxidized low-density lipoprotein (oxLDL) showed the highest secretion of IL-8 (>10-fold)-a surrogate marker of prostate inflammation--as well as IL-6, and bFGF. Co-treatment with DHT significantly inhibited oxLDL-induced secretion of IL-8, whilst an AR-antagonist, bicalutamide, reversed DHT effects. DHT suppresses oxLDL receptor (LOX-1) expression. CONCLUSIONS: Our data suggest that fats and insulin could have a detrimental effect on prostate health, boosting inflammation, a key pathogenic factor in BPH. Conversely, beneficial effects of DHT in counteracting lipid- and insulin-induced prostatic alterations, suggest that T-via its conversion into DHT-may have unexpected beneficial effects on prostate health.