Study of prostate smooth muscle hyperreactivity in insulin-resistance obese mice: Role of TLR4.

Abstract

The worldwide incidence of obesity has increased dramatically in the past 25 years, and today is considered one of the most major public health problems. Recent clinical studies indicate a strong correlation between obesity and genitourinary tract diseases, such as erectile dysfunction and overactive bladder syndrome. The benign prostate hyperplasia (BPH), which is characterized by an increase in both physical size (static component) and prostate smooth muscle tone (PSM) (dynamic component) has been also linked to obesity. Prostate from obese rats show increased cell proliferation with consequent prostate growth, development of intraepithelial neoplasms and reduction in androgen receptors. Obesity has also been associated with prostate hypercontractility in rats, secondary to BPH. Obese mice show increased prostate levels of NF-kB and expressions of gp91phox, p22phox and p47phox subunits of NADPH oxidase. Recent studies have shown an important role for Toll-like receptors (TLR), particularly subtype 4 (TLR4), in the exacerbation of the chronic inflammatory condition associated with obesity, which in turn has been implicated in genesis / maintenance of insulin resistance. A preliminary study in our group evaluated the effect of high-fat diet-induced obesity on mice PSM reactivity. The contractile responses to phenylephrine (±1-adrenoceptor agonist), potassium chloride and electrical stimulation (neurogenic response) were significantly higher in prostate from obese mice compared with control group. Furthermore, we also showed a significant increase (52%) in reactive oxygen species (ROS) basal levels in prostate from obese animals compared to control group. However, the literature is scarce about the pathophysiology of prostatic hypercontractility in conditions of obesity and / or insulin resistance. Thus, this project aims to characterize the functional and molecular alterations in prostate from obese mice, with emphasis on the role of TLR4 and its downstream signaling pathway in the pathogenesis of obesity-induced prostate hyperreactivity. Our hypothesis is that the prostate hypercontractility in obese individuals have in common the activation of TLR4 with consequent increase in intracellular signaling and increases in inflammatory factor production. In addition, activation of TLR4 could lead to increase in ERO and phosphorylation of key-proteins for intracellular insulin signaling, such as IRS-1, thus altering the balance between contractile and relaxing tone of PSM. If our hypothesis is correct, the pathway involved in the TLR4 activation will be ideal targets for new drugs to reducing prostate complications associated with obesity. Specifically, we will study the contractile response of PSM of control, high-fat diet obese and TLR4(-/-) mice by using electrical stimulation (neurogenic contraction) and direct activation of ±1 adrenoceptor (phenylephrine) and muscarinic receptor (carbachol). The relaxations mediated by activations of ²2-adrenoceptors and nitric oxide - cyclic GMP pathway will also be investigated here. In prostatic tissue of control, obese and (TLR4 -/-), we will perform RT-PCR and / or Western blot studies to evaluate the signaling proteins involved in TLR4 activation (MyD88, TRIAP, TRIF, TRAMP, IFR-3 and NF-kB) and insulin resistance (IRbeta, IRS-1 and Akt). Oxidative stress in prostatic tissue will also be investigated here by measurement of ROS production, the expressions of NADPH subunits (gp91phox and p47phox), SOD and catalase. Finally, pharmacological treatments with anti-TLR4 monoclonal antibody and eritoran (TLR4 antagonist) will be performed to understand the molecular and functional obesity-induced prostatic changes.