Effects of Smoking and moderate Consumption of Alcohol on the expression of Glucose Transporter GLUT4 and Cytokines in skeletal muscle of rats

Abstract

It is known that the consumption of alcohol and smoking has a strong correlation with the development and installation of obesity, insulin resistance and diabetes. These changes are well correlated with inflammatory state, characterized by increased circulating cytokines. An important consideration focuses on the fact that individuals who like smoking and drinking, in general, do not perform any type of physical activity, which contributes to worse the obesity and insulin resistance levels. Today it is known that physical training is an important accessible and cheaper tool to combat gain of bodyweight and to improve insulin sensitivity, in addition to the cardiovascular benefits and fight against metabolic alterations developed by alcoholism and smoking. During contractile activity a reduction in the ATP/ADP ratio occurs, activating a protein kinase, 5'-AMP-dependent (AMPK), which stimulates glucose uptake by a distinct intracellular insulin pathway. The main carrier of glucose in this action is GLUT4. Furthermore, in humans, it was verified that physical training has a positive effect on the inflammatory state even in obese subjects by reduced circulating cytokines. In infarcted rats it was verified that physical training in treadmill during 8 weeks was enough to improve inflammation on muscle, with reduced expression of Tnf and increased Il10 expression. The goal of this study is to verify the effect of smoking and alcoholism on the expression of Il-10, Tnf, Prkaa2, Slc2a4 genes, AMPK and GLUT4 protein content, and pAMPK in gastrocnemius skeletal muscle and to evaluate aerobic exercise on the alterations provoked by smoking and alcoholism. Eighty Wistar male rats, will be divided in: N: non-smoker and nonalcoholic; S: smoker; A: alcohol; SA: alcoholic smoker; E: exercise; SE: smoker exercise; AE: alcohol exercise; SAE: alcoholic smoker exercise groups. Smokers groups will be exposed to a cigarette smoke exposure protocol with combustion of 4 cigarettes, during 30 min, 2x/day, five days/week, during 60 days. In alcoholic groups alcohol will be administered in 10% v/v during 60 days. The exercised groups will be subjected to an aerobic exercise protocol in a treadmill, 9.75 m/min, 60 min, during 60 days. A insulin tolerance test, and glucose tolerance test(GTT),will be done for assessment to insulin sensitivy. Lactacidemia analysis will be made to characterize the physical activity performed. Gastrocnemius muscle will be removed after 24h the last training session under anesthesia with sodium thiopental (50mg/kg BW). RT-PCR techniques will be performed for the evaluation of Slc2a4 (which encodes the GLUT4), Prkaa2 (coding AMPKa2), Tnf (coding tumor necrosis factor alpha) and Il-10 (encoding Il-10) gene expression. For the quantification of GLUT4 and AMPK protein, and AMPK phosphorylation Western Blotting will be performed. Statistical analysis will be performed by comparison of medias, using the ANOVA test, parametric, withpost-test when needed (Bonferroni). The differences among groups will be considered significant when P < 0,05.