Effects Of Ethanol On Insulin Sensitivity: Role Of Insulin Signal Transduction Molecules

Changes in human behaviour and lifestyle over the last century have resulted in a dramatic increase in the incidence of type 2 diabetes mellitus worldwide. Now, type 2 diabetes is taking its place as one of the main threats to human health in the 21st century. Insulin resistance is a key pathophysiologic feature of type 2 diabetes and is brought on by environmental and behavioural factors such as smoking, pregnancy, overly rich nutrition, obesity and so on. In recent years, many epidemiological studies suggested there exists a U-shape relationship between alcohol consumption and the risk of diabetes. Chronic regular mild to moderate alcohol consumption among healthy people may be associated with increased insulin sensitivity and a reduced risk of type 2 diabetes, while excessive consumption can impair glycaemic control and increased the risk of type 2 diabetes. To date, several studies have shown the effects of ethanol intake on glucose intolerance in muscle, adipocytes, and the liver. However, a few studies have conducted on the mechanisms underlying alcohol impairment of insulin signaling with some inconsistencies in reported results apparent. Thus, the direct actions of alcohol on specific components of the pathways which mediates glucose disposal and the mechanisms underlying alcohol-induced insulin resistance remain to be clarified. Although the mechanisms underlying insulin resistance are complicated, it is uniformly agreed that insulin signal transduction molecules play a central role. Abnormality in the number and/or functions of insulin signal transduction molecules would impair glucose disposal into peripheral tissues. Skeletal muscle is one of the most important target organs of insulin, and also important for serum glucose balance and energy metabolism. Insulin resistance in skeletal muscle can influence the whole body insulin sensitivity and partly attributes to the cause of type 2 diabetes. In the present study, we observed the effect of alcohol on insulin sensitivity both in vivo and in vitro. At the same time, we investigated the potential mechanism involving. From this study, we hope to elucidate some understanding about the mechanism between alcohol and insulin resistance and provide scientific advice for healthy drinking. The experiments were designed and the main results were summarized as follows:PartⅠEffects of chronic ethanol administration on insulin sensitivity and insulin signaling pathways in skeletal muscle of ratsObjective To establish the rat model of chronic ethanol administration and examine the effects of ethanol on insulin signal transduction molecules expression in skeletal muscle. To explore the mechanism by which chronic ethanol administration influences the whole body insulin sensitivity.Methods After acclimatization for one week, 80 rats were randomly divided into four groups(n=20/group) on the basis of body weight: control(C) group, Low(L), moderate(M) and high(H) alcohol group and each group was comprised of 10 male and 10 female rats. Ethanol, at the dose of 0, 0.8, 1.6, 2.6 g?(kg?bw)-1?day-1, were given by means of intragastric gavage. 19 weeks later, rats were killed and serum was collected for testing of fasting glucose and insulin. Whole-body insulin sensitivity of rats was assessed from fasting glucose and insulin leves and by the previously validated homeostasis model assessment (HOMA) index as follows: HOMA-IR= fasting glucose (mmol/L)×fasting insulin (μU/ml)/22.5.The homeostatic indices of ?-cell secretory activity (HOMA ? cell) were calculated as: HOMA- ? cell = 20×fasting insulin (μU/ml)/ Fasting glucose (mmol/L)-3.5. Expression of IR, IRS-1, IRS-2, PI-3K (p85α) and total GLUT-4 in skeletal muscle were detected by RT-PCR and Western blot.Results1. In male rats, ethanol intake increased the levels of fasting serum glucose and insulin. Fasting serum glucose of H group, fasting serum insulin of L, M group and HOMA-IR index of all ethanol–fed groups were higher than those of C group(p<0.05) .There was no significant difference among the HOMA-βcell of the four groups. In female rats, the H group's fasting serum glucose was higher but fasting serum insulin was lower than those of C group(p<0.05). There was no significant difference among the HOMA-IR of the four groups. HOMA-βcell in H and M group were significantly (p<0.05) decreased compared with the C group.2. Compared with the control rats, the expression of IR,IRS-1mRNA were suppressed by 1.6, 2.6 g·(kg·bw)^-1·day^-1 ethanol loading in both gender rats (p<0.05) . IRS-1mRNA expression in L group of male rats was up-regulated(p<0.05), while there was no significant difference in IRS-1mRNA expression between the control group and low ethanol dose group in female rats. With the ethanol doses increasing, IRS-2mRNA expression was decreased, but there was no significant difference among the four groups(p>0.05).3. In male rats, the L group p85αmRNA and protein expression was up-regulated (p <0.05), while the M and H group p85αexpression were significantly suppressed (p<0.05). There were no significant differences in GLUT-4 mRNA and protein expression between the control group and low alcohol dose group, whereas the expression of GLUT-4 was significantly (p<0.05) reduced in M group and H group. In female rats, p85αand GLUT-4 mRNA and protein expressions were significantly (p<0.05) diminished in M and H group, whereas their expression was similar in L group compared with C group.Conclusion The present results indicated that chronic ethanol administration(>=0.8g·(kg·bw)^-1·day^-1) could induce insulin resistance and down-regulated the expression of IR, IRS-1, PI-3K(p85α) and GLUT-4 in skeletal muscle may be the molecular mechanism. Effect of ethanol on insulin sensitivity showed a sexual difference. Female rats were more sensitive to ethanol's influence than males.PartⅡInfluence of ethanol on glucose uptake and insulin signaling pathways in rat's primary skeletal muscle cellsObjective To examine the direct effect of ethanol on glucose uptake in rat's primary skeletal muscle cells. To investigate expression and activity of insulin signal transduction molecules and explore the possible mechanism of ethanol affects insulin sensitivity.Methods Rat's primary skeletal muscle cells were exposed to different doses of ethanol (0, 25, 50, 100, 200, 400mmol/L) for 24h and with 200mmol/L ethanol for different time (6h, 12h, 24h). The effect of ethanol on insulin sensitivity in skeletal muscle cells was tested by 2-deoxglucose uptake. The mRNA expression of PI-3K(p85α) and GLUT-4 were detected by RT-PCR. The protein content of PI-3K (p85α), Akt-2, GLUT-4 and the phosphorylation protein of PI-3K, Akt were detected by Western blot.Results1. After exposed to 200mmol/L, 400mmol/L ethanol for 24h, the basic and insulin-stimulated 2-deoxglucose uptake were inhibited in rat's primary skeletal muscle cells(p<0.05), while insulin-stimulated 2-deoxglucose uptake of cells was significantly increased in 50mmol/L and 100mmol/L ethanol-treated groups (p<0.05). Insulin-stimulated 2-deoxglucose uptake were increased significantly after treated with 200mmol/L ethanol for 6h (p <0.05), and then decreased. The level of 2-deoxglucose uptake was even lower than that of control group after terased with ethanol for 24h (p <0.05).2. Exposed ethanol for 24h: the expression of PI-3K (p85α) was decreased in 400mmol/L ethanol-treated group (p<0.05). GLUT-4 mRNA , protein content of Akt-2, GLUT-4 and levels of insulin-stimulated phospho-PI-3K, phospho-Akt were all suppressed by treatment at 200, 400mmol/L (p<0.05). Cells treated with 50,100mmol/L ethanol, GLUT-4 expression and insulin-stimulated phospho-PI-3K and phospho-Akt were up-regulated (p<0.05); Exposed to 200mmol/L ethanol: at 6h, GLUT-4mRNA and protein content of Akt-2, GLUT-4 were increased, then decreased with prolong of exposure time. At 24h, the decrease was dramatically compared to control group (p<0.05).Conclusion Ethanol could directly influence insulin sensitivity in rat's primary skeletal muscle cells. Chronic high dose ethanol (200,400mmol/L for 24h) induced insulin resistance; Ethanol also could directly affect PI-3K (p85α), Akt-2, GLUT-4 expression and PI-3K, Akt phosphorylation. Above all, the decreased of expression and activity of these signaling molecules may be the mechanism of ethanol impairs insulin sensitivity.