| Background:Excessive ethanol consumption may be among the causes leading to insulin resistance. However, epidemiological data relating ethanol consumption to glucose metabolism or insulin sensitivity are contradictory. For example, it was shown that regular moderate ethanol drinkers are more insulin sensitive than abstainers. On the other hand, continuous or high intake of alcohol reportedly leads to glucose intolerance. Taking these reports into consideration, it seems that the effect of alcohol intake on insulin sensitivity is somewhat dose-dependent.The mechanism by which chronic alcohol exposure impairs insulin sensitivity is still unclear. It has been demonstrated that impaired insulin stimulation of glucose transport is not associated with impaired activation of PI 3-kinase or Akt. This suggests the precise step impaired by ethanol consumption might lie on the downstream of PI 3-kinase or Akt.Recently, Rachdaoui and colleagues showed that the ethanol-induced impairment of insulin-stimulated glucose uptake in adipocytes was mediated by the decreased expression of Gll alpha, a member of the Gq family of heterotrimeric GTP-binding proteins, which is required for insulin-stimulated glut4 translocation. Thus, this finding also indicate that other mechanisms, which contribute to insulin-stimulated glucose transport and have effects on the downstream of insulin signal pathway, might be involved in ethanol-impaired insulin signaling.Gs alpha and Gi alpha are other heterotrimeric GTP-binding proteins and have been shown to be regulators of insulin sensitivity, probably via regulationof intracellular cAMP synthesis. Also, it has been demonstrated that the Gs alpha/ cAMP-mediated mechanism participates in the pathophysiology of many alcohol-related diseases, such as alcoholic hepatitis and alcoholic sedation, etc. But whether such a mechanism is also involved in ethanol-induced impairment of insulin signaling is unclear.AMPK is a key regulator of cellular energy metabolism, and has also been proved as a regulator of insulin sensitivity. It was reported that the down-regulated activity of AMPK by ethanol feeding in rats' hepatocyte was involved in the mechanism of alcoholic fatty liver. Whether such a mechanism is also involved in the ethanol-induced insulin resistance remains to be elucidated. Objective:?The effect of chronic ethanol feeding at different doses on insulin sensitivity of rats at both body level and skeletal muscle level;?The effect of chronic ethanol feeding on the expression of insulin receptor, insulin receptor substrate 1, insulin receptor substrate 2 and GluT4;?The changes of Gs alpha and Gi alpha expression after ethanol exposure and the possible cAMP-mediated pathway in ethanol-induced insulin resistance;?The alternations of AMPK expression and activity by ethanol feeding and the role of AMPK in ethanol-impaired insulin signaling. Methods:(D Animal feeding: Forty male Wistar rats, divided into four groups, received either distilled water (control group) or ethanol, which was administered by gastric tube with a single daily dose: 5 g ? kg"1 (large dose group), 2.5 g ? kg"' (middle dose group ) and 0.5 g ? kg"' (small dose group).(2) Evaluation of insulin sensitivity: Fasting plasma glucose levels were monitored monthly. After 5 months, fasting serum insulin levels were measured and oral glucose tolerance test was performed. To further explore insulin sensitivity at skeletal muscle level, the ability of insulin-stimulated glucose uptake of the rat muscle was detected.(3) Effects of ethanol on classic insulin signal pathway: mRNA and protein levels of insulin receptor, insulin receptor substrate 1, insulin receptor substrate 2 and GluT4 of rats' skeletal muscle were measured using Real-time PCR (or RT-PCR) and Western blot;? Effects of ethanol on Gs alpha and Gi alpha-mediated pathway: mRNA levels of Gs alpha and Gi alpha of rats' skeletal muscle were measured using Real-Time PCR and RT-PCR; Protein levels were further measured using Western blot; Intracellular cAMP levels were detected using ELISA.(5) Effects of ethanol on expression and activity of AMPK: mRNA levels of alpha 1 subunit and alpha 2 subunit of AMPK were measured using Real-Time PCR and RT-PCR; Protein levels of total AMPK alpha subunit were detected using Western blot; Activities of AMPK were evaluated by measuring the protein levels of phosphated AMPK alpha subunit using Western blot. Results:Evaluation of insulin sensitivity: After ethanol treatment for 5 months, no significant differences were detected of fasting plasma glucose, fasting serum insulin and 2h plasma glucose levels after oral glucose load; Chronic ethanol feeding at 5g ? kg"' and 2.5g ? kg"1 significantly decreased insulin-stimulated glucose uptake in isolated skeletal muscle (P<0.01), indicating alcohol exposure can impair insulin sensitivity.Effects of ethanol on classic insulin signal pathway of rats' skeletal muscle: The expression of IR,IRS1 and IRS2 increased in all three alcohol-treated groups, both at mRNA and protein levels (P<0. 05). The increment of the expression is likely a protective effect after alcohol feeding because the up-regulation trend is more significant in small dose alcohol-fed rats than that of middle and large doses ones. Ethanol feeding with daily doses of 5g ? kg"1 and 2. 5g ? kg"1 significantly decreased GluT4 expression (P<0. 05), whereas no significant changes of GluT4 expression was found in 0. 5g ? kg"1 alcohol-fed group.Effects of ethanol on Gs alpha and Gi alpha-mediated pathway of rats'skeletal muscle: Gs alpha expression (mRNA and protein) in skeletal muscle was significantly increased in all three ethanol-treated groups (P<0.05). cAMP levels were also increased by ethanol treatment (P<0. 05). Both the increments were dose-dependent. No significant changes of Gi alpha expression between control and ethanol-treated groups were detected.Effects of ethanol on expression and activity of AMPK: No differences were detected in mRNA levels of alpha 1 subunit and alpha 2 subunit of AMPK. Consistently, protein levels of total AMPK alpha subunit were not altered after ethanol feeding. While, the protein levels of phosphated AMPK alpha subunit significantly decreased after ethanol treatment (P<0.05), indicating ethanol exposure might impair AMPK activity. Conclusions:CD Chronic ethanol consumption could impair insulin sensitivity of rats' skeletal muscle, at least partly via decreasing GluT4 expression. Also, ethanol-induced insulin resistance was accompanied with increased expression of insulin receptor, insulin receptor substrate 1 and insulin receptor substrate 2. Taking relative researches into consideration, the increment of insulin receptor, insulin receptor substrate 1 and insulin receptor substrate 2 seems like a protective effect.(2) Chronic ethanol exposure decreased insulin-induced glucose uptake in skeletal muscle, which was associated with increased expression of Gs alpha and intracellular cAMP levels. Because Gs alpha is a negative regulator of insulin sensitivity, the alteration in Gs alpha expression may, at least partly, contribute to the ethanol-induced impairment of insulin signal transduction.(3) Ethanol treatment also impaired the activity of AMPK in rats' skeletal muscle. AMPK is a known positive regulator of insulin signal transduction, the changes of AMPK activity may also be invovled in the mechanism of ethanol-induced insulin resistance.
|
PDF Full Text Request