| Objective:Excessive ethanol consumption may cause many diseases such as alcoholic cardiomyopathy, fatty liver and hepatitis of ethanol. Moreover, epidemiological data showed that long-term ethanol consumption was intimate related to insulin resistance. In recent years, with raising standard of living and changes of life style, the incidence of type 2 diabetes increases. Insulin resistance (IR) is one of the important mechanisms in the development of type 2 diabetes mellitus and insulin signal transduction pathway play a key role in this course. Thus, the direct actions of ethanol on specific components of the pathways which mediates glucose disposal and the mechanisms underlying ethanol-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 functions of insulin signal transduction molecules would impair glucose disposal into peripheral tissues. Insulin produces a diverse array of metabolic action by binding to a heterotetrameric receptor protein consisting of two a and twoβsubunits. Phosphorylated insulin receptor binds to, and activates IR substrate (IRS), which in turn leads to translocation of glucose transporters to the cell surface via a complicated cascade of signaling events that include phosphatidylinositol 3-kinase (PI3K), Akt/PKB.Protein-tyrosine phosphatases (PTPases) play a critical role in the regulation of carbohydrate metabolism. PTP-1B is a member of the PTPases family. PTP-1B can inhibit the insulin-signaling pathway by dephosphorylating IR and IRS. PTP-1B has been reported to be elevated in diabetes and in insulin-resistant states. Transgenic overexpression of PTP-1B decreased glucose uptake in muscle. Ablation of PTP-1B, in contrast, improves glucose uptake. Mice lacking PTP-1B are hyper-responsive to insulin and resistant to diet-induced obesity.Skeletal muscle is the largest organ that utilizes glucose in an insulin-dependent manner, and therefore represents a key site for the pathogenesis of diabetes, regardless of the specific events that triggers glucose intolerance. The present study was designed to examine whether insulin intolerance induced by chronic ethanol exposure is correlated to PTP-1B expression/activity in the skeletal muscle in a rat model of chronic ethanol exposure. Molecular targets of PTP-1B (e.g. IR and IRS-1) and downstream molecules of the insulin signaling pathway (e.g. PI3K and Akt) were also examined.Method:1. Animal care and feeding:Sixty adult male Wistar rats were housed individually. Rats were acclimated for one week prior to receiving daily edible ethanol (H,5 g·kg-1·d-1; M,2.5 g·kg-1·d-1 or L,0.5 g·kg-1·d-1) or water(C, control,5 g·kg-1·d-1) via gastric gavage for 22 weeks (n=15 in each group).2. General parameters:Investigate the general parameters such as body weight, plasma ethanol concentrations, fasting blood glucose and insulin and calculate HOMA Value of rats.3. Effects of ethanol on classic insulin signal pathway of rats'skeletal muscle:(1) PTP-1B mRNA levels were measured using real-time PCR. PTP-1B protein levels were measured using western blotting. PTP-1B activity was assayed with a p-nitrophenol phosphate (PNPP) hydrolysis method, respectively.(2) Total IRβand IRS-1, as well as their phosphorylated forms, were measured using western blotting, respectively.(3) We examined the protein expression of Akt and P-Akt by western blotting.(4) We examined the protein expression of MAPK and P-MAPK by western blotting.(5) We also examined the protein expression of GLUT4 by western blotting.Result:1. Evaluation of insulin sensitivity:After ethanol treatment for 22 weeks, no significant differences were detected of fasting plasma glucose, fasting serum insulin and 2h plasma glucose levels after oral glucose load.2. Effects of ethanol on classic insulin signal pathway of rats'skeletal muscle:(1) Compared with the control group, the expression of PTP-1B was elevated in the skeletal muscle by ethanol at the dose of 2.5 and 5 g·kg-1·d-1, but not at a lower dose of 0.5 g·kg-1·d-1.(2) In this study, our results indicated that PTP-1B activities were significantly elevated in both group H (2-fold, P<0.01) and group M (1.6-fold, P<0.05), compared to PTP-1B activities in group C. However, a lower dose of 0.5 g·kg-1·d-1 did not change of PTP-1B activity in the skeletal muscles significantly.(3) Compared with the control group, total IRβ.and IRS-1, as well as their phosphorylated forms, was decreased by ethanol at the two higher doses. Moreover, ethanol exposure caused decline in P-IRβ/IRβprotein ratios dose-dependently (p<0.01 for high-dose, and p<0.05 for medium-and low-dose) and of P-IRS-1/ IRS-1 protein ratios significantly by high-(p<0.01) and medium-(p<0.05) dose, respectively.(4) Compared with the control group, chronic ethanol consumption resulted in significantly reduced levels of P-Akt (p<0.05 and p<0.01 for 2.5 and 5 g·kg-1·d-1, respectively). A lower dose of 0.5 g·kg-1·d-1 did not affect the expression. In contrast, the levels of total Akt proteins were similar among the four groups.(5) Compared with the control group, chronic ethanol consumption resulted in significantly reduced levels of P-MAPK (p<0.05 and p<0.01 for 2.5 and 5 g·kg-1·d-1, respectively). A lower dose of 0.5 g9·kg-1·d-1 did not affect the expression. In contrast, the levels of total MAPK proteins were similar in control and ethanol-treated groups.(6) Compared with the control group, the expression of GLUT4 was decreased in the skeletal muscle by ethanol at the dose of both 2.5 and 5 g·Kg-1·d-1, but not at a lower dose of 0.5 g·kg-1·d-1.Conclusions:1. Chronic ethanol consumption could impair insulin sensitivity of rats'skeletal muscle.2. Chronic ethanol intake at sufficient doses could down-regulate the expression of IRβ, P-IRβ, IRS-1, P-IRS-1 and P-Akt in rat skeletal muscle, possibly through increased PTP-1B activity. This increase in either the expression or the activity of PTP-1B was supposed to be related to the dysfunction of insulin which induced insulin resistance in rat skeletal muscle after chronic ethanol feeding.
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