| Background:Excessive ethanol consumption may cause many diseases such as hepatitis of ethanol, fatty liver and many nevers damages related to nueropathy, sometimes may induce insulin resistance, but there are no enough knowledge and theory about ethanol consumption with glycogen metabolism , epidemiological data relating the ethanol consumption to glucose metabolism or insulin sensitivity are contradictory or lack of evidence . For example, it was known that regular moderate ethanol drinkers are more insulin sensitive than abstainers. On the other hand, large dose ethanol might cause insulin resistance and hepatocirrhosis, alcoholic hepatitis and alcoholic sedation, fatty liver. Some researcher reported that lipid vacuoles and fattiness of liver tissue were found after long term intake of ethanol.The mechanism by which acute alcohol exposure impairs glycose metabolim of liver is underinvestigation, such as hypoglicemia, change of insulin sensitivity. It has been demonstrated that impaired metabolism balance of glucose transportation and its consumption was found, the ethanol may influences insulin cell signaling, whether the long term feeding of ethanol influence the glycogen and whether glycogen synthesis pathways(enzymes) play a important role remain unclear.GSK-3beta is one of the key role in insulin signaling, there are two isomers, which influences PI3-K/PKB, involves regulation and synthesis of glycogen and proteins. Also, it has been demonstrated that the GSK-3beta participates in diabetes, tumor and Alzheimer's disease. Some research found that inhibition of GSK-3beta is associated with decreasing of G6PD -kinase or PCK1, lowed their output of liver glucose, heterogeneous glucose and finally increase glycogen syntheses .AMPK is another key regulator for all cellular energy metabolism, and it has been proved as a regulator of insulin sensitivity. The investigation reported that the down-regulated activity of AMPK in rats' hepatocyte after ethanol feeding participate in the mechanism of alcoholic fatty liver. Recently finding also showed a beta -glycogen binding domain(beta-GBD) which may deals with glycogen motebolism at AMPK, Whether a mechanism is involved in the ethanol-induced glycogen depletion remains to be elucidated.GLUT4 mainly assistances glucose transportation in muscle, but it may play a role in whole body metabolism of glucose including glycogen, AMPK may contribute to insulin-stimulated glucose transport in this satuation. Recently, researchers found that an ethanol-induced impairment of insulin-stimulated glucose uptake in some tissues such as adipocytes. Whether such a mechanism is also involved in liver tissue and specially on glycogen metabolism remains to be confirmed.Objective:1) To investigate the influences of long-term ethanol feeding on body weight, liver weight, plasma glucose, plasma insulin and HOMA Value in WISTAR rats.2) Find the effect of chronic ethanol feeding on the glycogen content, H &E staining, PAS-staining in liver tissue of rats.3)Probe into the changes of GSK-3beta expression both IF and Western blot after ethanol exposure and its possible role on glycogen metabolism.4)Examine AMPK activity , expression both mRNA and protein level after long-term feeding of ethanol, and their correlations with other enzymes pathways dealing with glucose metabolism and transportation.5) Detect the alternations of GLUT4 expression and its impact on the glucose transport, insulin signaling after ethanol feeding.Methods:1) Animal feeding: Forty-eight male WISTAR rats, were divided into four groups, received either distilled water (control D group) or ethanol, which was administered by gastric tube with a single daily dose as 5 g·kg-1 (large dose C group), 2.5 g·kg-1 (middle dose B group ) and 0.5 g·kg-1 (small dose A group).2) General parameters: Investigate the general parameters such as body weight, liver weight, fasting blood glucose and insulin and calculate HOMA Value of these rats.3) Evaluation of liver tissue expression: Pathological characteristics of H & E staining and PAS staining was done after 5 months ethanol treatment.4)Measurement of glycogen content: with M2 analyser ,the glycogen in liver tissue of rats was measured by Colorimetry.5)Effect of ethanol on GSK-3beta:GSK-3beta was measured with both immunoflurence and Western blot methods on liver tissue of rats after ethanol exposure.6) Effects of ethanol on expression and activity of AMPK: The mRNA levels of AMPK-alpha were measured using Real-Time PCR and RT-PCR; Protein levels of total AMPK alpha subunit were detected using Western blot; Activities of phosphated AMPK alpha subunit were evaluated using Western blot.7) Effects of ethanol on GLUT4: GLUT4 (both mRNA and protein levels )of rats liver were measured using Real-time PCR and Western blot.Results:1) The evaluation on body parameters and glucose, insulin: After ethanol treatment for 5 months, significant differences were detected on body weight and liver weight, the more the ethanol, the less of the weight, which indicate a positive correlation between them. Fasting plasma glucose and fasting serum insulin showed no significant differences, HOMA value changes but showed no statistical difference.2) The characteristics of liver tissue staining: H & E staining showed lots of lipid vacuoles and inflammatory lymph cells in A, B and C groups compared with D group. On the other hand, PAS staining revealed a reduction of glycogen granule. Long-time intake of ethanol influences glycogen content and makes some damages on the structure of liver tissue.3) Glycogen levels of liver tissure: Compared with D group, A, B and C groups showed a lower levels of Glycogen, the glycogen reduction was in a ethanol dose-dependent manner.4)Effects of ethanol on GSK-3: The activities of GSK-3beta of rats liver were significant over-expression (both IF and Western blot) , The high signal markers mainly found at area surrounding vessels in A,B,C groups, and the expression is likely a positive result on rats after alcohol feeding.5) 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.6) Influence on GLUT4:Ethanol feeding with daily ethanol of all dosages significantly decreased GLUT4 mRNA expression , whereas a significant change of GLUT4 protein expression was found in 5g·kg-1 alcohol-fed group. Conclusions:1)Chronic ethanol consumption may lower body weight, liver weight, impair liver tissue both at structures and cellular levels (lipid vacuoles and inflammatory cells),on the other hand, glycogen content and glycogen granule greatly depleted.2)Ethanol may increase GSK-3beta expression which means inhibite glycogen syntheses activities at the same time, This could be the reason of glycogen depletion in rats.3) Ethanol treatment impaired the activity of AMPK in rats' liver, AMPK is a known positive regulator of multi metabolism including insulin signaling, energy balance and glycogen synthesis, AMPK might participate in the mechanism of ethanol-induced glycogen over-consumption.4)Ethanol could decrease GLUT4 expression in liver, the rats were accompanied with glucose intake and transport problems.5)All in all, the machanism of glycogen depletion might be the involvement of series problems such as glucose intake, transport, decresed glycogen syntheses .A lowed AMPK activities, might be the key role which relax the inhibition of GSK-3, decrease GLUT4 function and contribute to GS reduction, followed by glycogen depletion.
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