| Background:Excess of ethanol consumption has been a serious social problem and ethanol is the most abused drugs used at present.Epidemiological data showed that excess ethanol consumption may cause many diseases such as ethanolic hepatitis and fatty liver.Also,ethanol disturbs the metabolism of carbohydrate,fat and protein.Because of the importance of homeostasis of blood sugar,more and more researches focus on the effects on glucose metabolism caused by ethanol.It reported that acute ethanol treatment resulted both in severe hypoglycemia and hyperglycemia,which attributes to the glycogen deposition.It's important to know what happens to glycogen metabolism after ethanol exposure.Glycogen synthesis and glycogenolysis is one of the most important biochemistry functions of liver,which provides energy not only for liver but also for other parts of the body.Decrease in blood sugar enhances glycogenolysis and gluconeogenesis. Glycogen synthase(GS) is a key enzyme in glycogen synthesis and its phosphorylated form is inactive.Glycogen synthase kinase-3(GSK-3),a serine/threonine protein kinase,is one of the key roles in glucose metabolism.There are two isomers, GSK-3α/β,which involve in signal transduction.Under basal conditions,GS is maintained in a low-activity state principally through the continual phosphorylation by GSK-3α/βand GS is inactivated.Also,GSK-3's activity can be inhibited by phosphorylation at Ser21 of GSK-3αor Ser9 of GSK-3β,as a result,phosphorylation of GS is inhibited and glycogen synthesis is promoted.Actually,to measure the expression of GSK-3 reflect the activity of GS and studies focus on the enzyme of GSK-3β.Earlier studies have reported that the liver glycogen content decreased after 5 months ethanol treatment by intragastric administration and the activity and expression of GS in rat liver tissue decreased.There was no further discussion of GSK-3 caused by ethanol.What would happen to the glycogen content and GSK-3 if L02 cell were treated with ethanol in vitro should be further discussed.AMP-activated protein kinase(AMPK) is a key regulator of intracellular of energy homeostasis.AMPK is a heterotrimeric complex composed of a catalyticαsubunit and regulatoryβandγsubunits.The kinase is activated by an elevated AMP/ATP ratio due to cellular and environmental stress.Phosphorylation at Thr172 is required for AMPK activation.Ethanol is rich in energy(7.1 kcal/g) and it reported that ethanol induced decrease or increase in the phosphorylation of AMPK.What would happen to AMPK of human L02 cell after ethanol exposure and the relationship between AMPK and GSK-3 are sparely reported.Few investigations on effects of ethanol on glycogen synthesis were reported,let alone effects of normal human liver cell.Now,our experiment concentrates on the effects of ethanol treated L02 cell,the normal human liver cell.What would happen to the glycogen content,the protein expression of GSK-3βand P-AMPK after ethanol treatment? And we'll also study the correlation of the two kinds of protein.Objective:To investigate the effects of ethanol on glycogen content,glycogen synthase kinase-3β(GSK-3β) and phospho-AMP-activated protein kinase(P-AMPK) of human liver L02 cell in vitro.And to study whether there is a link between P-AMPK and GSK-3βafter AICAR or Compound C added which works as a selective agonist or inhibitor of AMPK respectively.Methods:L02 cells were cultured in RPMI 1640 medium with 10%Ovine Calf Serum at 37℃in the presence of 5%CO2 in a humidified incubator.Cells were treated with ethanol and were divided into 0,50 or 100 mmol/L group according to different ethanol concentration,while 0 mmol/L group treated with culture medium as before. After the treatment of ethanol for 24 hours,all cells were collected for further analysis. Glycogen content was measured by anthrone-sulfuric acid colorimetry and the expression of both GSK-3βand P-AMPK was observed by western blot analysis. After AICAR and Compound C which works as a selective agonist or inhibitor of AMPK respectively treated the cells for 1 h prior to ethanol,GSK-3βexpression was determined.SPSS11.5 software were used for statistics.Results:Compared with 0 mmol/L group,the glycogen content decreased by 41.3% (P＜0.01) and 52.8%(P＜0.01) respectively in 50 mmol/L and 100 mmol/L group. Protein expression of GSK-3βand P-AMPK increased in ethanol groups.The expression of GSK-3βincreased by 15.2%(P=0.011) and 20.1%(P=0.002) respectively in 50 mmol/L and 100 mmol/L groups,while P-AMPK by 8.99% (P=0.002) and 10.3%(P=0.001) respectively.No obvious change of GSK-3βexpression was observed after either AICAR or Compound C treatment in control and ethanol group.It showed statistical significance between control and ethanol groups, while no statistical significance between ethanol groups no matter in glycogen content or protein expression.Conclusions:Glycogen content of L02 cell decreased after ethanol exposure.This reduction might be due to the GSK-3β.Activated AMPK may be as a result of energy disturbance of L02 cell and might connect with decreased glycogen synthesis.No obvious correlation was observed between GSK-3βand P-AMPK.
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