【作者】 陈少华；

【导师】 赵家军；

【作者基本信息】 山东大学 ， 内分泌与代谢病， 2007， 博士

【摘要】 背景;酒精是目前现代世界各国社会中饮用广泛的饮料,具显著成瘾性,长期、大量的摄入酒精对机体健康产生多方面危害和影响,如酒精性肝炎、脂肪肝、酒精性精神依赖等。酒精对机体肝脏负面影响已经有众多研究证实,但酒精对于肝脏内糖代谢以及糖原代谢有何种影响以及作用的环节则一直没有定论。流行病学资料提示,长期、少量规律饮酒可以增加胰岛素敏感性,降低2型糖尿病的发病率;而长期和大量饮酒则可诱导胰岛素抵抗,损害机体糖耐量。多项研究证实,大量饮酒对如酒精及其代谢产物可引起肝脏的脂肪变、炎症及酒精性肝硬化等病变,酒精肝和脂肪肝一样是人类重要疾病之一,长期过量酒精（乙醇）的摄入能使肝细胞的脂肪的代谢紊乱,造成肝内脂肪积聚,逐渐形成酒精性脂肪肝。长期饮酒对肝脏的损伤影响逐渐被研究者所关注,如酒精及其代谢产物可引起肝脏的脂肪变,炎症及酒精性肝硬化等病变。酒精有可能通过对其氧化代谢作用影响肝脏,使糖代谢异常,糖异生障碍。国内外关于酒精对糖、脂代谢的研究虽然逐渐增多,但多集中在糖转运信号通路方面,而对于能量储存中的重要环节糖原代谢影响,酒精干预情况下的相关代谢酶和途径的研究较少,需要进行探讨。糖原合成激酶-3（GSK-3）是一个在信号转导中的关键酶,为丝/苏氨酸蛋白激酶,有两个同功异构体,分别是GSK-3alpha、GSK-3beta,目前发现参与多种代谢,GSK-3参与并影响磷脂酰肌醇-3激酶（PI3-K）/蛋白激酶B（PKB）许多下游事件,包括胰岛素诱导的糖原和蛋白质合成调控,细胞生长分化,抗凋亡过程等。GSK-3在2型糖尿病患者的骨骼肌中活性升高,有学者证实在STZ鼠肝脏中,GSK-3alpha mRNA是对照组的143%,在DM动物模型中,抑制了GSK-3的活性可以提高胰岛素的敏感性。肝组织糖原合成、储存是保障机体生存的重要生化过程,糖原合成酶（GS）是这一过程的关键酶,而它的酶活性是受GSK-3的负调控的。GSK-3通过催化GS磷酸化使之失活而阻止糖原合成,而活化的PKB可解除GSK-3对GS的负调节作用,使GS磷酸化减少,活性增加,从而促进糖原合成过程。另有实验证明GSK-3还能直接磷酸化胰岛素受体底物（IRS-1）对胰岛素信号通路起负调节作用,间接抑制葡萄糖转运摄取,而PI3-K/PKB使GSK-3失活,解除上述抑制作用,从而促进葡萄糖转运进行,但目前对此观点还有很多争议。目前研究还发现,抑制GSK-3选择性能减少葡萄糖-6-磷酸酶及磷酸稀醇丙酮酸羧激酶基因表达,从而减少糖异生和肝糖输出,提高了糖原合成。腺苷酸激活的蛋白激酶（AMPK）是对细胞内糖、脂代谢都有重要调节作用的因子,早期离体肌肉实验中就发现,AMPK激活剂刺激的AMPK可以增加细胞内糖原浓度,同时也发现AMPK对糖原合成酶和糖原磷酸化酶有磷酸化作用,也有认为糖原增加是AMPK作用于糖原代谢酶的缘故。目前估计,AMPK增加葡萄糖转运和己糖激酶Ⅱ的活性,从而使6-磷酸葡萄糖增加,6-磷酸葡萄糖可负反馈抑制糖原磷酸化酶和正反馈激活糖原合成酶,另一方面它同时作为糖原合成的底物存在。AMPK的beta亚单位被发现含有的beta-GBD（beta-糖原结合区）,尽管尚缺乏有力的研究证据,但AMPK是调节糖原的重要代谢环节,最终结果依然是AMPK促进糖原合成的途径得到大家共识。GLUT4是葡萄糖转运的重要载体,对糖和糖原代谢都是不可或缺的部分,在机体代谢过程中与AMPK关系紧密,AMPK活性表达对GLUT4起到正面调节作用,对于GLUT4的认识也是糖原代谢通路认识的深化。近年来研究发现,AMPK可以作用于GSK-3,通过降低GSK-3的活性而调高糖原的合成,还可通过多种方式调节机体胰岛素敏感性,调节GLUT4的表达等。酒精可降调肝脏组织中AMPK的活性,这是酒精诱导的脂肪肝的发生机制之一,是否也是肝脏糖原合成降低的原因?目的;1)观察长期饮酒对大鼠机体水平体重、肝脏重、血糖和胰岛素以及胰岛素敏感性指标影响,2)观察长期饮酒对大鼠苏木素伊红（H&E）染色肝脏组织结构和细胞损伤的特征。3)观察长期饮酒对大鼠肝脏组织糖原含量以及肝脏组织糖原颗粒分布状况的影响4)测定影响糖原代谢的关键酶---糖原合成激酶-3（GSK-3）活性,探讨长期饮酒以后,GSK-3活性的变化是否与糖原水平的变化密切相关。5)观察长期饮酒对肝脏中AMPK表达与活性的影响,探讨AMPK在长期饮酒的大鼠肝脏糖原代谢中的作用,同时观察肝GLUT4表达,探讨酒精干扰葡萄糖转运的可能性。方法;1)动物分组及喂养;雄性Wistar大鼠48只,按体重随机分为4组,每组12只,即小剂量饮酒A组;(酒精量0.5 g·kg^-1·d^-1);中剂量饮酒B组(酒精量2.5 g·kg^-1·d^-1);大剂量饮酒C组(酒精量5g·kg^-1·d^-1);对照D组(蒸馏水;5g·kg^-1·d^-1)。每周测体重,据体重变化调整饮酒剂量,喂养时间为5个月。2)大鼠机体一般状况指标和血液测定;大鼠试验前以及试验结束时体重,肝重,血糖,血胰岛素等指标测定,并测算相应胰岛素敏感性指标（HOMA指数）。3)酒精对大鼠肝组织结构以及糖原颗粒的影响;常规H&E染色处理动物肝脏组织切片,观察对组织结构和细胞病变的特征;糖原染色（PAS）染色处理组织切片,观察肝细胞内糖原颗粒疏密分布。4)酒精对大鼠肝脏糖原含量影响;M2酶标仪检测处理组织的光密度值,采用光电比色原理应用不同梯度标准葡萄糖浓度协助判定肝组织浆糖原含量。5)酒精对肝GSK-3beta表达的影响;组织免疫荧光法和Western blot方法检测检测大鼠肝脏组织GSK-3beta活性表达水平,判定其对糖原合成影响。6)酒精对肝脏AMPK表达的影响;RT-PCR、Real-Time PCR检测AMPKa1亚单位和AMPKa2亚单位的mRNA水平;Western blot检测总AMPKa亚单位和磷酸化的AMPKa亚单位的蛋白水平。7)酒精对肝脏GLUT4mRNA影响;Real-Time PCR检测肝脏内GLUT4的mRNA水平;同时Western blot检测上述分子的蛋白水平。结果;1)机体一般性指标影响评价;5月酒精灌胃后,与对照D组比较,服酒精A、B、C组大鼠的体重和肝脏重量有不同程度的下降,以酒精灌胃量大的C组下降最明显;酒精灌胃量与体重（P＜0.001）,肝脏重量（P＜0.001）均呈明显负相关。酒精组HOMA-IR有轻度增加,但未见统计学差异。2)肝组织H&E染色特征;酒精A、B、C组肝结构与正常对照比较呈明显松散,细胞间质减少,以高酒精剂量C组最为显著,同时有脂肪变性空泡（脂粒）,伴汇管区炎性细胞浸润。3)酒精对肝糖原水平影响;A、B、C各组酒精鼠糖原水平显著低于对照D组水平,各酒精剂量组糖原含量呈递次降低的趋势;行直线相关分析显示,糖原含量与酒精灌胃量呈明显负相关（P＜0.001）;糖原含量分别还与肝脏重量（P＜0.05）、体重（p＜0.01）均呈正相关。4)PAS染色特征;高酒精剂量B、C组细胞形态显著变形,排列紊乱,细胞问、细胞内PAS染色相对单薄,呈不均匀样;而对照D组细胞结构致密完整,排列较整齐,无裂隙,细胞间、细胞内PAS染色饱满均匀。5)肝组织GSK-3beta表达水平;肝组织免疫荧光显示明显A、B、C各酒精组肝组织均显示GSK-3染色表达强度增高。GSK-3染色表达以肝内汇管区域为著,B、C组为明显,A组强度略降低,呈围绕汇管区分布,强度与应用酒精剂量相关,对照D组无此表现。Western blot蛋白水平表达也提示服用酒精组表达较强。6)肝组织AMPK表达与活性结果;未见酒精影响AMPKa1亚单位和AMPKa2亚单位的mRNA水平;但酒精显著降调节了酒精大鼠肝组织磷酸化AMPK的蛋白水平（P＜0.05）,提示长期饮酒可抑制AMPK活性。7)肝组织GLUT4表达检测;酒精鼠肝组织GLUT4mRNA和蛋白表达水平均比正常对照组降低。结论;1)长期饮酒削弱大鼠体质,减低体重和肝重,使大鼠肝脏呈脂肪空泡和炎性病变,有可能降低其胰岛素敏感性;酒精显著降低其肝脏糖原储备水平,糖原颗粒明显稀疏减少。2)饮用酒精造成大鼠肝糖原合成关键酶GSK-3beta上调表达,而其活性提高意味着糖原合酶活性受抑,这种糖原合酶功效下降可能是酒精致大鼠糖原耗竭的原因之一。3)长期酒精摄入也导致AMPK-P肝脏表达降低,提示能量代谢核心酶AMPK的环节被干扰,无法顺利进行糖原合成和能量的储存。4)长期饮酒还同时降低了肝GLUT4表达水平,可能通过影响葡萄糖转运而减少了糖原。5)长期饮酒造成糖原储备减少的机制可能是肝葡萄糖转运被干扰、AMPK活性下降,从而减弱了对GSK-3的抑制,过高GSK-3活性使糖原合酶活性受抑制,糖原合成受阻,是多个环节共同影响糖原合成代谢。更多还原

【Abstract】 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.更多还原