Van Auken M, Rulfs J, Buckholt M A, Garnache A K, Miller T B
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655, USA.
J Cell Physiol. 1996 Jan;166(1):208-16. doi: 10.1002/(SICI)1097-4652(199601)166:1<208::AID-JCP22>3.0.CO;2-9.
When 3-4-week-old rats (young rats) are used as a source of hepatocytes, primary culture cells express the adult, differentiated, liver-specific isoform of glycogen synthase. Synthase enzyme protein levels are relatively stable over a 3 day culture period in young but not in adult (> 150 g rat) hepatocyte cultures. Corresponding synthase enzyme activity and mRNA levels decrease over time in culture in adult but not in young hepatocyte cultures. Young rat hepatocytes also have the ability to proliferate in chemically defined medium in the absence of added mitogens. A diabetes-induced increase in total synthase activity has been demonstrated by our lab and others, using cultured hepatocytes, liver homogenates, and perfused livers. In the present study, utilizing synthase-specific antibody and primary cultures of cells from young normal and alloxan diabetic rats, we found that greater total synthase activity in the diabetic cells was associated with higher levels of enzyme protein. Immuneprecipitation of 35S methionine-labeled freshly plated cells demonstrates an increase in the rate of protein synthesis in diabetic as compared with normal cells. Synthase mRNA levels are correspondingly increased in the diabetic relative to normal cells. Chronic exposure of young, normal hepatocytes to increasing levels of glucose induces a dose-dependent increase in total synthase activity, total synthase protein, and synthase message levels. By comparison, cells from diabetic animals do not respond by any of these measures to increased glucose concentrations. We conclude that this defined primary culture system represents a useful model for investigating the regulation of hepatic glycogen synthase and the defects which occur as a result of diabetes.
当使用3 - 4周龄的大鼠（幼鼠）作为肝细胞来源时，原代培养细胞表达糖原合酶的成年、分化的肝脏特异性同工型。在幼鼠肝细胞培养中，合酶蛋白水平在3天的培养期内相对稳定，而在成年（体重>150 g大鼠）肝细胞培养中则不然。在成年肝细胞培养中，相应的合酶活性和mRNA水平随培养时间下降，而在幼鼠肝细胞培养中则没有这种情况。幼鼠肝细胞也有能力在无添加促有丝分裂原的化学限定培养基中增殖。我们实验室和其他研究小组利用培养的肝细胞、肝脏匀浆和灌注肝脏，已证明糖尿病会导致合酶总活性增加。在本研究中，利用合酶特异性抗体以及来自正常幼鼠和四氧嘧啶糖尿病幼鼠的原代细胞培养，我们发现糖尿病细胞中更高的合酶总活性与更高水平的酶蛋白相关。对新鲜接种的35S甲硫氨酸标记细胞进行免疫沉淀显示，与正常细胞相比，糖尿病细胞中的蛋白质合成速率增加。糖尿病细胞中的合酶mRNA水平相对于正常细胞也相应增加。将幼龄正常肝细胞长期暴露于不断升高的葡萄糖水平下，会导致合酶总活性、合酶总蛋白以及合酶信息水平呈剂量依赖性增加。相比之下，来自糖尿病动物的细胞对葡萄糖浓度升高没有这些反应。我们得出结论，这种明确的原代培养系统是研究肝糖原合酶调节以及糖尿病导致的缺陷的有用模型。
