苹果酸酶在葡萄糖和胰岛素诱导鹅肝脂肪变性中的作用研究

本研究旨在研究不同浓度葡萄糖和胰岛素诱导鹅肝脂肪变性过程中苹果酸酶(Malic enzyme,ME)的活性及其mRNA表达情况,以探讨ME在鹅肝脂肪变性中的作用。根据鸡ME基因序列保守区设计引物,通过RT-PCR、克隆测序等技术扩增鹅ME基因;分离、培养四川白鹅原代肝细胞,添加不同浓度葡萄糖和胰岛素诱导肝细胞脂肪变性,检测细胞内甘油三酯(TG)含量、ME活性及其mRNA表达情况。结果发现,鹅ME基因跟原鸡同源性最高;与对照组相比,葡萄糖及其与胰岛素协同作用均能显著促进肝细胞内甘油三酯(TG)沉积,且呈现出剂量递增效应,而胰岛素对细胞内TG含量影响不明显;与对照组比较得出:ME的活性和mRNA表达水平随着葡萄糖浓度升高而增加,30mmol.L-1葡萄糖具有明显的促进作用(P<0.05);低浓度(50nmol.L-1)胰岛素显著增加ME的活性和mRNA表达水平(P<0.05),当胰岛素浓度达到200nmol.L-1时,ME的活性和mRNA表达水平受到一定抑制;葡萄糖和低浓度胰岛素(50nmol.L-1)协同能促进ME的活性和mRNA表达水平。本研究扩增获得了鹅ME基因部分序列,且发现葡萄糖和胰岛素协同可以增强ME的活性和mRNA表达水平,从而显著增加肝脏中TG的含量,诱导鹅肝脂肪变性。

The Role of Malic Enzyme in Goose Hepatic Steatosis Induced by Glucose and Insulin

The experiment was conducted to study the enzyme activity and mRNA expression of malic enzyme(ME) induced by glucose and insulin in goose primary hepatocyte,and identify the role of malic enzyme in goose hepatic steatosis.In this study,partial sequence of ME gene was cloned using Sichuan White goose.The goose primary hepatocyte was cultured and hepatic steatosis was induced by means of adding different concentrations of glucose and insulin,malic enzyme activity and mRNA expression and the triglyeride(TG) content in cells tested were detected.The goose ME gene sequence had highest similarity with Gallus gallus.Compared with the control group,glucose and its synergy with insulin could significantly increase the TG content in a dose dependent manner,however,insulin alone failed to notably affect TG content;the enzyme activity and mRNA expression of ME gradually increased with glucose concentrations increasing,and 30 mmol·L-1 glucose could significantly increase those parameters(P<0.05);low contents of insulin(50 nmol·L-1)could largely elevate the enzyme activity and mRNA expression of ME(P<0.05),which could be suppressed by 200 nmol·L-1 insulin.In addition,the synergy of glucose with lower content of insulin(50 nmol·L-1) could significantly increase the enzyme activity and gene expression of ME.In this study,the partial sequence of goose ME gene were cloned.Meanwhile,glucose and insulin could coordinately promote the mRNA expression and enzyme activity of ME in goose liver resulting in TG accumulation.