高脂饮食诱导胰岛素抵抗过程中肝脏能量代谢特征的研究

Study on the Characteristics of Liver Energy Metabolism during the Induction of Insulin Resistance by High Fat Diet

本研究旨在通过建立胰岛素抵抗全过程的小鼠模型,研究在这个过程中,肝内线粒体功能和能量代谢的变化情况。试验选取60只雄性C57BL6小鼠,随机选取30只饲喂普通维持饲料作为对照组(Con组),另外30只饲喂高脂饲料作为高脂饮食组(HFD组)进行造模,分别于饲喂的第4、6、8、10和12周,各组随机选取6只小鼠进行处理,经胰岛素抵抗评估和病理组织学检查确认成功建立胰岛素抵抗全过程的模型。在第8和12周,采集鼠尾血液,进行葡萄糖耐受试验(glucose tolerance tests,IGTT)和胰岛素耐受试验(insulin tolerance tests,ITTs)。在第4、6、8、10和12周,采集小鼠的血清和肝组织样品进行试验:1)检测空腹血糖和血清含量;2)ELISA法检测肝组织中的Srebp-1c酶、PFK1和COX-Ⅰ含量;3)生化检测血清中的ALT和AST含量;4)Western blot法检测肝组织中的PI3K、Akt、P-Akt、GLUT4、GSK-3β、P-GSK-3β、FOXO1、P-FOXO1、SIRT1、AMPK、P-AMPK、PGC-1α蛋白表达量;5)采用透射电镜、HE染色、油红O染色、PAS染色检测肝病理组织和结构的变化。结果表明:1)12周的高脂日粮引起小鼠肥胖、HOMA-IR增加,油红O染色显示,明显的脂肪沉积,成功诱导小鼠胰岛素抵抗全过程模型;2)胰岛素抵抗过程中,小鼠ALT和AST增加,HE结果显示,明显的脂滴空泡、肝细胞结构紊乱;3)在饲喂高脂日粮的第4周之后,肝胰岛素上游信号开始受到影响;4)与Con-6组相比,HFD-6组小鼠肝内的GLUT4蛋白表达量和P-FOXO1/FOXO1比值、PFK1含量显著下降(P<0.01);与Con-4组相比,HFD-4组小鼠肝内的GSK-3β磷酸化、Srebp-1c酶水平逐渐降低(P<0.05);PAS结果显示从第8周开始,HFD组小鼠肝细胞内糖原含量减少;5)与Con-4组相比,HFD-4组小鼠肝的AMPK、P-AMPK、SIRT1含量显著降低(P<0.01和P<0.001);6)HFD组小鼠肝中Mfn2在第10周出现上升,Drp1蛋白的表达呈现先降低后升高的趋势;7)与HFD-6组相比,HFD-6组小鼠肝中的PINK1和Parkin蛋白明显下降(P<0.05)。综上表明,肝作为代谢的主要器官,在饲喂60%高脂饲料后,会发生选择性胰岛素抵抗,在抵抗后整体上能量分解代谢减弱、线粒体损伤增强,而清除损伤线粒体的自噬功能却减弱,另外在第8、10周时出现了短暂的线粒体生物发生与融合增强现象,说明由于肝能量缺乏导致了短暂的代偿作用。本研究从发生全过程的角度阐释肝胰岛素抵抗中的能量代谢特征和规律,为进一步深入研究营养过剩影响细胞能量代谢的机制提供参考。

The purpose of this study is to investigate the changes in mitochondrial function and energy metabolism in the liver during the entire process of insulin resistance by establishing a mouse model.A total of 60 male C57BL6 mice were randomly selected and divided into three groups:a control group(Con group)fed with normal maintenance feed;a high-fat diet group(HFD group)fed with high-fat feed.The model was established by randomly selecting six mice from each group at weeks 4,6,8,10,and 12 of feeding.The successful establishment of the whole-process model of insulin resistance was confirmed by insulin resistance assessment and histopathological examination.At the 8th and 12th weeks of the experiment,blood was collected from the tail of the mice for IGTT and ITTs.At weeks 4,6,8,10,and 12 of the experiment,serum and liver tissue samples were collected from the mice for testing:1)fasting blood glucose and serum content were measured;2)The contents of Srebp-1c enzyme,PFK1,and COX-I in liver tissues were detected by ELISA.3)Biochemical detection of ALT and AST levels in serum;4)Western blot was used to detect expression level of PI3K,Akt,P-Akt,GLUT4,GSK-3β,P-GSK-3β,FOXO1,P-FOXO1,SIRT1,AMPK,P-AMPK,and PGC-1αprotein in liver tissue;5)Transmission electron microscopy,HE staining,oil red O staining,and PAS staining were used to detect changes in the pathological organization and structure of the liver.The results showed that:1)12 weeks of high-fat diet caused obesity and increased HOMA-IR in mice,and oil red O staining showed significant fat deposition,successfully inducing a model of insulin resistance in the entire mouse.During insulin resistance,the levels of ALT and AST in mice increased,and HE results showed significant lipid droplet vacuoles and structural disorder of liver cells.3)After the fourth week of feeding a high-fat diet,the upstream signaling of insulin in the liver began to be affected;Compared with the Con-6 group,the expression of GLUT4 protein and the ratio of P-FOXO1/FOXO1 as well as the content of PFK1 in the liver of HFD-6 group mice significantly decreased(P<0.01);Compared with the Con-4 group,the GSK-3βexpression in the liver of the HFD-4 group mice Phosphorylation and Srebp-1c enzyme levels gradually decreased(P<0.05);The PAS results showed that from the 8th week,the glycogen content in the liver cells of HFD mice decreased;Compared with the Con-4 group,the AMPK,P-AMPK,and SIRT1 contents in the liver of the HFD-4 group significantly decreased(P<0.01 and P<0.001);6)In the HFD group,the expression of Mfn2 in the liver increased at week 10, while the expression of Drp1 protein showed a trend of decrease first and then increase. Compared with the HFD-6 group, the PINK1 and Parkin proteins in the liver of the HFD-6 group mice decreased significantly ( P <0.05). In summary, the liver, as the main organ of metabolism, undergoes selective insulin resistance after feeding 60% high-fat feed. After resistance, overall energy catabolism decreases, mitochondrial damage increases, and autophagy, which clears damaged mitochondria, weakens. In addition, there is a brief increase in mitochondrial biogenesis and fusion at weeks 8 and 10, indicating a temporary compensatory effect due to energy deficiency in the liver. This study explains the characteristics and regularities of energy metabolism in hepatic insulin resistance from the perspective of the entire process, providing a reference for further studying the mechanism of excess nutrients affecting cellular energy metabolism.