饮食诱导的小鼠非酒精性脂肪性肝病进展中糖代谢酶的变化

Changes in glucose metabolism enzymes during progression of diet-in⁃duced nonalcoholic fatty liver disease in mice

目的:利用小鼠非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)模型明确非酒精性脂肪肝(nonalcoholic fatty liver,NAFL)向非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)进展中糖代谢酶蛋白表达水平的变化。方法:将48只C57BL/6J小鼠分为对照饮食(control diet,CD)组和高脂饮食(high-fat diet,HFD)组,每组24只,均在喂养后第8、12、16和20周处死6只小鼠。监测各组小鼠体重、肝重和血清指标的变化;HE染色和油红O染色明确肝脏脂肪变性情况并进行NAFLD活动度评分(NAFLD activity score,NAS),以明确NAFL向NASH进展的时点;Western blot实验检测糖代谢酶蛋白水平变化并与NAS进行相关性分析,明确糖代谢酶蛋白水平与NAFLD进展的关系。结果:相比CD组,HFD组小鼠在喂养4周后体重开始增加,肝重在16周时明显增加,血清丙氨酸转氨酶和血糖在12周后开始上升,总胆固醇在16周后开始升高(均P<0.05)。肝组织HE染色、油红O染色及NAS提示,小鼠在16周时大部分进展为NASH。Western blot结果提示,喂养16周后,与CD组相比,HFD组小鼠肝脏糖酵解相关酶己糖激酶1(hexokinase 1,HK1)、HK2、丙酮酸激酶M2(pyruvate kinase M2,PKM2)和乳酸脱氢酶A(lactate dehydrogenase A,LDHA)蛋白表达水平显著升高(均P<0.05),而糖异生相关酶葡萄糖-6-磷酸酶催化亚基(glucose-6-phosphatase catalytic subunit,G6PC)、果糖-1,6-二磷酸酶1(fructose-1,6-bisphosphatase 1,FBP1)和磷酸烯醇式丙酮酸羧激酶1(phosphoenolpyruvate carboxykinase 1,PCK1)蛋白表达水平显著下降(均P<0.05);HFD组HK2、PKM2、LDHA、G6PC、PCK1和丙酮酸羧化酶(pyruvate carboxylase,PC)的蛋白水平在第8周进展到第20周时发生明显变化(均P<0.05),且与NAS存在明显相关性,其中PKM2和PC与NAS存在强正相关,而PCK1与NAS存在强负相关。结论:糖代谢酶G6PC、PCK1、PC、HK2、PKM2和LDHA的蛋白表达水平在NAFL向NASH的进展过程中发生明显变化,对这些酶的干预可能有助于抑制NAFLD的进展。

AIM:Utilizing a murine model of nonalcoholic fatty liver disease(NAFLD),we investigated alterations in hepatic protein expression of glucose metabolism enzymes during the progression from nonalcoholic fatty liver(NAFL)to nonalcoholic steatohepatitis(NASH).METHODS:Male C57BL/6J mice were randomly divided into control diet(CD)group(n=24)and high-fat diet(HFD)group(n=24).Six mice in each group were euthanized at 8,12,16 and 20 weeks.Changes in body weight,liver weight and serum biochemical parameters were monitored.Hepatic steatosis was evaluated by HE staining and oil red O staining.The NAFLD activity score(NAS)was determined to identify the time‑point of NAFL to NASH transition.Western blot analysis was conducted to examine alterations in glucose metabolism en-zyme expression.Correlations between the protein levels and NAS were analyzed to elucidate the associations with NAFLD progression.RESULTS:Compared with CD-fed mice,HFD-fed mice exhibited increased body weight starting at 4 weeks,markedly elevated liver weight at 16 weeks,increased serum alanine aminotransferase and glucose levels after 12 weeks,and increased serum total cholesterol level after 16 weeks(all P<0.05).Histological assessments including HE staining,oil red O staining and NAS determination signified that most HFD-fed mice developed NASH at 16 weeks.Com-pared with CD-fed mice,HFD-fed mice displayed up-regulated hepatic protein expression of glycolysis-related enzymes,hexokinase 1(HK1),HK2,pyruvate kinase M2(PKM2)and lactate dehydrogenase A(LDHA),at 16 weeks(all P<0.05),while gluconeogenesis-related enzymes,glucose-6-phosphatase catalytic subunit(G6PC),fructose-1,6-bisphos-phatase 1(FBP1)and phosphoenolpyruvate carboxykinase 1(PCK1),were decreased(all P<0.05).Remarkably,HK2,PKM2,LDHA,G6PC,PCK1 and pyruvate carboxylase(PC)protein levels exhibited significant alterations between 8 and 20 weeks in HFD-fed mice(all P<0.05)and displayed marked correlations with NAS,among which PKM2 and PC showed strong positive correlation,while PCK1 exhibited robust negative correlation.CONCLUSION:The protein ex-pression levels of glucose metabolism enzymes G6PC,PCK1,PC,HK2,PKM2 and LDHA are notably altered during NAFL to NASH progression,implying that targeting these enzymes may aid in impeding NAFLD progression.