The Effect Of Hepatocyte-specific Nrf2 Knockout On Hepatic Glucose And Lipid Metabolism Dysfunction In Mice Induced By Dietary Energy Excess

Objective: As the main energy source of the human body,glucose and fatty acids are often mutually regulated in terms of metabolism.Glucose can be converted into fatty acids and cholesterol through the pathway of lipid synthesis,and excessive lipid secretion or lipid substances stored in lipid droplets will regulate the decomposition of glycogen.With the progression of science and technology and the improvement of living standards,high-fat and high-sugar diets are available,gradually change to the role of a health killer.In fact,people have to face new health challenges: excessive dietary nutrition,especially the resulting excess dietary energy,when the body’s energy intake and utilization level is seriously imbalanced,will cause glucose and lipid metabolism disorders,which promotes non-alcoholic fatty liver disease(NAFLD),obesity,diabetes,hyperlipidemia,high cholesterol and other chronic diseases to increase.Paying attention to the impact of dietary energy excess on the liver glycolipid homeostasis has important public health significance for maintaining the health.Excess energy intake can lead to obesity and insulin resistance,which have been identified as important risk factors for hepatic glucose and lipid metabolism disorders.Nuclear factor erythroid-derived 2-like 2(NFE2L2,Nrf2)has important regulatory functions in mitochondrial biogenesis,adipocyte differentiation and liver energy metabolism.Recent studies have shown that the Nrf2/ARE pathway plays an important role at the intersection of energy metabolism and nutrient intake.Genomic,protein,and physiological data from several laboratories also demonstrate the involvement of Nrf2 in energy metabolism.The liver,as the center of the body energy metabolism network,should receive high attention during this process.However,most of the current research focuses on the effect of Nrf2 on the overall metabolism of the body.In the current study,by establishing a hepatocyte-specific Nrf2 knockout mouse model and giving excessive dietary energy intake,intuitively study the regulation of hepatic Nrf2 on the homeostasis of glucose and lipid metabolism in dietary energy excess.Provide experimental basis and theoretical basis for the prevention and treatment of chronic diseases related to dietary energy excess.Methods:1.Hepatocyte-specific Nrf2-specific knockout mice model of hepatic glucose and lipid metabolism disorder caused by excessive energy intake: Hepatocyte-specific Nrf2knockout(Nrf2(L)-KO)mice and littermate control(Nrf2-Lox P)mice.The obtained two kinds of mice were crossed with high food intake mice,that is,OB/+ mice,and crossed for more than two generations to obtain stable Nrf2 knockout(Nrf2(L)-KO:ob/ob)mice in the context of high food intake and littermate control(Nrf2-Lox P:ob/ob)mice.We fed the wild-type group(Nrf2(L)-KO:WT,Nrf2-Lox P:WT)and the high food intake group(Nrf2(L)-KO:ob/ob,Nrf2-Lox P:ob/ob)adequate normal feed,a total of four groups of 6-8 each.Each group was fed for 20 weeks,the data of diet and drinking water were dynamically monitored,the body weight of the mice was measured every week,and blood sugar indicators of the mice were monitored.At the end of observation,mouse plasma and various vital organ tissue samples were collected.2.Sample processing: the collected liver tissue was fixed with 4% paraformaldehyde and then stained with H&E or oil red.The kit was used to detect triglyceride,low-density lipoprotein,high-density lipoprotein and total cholesterol;For the frozen liver tissue,take a part to extract the total RNA of the liver tissue,and test the knockout effect of Nrf2 gene;Extract the total protein of the liver tissue to detect the level of key proteins such as the regulation of glucose,lipid and lipid metabolism,etc.3.Using RNA-seq to detect the effect of hepatocyte-specific Nrf2 knockout on the homeostasis of liver glucose and lipid metabolism in the context of excessive energy intake:we took normal dietary energy group(Nrf2(L)-KO:WT,Nrf2-Lox P:WT)mice and excess energy intake group(Nrf2(L)-KO:ob/ob,Nrf2-Lox P:ob/ob)mice were fed with adequate normal feed.Liver tissues of mice were collected for high-throughput genomics sequencing to explore the effect of hepatocyte-specific Nrf2 knockout on glycolipid homeostasis in hepatic steatosis induced by excess energy intake.Results:1.The adipose tissue weight of Nrf2(L)-KO mice was reduced under the background of excess dietary energy.Under excessive dietary energy feeding,the body weight of the 14-week-old Nrf2-Lox P: ob/ob group mice was significantly higher than that of the Nrf2(L)-KO: ob/ob group(P< 0.05),and the body weight of the mice in the two groups at other time points No significant difference.The paragonadal fat and subcutaneous fat in the Nrf2(L)-KO group were lower than those in the Nrf2-Lox P group,and the difference was statistically significant(P< 0.05).There were no differences in other organs between the two genotypes.There was no significant difference between the two genotypes in basic indicators such as food and water intake and blood sugar level.2.The blood lipid excretion of Nrf2(L)-KO mice decreased under excessive dietary energy feeding,but there was no significant difference in hepatic steatosis.There was no difference in plasma TG levels among the four groups of mice.The levels of total cholesterol,very low-density lipoprotein and high-density lipoprotein in plasma of Nrf2-Lox P group were significantly higher in ob/ob mice than in WT mice(P<0.05),while in plasma of ob/ob mice The levels of total cholesterol and very lowdensity lipoprotein in Nrf2(L)-KO group were significantly lower than those in Nrf2-Lox P group(P<0.05).The results of H&E staining and Oil Red O staining showed no significant difference in liver fattyness.The TG level in the liver of the ob/ob group was significantly higher than that of the WT group(P < 0.05),while no significant changes were observed within each group.The level of T-CHO in liver of Nrf2-Lox P mice in ob/ob group was significantly higher than that in WT group(P< 0.05),but no similar change was seen in Nrf2(L)-KO group.Glycogen levels in liver of mice in ob/ob group were significantly lower than those in WT group(P< 0.05),while no significant changes were observed within each group.3.Deletion of Nrf2 in hepatic cells affects carbohydrate metabolism in the liver.Acetaldehyde dehydrogenase(ALDH1A1),carbohydrate response element binding protein(Carbohydrate response element binding protein,CHREBP),acetylated superoxide dismutase SOD2(Acetyl-SOD2)in mouse liver under excessive dietary energy feeding,protein expression of β-oxidation-related protein PPARα,and the first rate-limiting enzyme glucokinase(GCK)catalyzing glycolysis were significantly higher than those in the WT group(P< 0.05),protein expression of lipid transport-related proteinuria protein(Urinary protein,MUP)was significantly lower than those in the WT group(P< 0.05).The ratelimiting enzyme glucose-6-phosphatase(G6Pase),which catalyzes hepatic gluconeogenesis,and acetaldehyde dehydrogenase(ALDH1A1),which affects the pathway of hepatic gluconeogenesis,were significantly lower in hepatocytes after knockout of Nrf2 in ob/ob background.In the same group as the control(P< 0.05),forkhead box protein O1(FKHR/FOXO1)had no significant change among the groups.The protein acetylation levels in the liver of mice in each group tended to increase but not significantly.However,there were no significant differences in apolipoprotein B48(APOB48)and fatty acid transmembrane protein(Platelet glycoprotein 4,CD36)among the genotypes.The sterol regulatory element binding protein 1(SREBP1),which regulates lipid production,was slightly increased in ob/ob mouse liver,but it was significantly lower than that of the control group after Nrf2 knockout in hepatocytes(P< 0.05).There was no significant difference in enhancer-binding protein C/EBPα in CAAT region among all genotypes.While C/EBPβ was significantly decreased in WT mice with hepatic Nrf2 knockout,but this difference was not seen in ob/ob mice(P< 0.05).Hepatocyte nuclear factor 4α(HNF4α)in the ob/ob mouse liver was significantly higher than that of the control group after Nrf2 knockout(P< 0.05).Conclusions:1.Under excessive dietary energy feeding,mice with elevated plasma lipids and liver lipids developed severe hepatic steatosis.2.The adipose tissue weight and blood lipid excretion of hepatic cell-specific Nrf2 knockout mice were reduced under excessive dietary energy feeding,but there was no significant difference in the degree of hepatic steatosis.3.Nrf2 in hepatocytes regulates carbohydrate and lipid metabolism in the liver,and increases the acetylation level of liver proteins to a certain extent.4.Under excessive dietary energy feeding,Nrf2 in hepatic cells may regulate the homeostasis of glucose and lipid metabolism in the liver by affecting the four major metabolic pathways,including lipid metabolism,retinal metabolism,glutathione metabolism,and cytochrome P450 metabolism.