Effects Of Nrf2 And Sirt3 Deficiency On Hepatic Lipid Metabolism In The Fed-fasted Cycle

Objective:Coordination of lipid metabolism in the fed-fasted cycle is essential for health.In the feeding and subsequent state,dietary fats are digested and packaged into chylomicron(CM),which can be transported from the intestines to tissues such as fat through circulation.In the fasted state,fat tissue lipolysis is enhanced,fatty acid(FA)is released into the circulation and transported to the liver for energy supply and synthesis of triglyceride(TG),which in turn can be secreted in the form of very low density lipoprotein(VLDL)-TG.Dysregulation of lipid metabolism in the fed-fasted cycle may lead to obesity,hyperlipidemia,and nonalcoholic fatty liver disease.Physiologically,the rate of FA obtained by liver uptake from plasma and De novo synthesis(DNL)by the liver is balanced with the rate of oxidation and secretion of FA in the form of VLDL-TG,which make the content of hepatic TG in the steady state is less than 5%.Thus,during the transition from fasting to eating,gene expression in the liver undergoes significant changes to maintain lipid homeostasis.Nuclear factor erythroid-2-related factor 2(NRF2)is an important transcription factor in regulating adaptive antioxidant response,and studies have proved that it can regulate lipid metabolism related factors.Fasting can increase NRF2 expression levels.Sirtuin 3(SIRT3)is a nicotinamide adenine dinucleotide(NAD~+)-dependent deacetylase,which plays an important role in various metabolic processes such as fatty acid oxidation and ketone body production.At the same time,during a certain fasting period of the body,the expression of SIRT3 in the liver will increase with the extension of fasting time.In this study,a mice model of Nrf2 or/and Sirt3 knockout was established,in order to further explore the regulation and related mechanisms of NRF2 and SIRT3 on liver lipid metabolism in the fed-fasted cycle.Methods:1.Basic and metabolic indexes:Whole-body Nrf2 and Sirt3 double knockout(DKO)mice,Nrf2 whole-body knockout(Nrf2-KO)mice,Sirt3 whole-body knockout(Sirt3-KO)mice and control(WT)mice were obtained by hybridization of Mice with both Nrf2 and Sirt3 Het(Nrf2-Het,Sirt3-Het).All four groups of mice had females and males and ate and drank normally.Mice were measured for weight and blood glucose,and a glucose tolerance test(GTT)was performed.The blood ketones and body components of mice in WT group and DKO group were further detected,lipid efflux experiments and exogenous lipid clearance experiments were performed,and the basic metabolic indexes of relevant mice were analyzed by small animal metabolism measurement and analysis system.For30 weeks,mice were collected in two different feeding states,overnight fasted and 8 h refed after fasted(Refed 8 h).2.Blood lipid and liver lipid detection:The kit was used to determine TG in serum/plasma in different eating states of mice in the WT and DKO group,and further detected female mice’s glycerol,non-esterified fatty acids(NEFA),total cholesterol(T-CHO),and low-density lipoprotein cholesterol(LDL-C)and high-density lipoprotein cholesterol(HDL-C)were extracted from the livers of mice and TG were measured.3.Pathological staining:HE staining and Oil red O staining were performed on the liver tissues of four groups of mice.4.Protein expression level detection:Four groups of mice liver proteins were extracted for Western blot to detect the expression levels of relevant proteins.Results:1.The results of regular weight monitoring of mice in WT,Nrf2-KO,Sirt3-KO and DKO showed that there was no significant difference in weight change between the groups.In addition,the blood glucose of mice was detected,and the results showed that there was no significant difference in the same feeding state of each group.After the mice were collected and the organ coefficients of the mice were compared,the results showed that there was no significant difference in the important organ coefficients between the mice.2.The mice were monitored for food intake and water intake,and the results showed that the female mice in the DKO group drank significantly more water than the control mice in the control group,and the difference was statistically significant.There was no significant difference in the amount of food eaten between the groups.3.The liver TG of mice in each group was extracted and tested,and the results showed that the liver TG content of female mice in the DKO group was significantly lower than that in the other three groups of mice in the O/N Fasted state.There was no significant difference in liver TG content between male mice.Female mice liver Oil red O staining also showed that the liver red staining of mice in the O/N Fasted state decreased.4.The four groups of mice with O/N Fasted and the two groups of female mice with Refed 8 h were stained,and there was no significant difference in liver tissue morphology between the four groups in the O/N Fasted state.There was also no significant difference in liver tissue morphology between the two groups of female mice at Refed 8 h.However,comparing O/N Fasted and Refed 8 h mouse liver tissue,it was shown that the cytoplasm of Refed 8 h mouse hepatocytes was empty,and a large amount of glycogen accumulated.5.The basal metabolic indexes of female mice in WT group and DKO group were analyzed by small animal metabolism measurement and analysis system,and the results showed that the energy balance of female mice in the DKO group tended to decrease,and there was no obvious difference between other basal metabolic indexes.6.The results of serological indexes related to lipid metabolism in mice showed that there was no significant difference in exogenous lipid clearance and liver lipid efflux between mice in WT group and DKO group.The serum/plasma lipid levels of DKO female mice were higher in O/N Fasted than those in the WT group,while the T-CHO,LDL-C and HDL-C were lower than those in the WT group in the Ad libitum state,and the difference was statistically significant.In addition,male mice were tested,and there was no significant difference in TG levels between the groups under different feeding conditions.7.The liver protein acetylation level of Sirt3-KO and DKO group mice was increased,and the liver protein acetylation level of female mice in the DKO group was higher than that in Sirt3-KO group,and there was no significant difference in liver protein acetylation between male mice in Sirt3-KO group and DKO group.Conclusion:The deletion of Nrf2 and Sirt3 mainly affected the process of liver lipid metabolism in the fed-fasted cycle of female mice,and the double deletion of Nrf2 and Sirt3 may affect the lipid metabolism process by regulating the level of liver protein acetylation.