The Role And Mechanism Of Palmitoleic Acid In Regulating Hepatic Gluconeogenesis Induced By High Fat Through SIRT3

Objective:At present,the number of patients with diabetes,especially type 2 diabetes,is increasing year by year in China and even in the world,and it is expected to reach 700 million by 2045.Finding nutritional interventions is positive significance for the prevention and treatment of diabetes.Increased gluconeogenesis and increased liver glucose output are important mechanisms of early abnormal glucose metabolism,and also one of the important links in the occurrence and development of diabetes.Therefore,gluconeogenesis can be used as a research direction for treatment.Our previous research found that palmitoleic acid can not only increase insulin sensitivity,but also reduce hepatic gluconeogenesis to maintain blood glucose homeostasis.We have confirmed that the effect of increasing systemic insulin sensitivity is achieved by reducing the inflammatory response.Therefore,we want to observe the effect and mechanism of palmitoleic acid in regulating hepatic gluconeogenesis induced by high fat.Methods:1.Effect of palmitoleic acid on hepatic gluconeogenesis in mice 60 C57BL/6N SPF male mice of 5-6 weeks age were fed with low-fat(LFD)or high-fat diet(HFD)feed for 12 weeks,and then were given bovine serum albumin(BSA)(control),oleic acid(OL),and palmitoleic acid(PO)by gavage.Monitor the change of body weight during the period.Glucose,insulin,and pyruvate tolerance tests were performed at the 15th,16th,and 17th weeks of LFD or HFD feeding.The blood and tissues of mice were collected at the 18th week,and the liver was used to detect the expression of SIRT3 and gluconeogenesis-related enzymes2.The effect of liver specific SIRT3 overexpression on gluconeogenesis in mice 20 C57BL/6N SPF male mice of 5-6 weeks age were injected adeno-associated virus AAV-GFP as control,while another 20 C57BL/6N SPF male mice were injected AAV-SIRT3 vector to overexpress SIRT3 specific in liver.Mice were randomly divided into 4 groups,low-fat diet control group(LFD AAV-GFP),low-fat diet liver specific SIRT3 overexpress group(LFD AAV-SIRT3),high-fat diet control group(HFD AAV-GFP),high-fat diet liver specific SIRT3 overexpress group(HFD AAV-SIRT3),feed for 12 week s,record body weight and food intake,perform glucose tolerance,insulin tolerance,and pyruvate tolerance tests at 13th-15th weeks of LFD or HFD feeding.When detecting liver SIRT3 overexpression,the expression of key enzymes PEPCK,PCX,MDH2 in liver gluconeogenesis and their enzymatic activity and acetylation were measured.Immunoprecipitation was used to detect the binding effect of SIRT3 on gluconeogenesis-related enzymes.Separated mitochondria and cytoplasmic proteins was observe the changes in the location of SIRT3.Results:1.Under high-fat feeding conditions,palmitoleic acid increases insulin sensitivity,but does not alter the expression of gluconeogenesis pathway related proteinsGTT,ITT and PTT experiments were carried out in mice with palmitoleic acid intervention.Under high fat feeding condition,GTT showed that the glucose level of palmitoleic acid-treated mice decreased at multiple time points compared with that of BSA group and oleic acid group,with statistical significance(P<0.05),indicating that palmitoleic acid significantly reduced the increase of glucose level induced by high fat.ITT showed that palmitoleic acid-treated mice were more sensitive to insulin than BSA and oleic acid-treated mice,further suggesting that palmitoleic acid-treated mice could increase systemic insulin sensitivity and reduce blood glucose levels.PTT showed that palmitoleic acid reduced liver gluconeogenesis in obese mice fed high fat diet.However,western blotting showed that palmitoleic acid did not decrease the expression of proteins associated with liver gluconeogenic pathway,PEPCK,PCX,and MDH22.SIRT3 overexpression in liver increases gluconeogenesis and MDH2 protein levelsGTT and PTT experiments were performed in mice with SIRT3 overexpression.GTT showed that high fat feeding significantly increased glucose levels in both fasting and glucose injection mice(15 min and 30 min).In the high-fat fed group,SIRT3 overexpression significantly increased blood glucose levels 15 and 30 min after glucose injection,and was significantly different compared with the control group.PTT showed that high-fat feeding significantly increased liver gluconeogenesis,as shown by increased blood glucose levels at 15,30,and 60 min after pyruvate injection compared with low-fat feeding.SIRT3 overexpression increased liver gluconeogenesis in both high-fat and low-fat diets.In primary hepatocytes of mice fed with high fat for 12 weeks,overexpression of SIRT3 increased glucose production in hepatocytes,while treatment of primary hepatocytes with SIRT3 inhibitors decreased glucose production in hepatocytes.Western blotting showed that SIRT3 overexpression increased the expression of MDH2,but did not change the expression of PEPCK and PCX.3.Palmitoleic acid reduces SIRT3 expression and glucose production in liver cellsPalmitoleic acid interfered with mice,and the liver tissues were subjected to western blotting.There was no difference in the expression of SIRT3 in the livers of mice fed low fat diet.The expression of SIRT3 in liver of mice fed with palmitoleic acid was significantly reduced in high fat diet.Cell experiment showed that primary hepatocytes of mice fed high-fat for 12 weeks were treated with palmitoleic acid.Palmitoleic acid reduced the expression of SIRT3 in primary hepatocytes and reduced the increase in liver cell glucose production due to overexpression of SIRT3.4.Protein interactionsProtein immunoprecipitation showed that SIRT3 had protein-protein binding with PEPCK,PCX and MDH2.The acetylation level of lysine residue in Western blotting showed that SIRT3 could deacetylate PCX and MDH2.Conclusion:Palmitoleic acid may reduce the expression of SIRT3 in hepatocytes,increase the acetylation level of PCX and MDH2,reduce the enzyme activity of PCX and MDH2,and thus decrease the gluconeogenesis in the liver.