Regulation Of SIK1 On Glucose And Lipid Metabolism In Liver Cells And The Intervention Mechanism Of Zhenqing Recipe On Glucose And Lipid Metabolism Disorder Induced By High Glucose

BackgroundType 2 diabetes mellitus with Nonalcoholic fatty liver disease(NAFLD)is mainly characterized by the disorder of glucose and lipid metabolism.Salt-inducible kinase 1(SIK1)plays an important role in regulating glucose and lipid metabolism.It can phosphorylate c AMP Regulated Transcriptional Coactivator 2(CRTC2)and Sterol regulatory Element-Binding Protein 1c(SREBP-1c),thereby affecting hepatic gluconeogenesis and lipid synthesis.CRTC2 is a key regulator of fasting blood glucose,which is fixed in the cytoplasm in basal state.During fasting,CRTC2 is dephosphorylated and enters the nucleus,where it binds to the c AMP-Response Element Binding Protein(CREB)and upregulates the transcription of Phosphoenolpyruvate carboxykinase(PEPCK)and Glucose-6-phosphoatase(G6Pase)leading to elevated fasting blood glucose.As an important transcription factor related to hepatic lipid synthesis,SREBP-1c is integrated with Sterol Response Elements(SREs)after phosphorylation into the nucleus,and activates the transcription of Acetyl Co A Carboxylase(ACC)and Fatty Acid Synthase(FAS)which catalyze lipid synthesis.Studies have shown that the activity of SIK1 can promote the phosphorylation of CRTC2 and srebp-1c,causing them to remain fixed in the cytoplasm and reducing the transcription of their downstream target genes.In our previous experiments,it was found that the expression and activity of SIK1 in hepatocytes of type 2 diabetic model rats and Hep G2 cells after high glucose stimulation were inhibited,and the expression of lipid synthesis genes was up-regulated.ObjectiveWe hypothesized that hyperglycemia may be an important mechanism for the progression of NAFLD in type 2 diabetes mellitus,resulting in the disorder of glucose and lipid metabolism caused by the inhibition of SIK1 activity.However,Zhenqing Recipe(ZQF),a clinically verified prescription,can reduce the blood glucose and lipids of rats with type 2 diabetes mellitus(NAFLD),and up-regulate the expression of SIK1.We will further investigate the intervention mechanism of ZQF and oleanolic acid on glucose and lipid metabolism disorders induced by high glucose in hepatocytes from the cellular level.MethodsIn order to determine the effect of high glucose on SIK1 and glucose metabolism,we cultured Hep G2 cell lines and primary mouse liver cells with high glucose medium(25 mmol/l glucose),simulated the hyperglycemic environment,and detected the expression of SIK1 and glucose metabolism genes in cells.Moreover,under normal glucose environment(5.5 mmol/l glucose),the cells were treated with the SIKs inhibitor HG-9-91-01 and adenovirus ad-sik1 to observe the effect of inhibition and up-regulation of SIK1 on CRTC2,SREBP-1c and downstream genes related to glucose and lipid metabolism.In order to clarify the intervention mechanism of Zhenqing Recipe on glucose and lipid metabolism disorders induced by high glucose,we used metformin,Zhenqing Recipe and olenic acid to intervene Hep G2 cells in high glucose environment,respectively,to observe the changes of SIK1,CRTC2,SREBP-1c and the expression of downstream genes related to glucose and lipid metabolism,as well as the changes of glucose content in culture medium and TG content in cells.Results1.High glucose stimulation had no significant effect on the viability of Hep G2 cell lines.2.High glucose stimulation promoted the expression of CRTC2,G6 Pase and PEPCK in both Hep G2 cells and primary mouse liver cells.3.High glucose stimulated nuclear translocation of CRTC2 in Hep G2 cells.4.High glucose stimulation of primary mouse hepatocytes resulted in increased expression of SIK1 nuclear protein.5.HG-9-91-01 can cause the decrease of SIK1 protein and activity in both Hep G2 and primary mouse liver cells,and the significant increased expression of CRCT2,G6 Pase,PEPCK,srebp-1c,FAS and ACC proteins.6.After the treatment of primary mouse hepatocytes with HG-9-91-01,the glucose content in cell culture medium was higher than that in the control group.7.The nuclear translocation of SIK1,CRTC2 and SREBP-1c was observed after HG-9-91-01 treatment of primary mouse hepatocytes.8.After the over-expression of adenovirus ad-sik1 was constructed and infected with primary mouse liver cells,the m RNA of SIK1 significantly increased,but there was no significant change in its protein expression,and the protein expressions of CRTC2,G6 Pase,PEPCK,SREBP-1c,FAS and ACC were also not significantly different from those of the control group.9.The primary mouse hepatocytes were treated with HG-9-91-01 and then infected with ad-SIK1.It was found that the expression of SIK1 protein decreased after HG-9-91-01 treatment,and the expression of SIK1 protein at such a low level could be upregulated to a normal level by ad-SIK1 infection.Under the condition of HG-9-91-01 treatment,CRTC2,SREBP-1c,G6 Pase,PEPCK,FAS and ACC in the ad-SIK1-infected group were inhibited,and the glucose content in the cell culture medium decreased and the lipid droplets of the cells decreased,compared with the cell group not infected with ad-SIK1.10.High glucose culture could up-regulate the expression of gluconeogenesis and lipid synthesis genes in Hep G2 cells,and increase the glucose content in cell culture medium and intracellular TG content.Intervene with metformin,oleanolic acid or Zhenqing formula can inhibit these changes and reduce the gluconeogenesis and the upregulation of lipid synthesis induced by high glucose.11.In Hep G2 cells cultured with high glucose,SIK1 protein expression and phosphorylation activity of SIK1 protein were decreased,and SIK1,CRTC2 and SREBP-1c were transferred into the nucleus.The intervention of oleanolic acid or Zhenqing formula could inhibit the above nucleation phenomenon and keep SIK1,CRTC2 and SREBP-1c in the cytoplasm,thus inhibiting the expression of glycolipid metabolism genes downstream of CRTC2 and SREBP-1c.ConclusionsBoth the high-glucose environment and the SIKs inhibitor hg-9-91-01 can lead to decreased SIK1 activity and nuclear translocation in Hep G2 cell lines and primary mouse hepatocytes.High-glucose environment promoted the expression of glucose-related genes in Hep G2 cell lines and primary mouse liver cells.HG-9-91-01 was found for the first time to cause nuclear translocation of primary mouse hepatocytes SIK1,CRTC2 and SREBP-1c,and promote gluconeogenesis and lipid synthesis of Hep G2 cell lines and primary mouse hepatocytes.In the case of low expression of SIK1 protein in primary mouse hepatocytes,the activated gluconeogenesis and lipid synthesis were inhibited by upregulation of the expression of SIK1.The intervention of metformin,Zhenqing prescription and oleanolic acid could improve the disorder of glucose and lipid metabolism in Hep G2 cells induced by high glucose environment.