Role Of Salt Inducible Kinase Lin High Glucose-induced Lipid Accumulation In HepG2 Cells And Metformin Intervention

ObjectiveThe liver is important for systemic energy homeostasis achieved by regulating glucose and lipid metabolism.Hepatic lipogensis is closely related to glucose metabolism.When hepatic glucose levels exceed the liver's capacity for glycogen storage,glucose is converted into fatty acids and leads to fatty liver disease.In the experimental hepatic steatosis has been reported in human hepatoma HepG2 cells exposed to high glucose,and high glucose diet induced non-alcoholic fatty liver disease(NAFLD)animal model,were accompanied by high levels of lipogenic factor,sterol regulatory element-binding protein-lc(SREBP-1c),fatty acid synthase(FASN),and acetyl CoA carboxylase ?(ACC1)expression,and increased triglyceride(TG).Metformin,activates AMP-activated protein kinase(AMPK).Activation of AMPK,represented by phosphorylation at Thr172(pThr172),is inhibited in HepG2 cells,when the cells are treated with high concentrations of glucose,and was restored by metformin.And metformin also reduced the high glucose induced-lipid accumulation.Salt induced kinase 1(SIK1),belongs to the AMPK family,involves the regulation of lipogenesis,and regulates srebp-1c expression and activity.SIK1 downregulation in hepatocytes enhancesSREBP-1cexpression followed by TG accumulation in a hepatic steatosis model in experimental type 2 diabetes mellitus(T2DM)rats.However,SIK1 overexpression also suppresses SREBP-1cmRNA expression and the expression of the downstream FASN and ACC 1 genes.We have also recently reported that suppressed SIK1 mRNA and protein levels,also reduced SIK1 kinase activity,as measureas by the level of phosphorylation at Thr182(pThrl82 corresponds to pThr172 of AMPK),and increased the cells,proliferation in rat mesangial cells HBZY-1,when cultured under high glucose conditions.Liver kinase B1(LKB1),an upstream activator of AMPK family kinases,has been found to phosphorylate Thr182of SIK1.The LKB1-AMPK axis regulates hepatic lipogenesis by sensing glucose influx,and has been proposed to be the metformin target.However,roles of salt inducible kinase(SIK1)in high glucose-induced triglyceride accumulation in human hepatoma HepG2 cells as well as in the molecular mechanism by which metformin-mediated regulation of high glucose-induced lipogenesis,remain unclear.Therefore,a cell model for high glucose-induced hepatic steatosis was prepared by exposing HepG2 cells to high glucose.For the first time to explore the role of SIK1 in high glucose-induced lipid accumulation in HepG2 cells,and to further investing whether SIK1 is involved in metformin-mediated regulation of high glucose-induced lipid accumulation in HepG2 cells by overexpression of SIK1 and metformin intervention in HepG2 cells.Methods1.HepG2 cells were cultured in DMEM medium containing normal glucose(5.5 mmol/L D-glucose),supplemented with 10%foetal bovine serum(FBS),100 ?g/mL streptomycin and 100 U/mL penicillin.Cells were maintained at 37? with 5%CO2 in a humidified atmosphere.When HepG2 cells were grown to 80%confluence,the medium was changed to serum-free medium for 24 h.Then,the cell model for hepatic steatosis was prepared by exposing the cells to high glucose(25 mmol/L D-glucose)for 24 h.Intracellular triglycerides were visualized by Oil Red O and measured using a triglyceride assay kit.The mRNA levels of SIK1,SREBP-lc,FASN,and ACC1 were measured by reverse transcription-polymerase chain reaction(RT-PCR),and protein levels were measured by western blot.The protein expression level of SIK1 pThr 182(SIK1 activity)was determined by western blot.The cellular distribution of SIK1 were processed for immunofluorescence,images were visualized with Olympus confocal microscope.2.To directly confirm the relevance of SIK1 to lipogenesisinduced by high glucose,recombinant plasmid GV320-SIK1 was constructed.HepG2 cells were transient transfected with GV230-SIK1 in normal glucose medium for 48h.To evaluate transfection efficiency,RT-PCR and western blot were used.Transformed cells were treated with high glucose for 24 h.Intracellular triglycerides were visualized by Oil Red O and measured using a triglyceride assay kit.The mRNA levels of SIK1,SREBP-1c,FASN,and ACC1 were measured by reverse transcription-polymerase chain reaction(RT-PCR),and protein levels were measured by western blot.The protein expression level of SIK1 pThr 182(SIK1 activity)was determined by western blot.The cellular distribution of SIK1 were processed for immunofluorescence,images were visualized with Olympus confocal microscope.3.To gain insight into the clinical relevance of SIK1-mediated regulation of high glucose-induced lipogenesis,we decided to examine the effect of metformin on SIK1-mediated regulation of high glucose-induced lipogenesis in HepG2 cells.HepG2 cells were cultured in DMEM medium,when HepG2 cells were grown to 80%confluence,the medium was changed to serum-free medium for 24 h.The cells were pre-incubated with or without 0.5 mmol metformin in serum-free medium with normal glucose for 1 h followed by incubation with high glucose for 24 h.The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)method was used to evaluate the effects of metformin on the viability of HepG2 cells.Intracellular triglycerides were visualized and measured.The mRNA levels of SIK1,SREBP-1c,FASN,and ACC1 were measured.The protein expression level of SIK1 pThr 182(SIK1 activity)was determined.The cellular distribution of SIK1 were visualized with Olympus confocal microscope.Results1.Lipid accumulation in HepG2 cells was obvious after treatment with high glucose(25 mmol/L D-glucose)for 24 h.TG and the lipogenic factors,SREBP-1c,FASN,and ACC1 expression were increased.2.SIK1 expression and activity were surppressed in HepG2 cells after treatment with high glucose(25 mmol/L D-glucose)for 24 h.And SIK1 was transferred from the cytoplasm to the nucleus.3.Overexpression of SIK1 in HepG2 cells,suppressed lipogenesis and lipogenic factors SREBP-1c,FASN,and ACC 1 expression,even in high glucose conditions.4.Metformin suppressed lipogenesis and lipogenic factors SREBP-1c,FASN,and ACC1 expression,even in high glucose conditions.5.Furthermore,treatment with metformin upregulated SIK1 mRNA and protein levels,as well as the active form of SIK1.And metformin induced part of SIK1 was transferred from the nucleus to the cytoplasm.ConclusionsHigh concentration of glucose(25 mmol/L D-glucose)could induce lipid accumulation in human hepatoma HepG2 cells.SIK1 expression levels and activity were suppressed in high glucose induced-lipid accumulation in HepG2 cells,which was negatively correlated with that of lipogenic factors and lipid accumulation in HepG2 cells.We observed that overexpression of SIK1 suppressed high glucose induced-lipid accumulation and lipogenic factors in HepG2 cells.And metformin suppressed high glucose induced-lipid accumulation in HepG2 cells via the induction and activation of SIK1.