Metformin Inhibits Hepatic Triglyceride Synthesis Through The XBP1/DGAT2 Pathway

Background:Increasing hypertriglyceridemia(HTG)and non-alcoholic fatty liver disease(NAFLD)have emerged pandemically worldwide and significantly increase risk for diabetes,obesity,and atherosclerotic cardiovascular disease(ASCVD).NAFLD is one of the most common chronic liver diseases characterized by the accumulation of triglycerides(TG)in liver.Treatment to lower HTG and intrahepatic triglyceride deposition is regarded as an important strategy to improve the prognosis of ASCVD,NAFLD,and diabetes.Metformin is recommended as the first choice for monotherapy for Type 2 diabetes mellitus,and metformin also exhibits protective benefits in lipid metabolism,tumorigenesis,and cardiovascular protection.Clinical research has indicated that there were obvious clinical benefits of improved hypertriglyceridemia in diabetic and nondiabetic patients.The AMP-activated protein kinase(AMPK)signaling pathway is involved in the regulation of metformin-mediated lipid and glucose metabolism.Previous studies demonstrated that AMPK decreases triglyceride(TG)and cholesterol synthesis by regulating acetyl-CoA carboxylase(Acc),sterol regulatory elementbinding protein-lc(SREBP-lc),and stearyl-coenzyme A desaturase 1(Scd1)activity.Diacylglycerol O-acyltransferase 2(DGAT2)catalyzes the final and the committed step in the synthesis of TG.Mice with hepatic overexpression of DGAT2 exhibit profound hepatic insulin resistance and steatosis.Inhibition of DGAT2 expression has been shown to improve insulin resistance and hepatic steatosis in T2DM rats,suggesting that DGAT2 plays an important role in hepatic steatosis and hypertriglyceridemia in T2DM.X-box binding protein 1(XBP1)functions as a transcription factor in response to endoplasmic reticulum(ER)stress,adipocyte differentiation,and fatty acid and TG synthesis,while DGAT2 serves as a downstream target gene of XBP1.Objectives:The present study investigated whether metformin improves the de novo synthesis pathway of TG via regulation of DGAT2 or XBP1 in the liver and whether AMPK is involved.The results could facilitate the development of a new therapeutic agent for treating HTG,NAFLD,and ASCVD with HTG.Methods:1.In Vivo,male C57/BL6 mice were fed with control diet(Research Diets D12450)or high-fat diet(HFD,Research Diet D12492),or HFD+metformin(300 mg/kg/d)treatment for 5 weeks.The body weight and food intake of the mice were measured every week.2.In Vitro,the HepG2 cells were treated with different concentration of metformin(2 mmol/L 或 5 mmol/L),or 200 μM palmitic acid(PA),or small interfering RNAs targeting XBP1(XBP1 siRNA),or dominant negative(DN)-AMPKal plasmid,or recombinant adenovirus expressing XBP1(Ad XBP1)for 24 h or 48 h,respectively.DGAT2 protein level was observed.3.TG determination:TG content in liver was tested by glycerol-phosphooxidaseperoxidase method and TG detection kit.4.Real-time quantitative PCR was used to detect the expression of XBP1 and DGAT2 genes.5.Western Blotting was used to detect the protein level of AMPK,XBP1 and DGAT2.6.Immunofluorescence was utilized to detect the expression and distribution of XBP1 protein in hepatocytes.7.Dual-luciferase reporter assay was performed to measure the expression of DGAT2.Results:1.Metformin treatment reduced hepatic TG content in mice.To determine if metformin affects the cellular TG content in the liver,mice were fed a HFD or HFD with metformin for 5 weeks.A significant body weight gain was observed in HFD-fed mice compared with mice in the control group,but metformintreated mice lost weight.There was no significant difference in food take between the control,HFD-fed,and HFD with metformin groups.The cellular TG content in the liver was increased in HFD-fed mice,but metformin treatment exhibited an inhibitory effect on TG accumulation in the liver of HFD-fed mice.2.Metformin decreased DGAT2 expression in the liver of mice and in HepG2 cells.HFD-fed induced a significant increase of lipogenic genes expression including Fas,Scdl,Accl,Dgat2,and Gpatl in the liver of mice.Though metformin treatment led to a slight decrease of Fas,Scdl,Acc1,and Gpatl expression,there was no significant difference in the liver between metformin-treated mice and HFD-fed mice.While the mRNA of DGAT2 represented a significant decrease in the liver of metformin-treated mice compared to the HFD-fed mice.The protein level of DGAT2 was significantly increased in the liver of HFD-fed mice,but metformin treatment alleviated this increase.To confirm this finding,HepG2 cells were stimulated with different concentrations of metformin,which resulted in a dose-dependent significant decrease in the mRNA level and luciferase activity of DGAT2.3.Metformin decreased nuclear and cytoplasmic XBP1 protein levels in the liver.A prior study demonstrated that XBP1 is an upstream regulator of DGAT2.Two forms of XBP1 have been identified unspliced form(XBP-lu)and spliced form(XBP1s),XBP-lu undergo a unconventional cytoplasmic splicing processing under stress conditions to converse a XBP-1s form,which translocates into nucleus as an active transcription factor.Using an anti-XBP1 antibody that recognizes both isoforms of XBP1,we analyzed the expression of nuclear and cytoplasmic XBP1 protein in the liver of mice and HepG2 cells after metformin treatment,XBP1 expression was increased in the liver of HFD-fed mice,but metformin treatment reduced the XBP1 mRNA level.The levels of both the nuclear and cytoplasmic forms of XBP1 protein were also significantly increased in the liver of HFD-fed mice,but metformin treatment alleviated this effect.Consistent with the in vivo data,nuclear and cytoplasmic XBP1 expression exhibited a dose-dependent decrease in HepG2 cells stimulated with metformin in vitro.4.Metformin decreased TG synthesis through regulating XBP 1-mediated DGAT2 expression.To test the regulatory effect of XBP 1 on DGAT2 expression,XBP1 siRNA was transfected into HepG2 cells.Levels of DGAT2 protein and mRNA were decreased compared with scrambled siRNA transfectants or control cells.DGAT2 luciferase reporter assay results supported the above results.There was no significant difference in hepatic TG content between XBP 1 siRNA-treated HepG2 cells and cells treated with mtformin and XBP1 siRNA.To further explore the role of XBP1 in metformin-induced effect on DGAT2 and TG,XBP1 knockout AML 12 hepatocytes were treated with 2 mmol/L metformin for 48 h or 200 μM palmitic acid(PA)with or without metformin for 48 h.The TG content was significant increased after PA incubation,but metformin treatment did not have an effect on TG content in XBP1 knockout AML 12 hepatocytes after PA incubation.In addition,DGAT2 protein was significant increased in XBP1 knockout AML 12 hepatocytes when treated by PA,metformin treatment exerted a mild inhibitory effect on DGAT2,but did not completely inhibit the PA-induced increase of DGAT2 protein.5.The effect of metformin on TG synthesis via the XBP1-mediated DGAT2 pathway involved AMPKMice were fed a control diet,HFD,and HFD with metformin for 5 weeks.There was no significant difference in total-AMPK level between the control,HFD-fed,and HFD with metformin groups.There was a significant decrease of phosphorylatedAMPK(p-AMPK)level in the liver of HFD-fed mice,while metformin increased pAMPK levels and stimulated AMPK activity.To verify the effect of AMPK activity on XBP1 and DGAT2 expression,HepG2 cells were transfected with dominant-negative mutant AMPKα1(DN-AMPKal)plasmid for 48 h in vitro.Inhibition of AMPK activity led to upregulation of XBP1 and DGAT2 expression in HepG2 cells.Immunofluorescence staining clearly illustrated that XBP1 protein expression was enhanced by DN-AMPK treatment,and metformin attenuated this change.DGAT2 protein levels were also increased by inhibition of AMPK activity,and this effect was also suppressed by metformin treatment.In order to further determine whether AMPK activity was involved in the metformin-induced regulation of the XBP1/DGAT2 pathway,DN-AMPK plasmid and recombinant adenoviruses expressing XBP1(Ad XBP1)were co-transfected into HepG2 cells.The DGAT2 protein level was significant decreased after metformin treatment,and overexpression of XBP1 and inactivation of AMPK significantly increased DGAT2 protein level.Conclusions:1.Metformin decreases DGAT2 expression and the hepatic TG synthesis.2.Metformin activates AMPK pathway to inhibit XBP1/DGAT2-mediated TG synthesis.3.XBP1 may be a new metabolic mediator for metformin in regulation of hepatic TG synthesis.