Roles And Underlying Mechanism Of PTG In Hepatic Glucolipid Metabolism Disorders

?Objective?Type 2 diabetes mellitus(T2DM)is a global public health problem nowadays.Current studies have shown that hepatic insulin resistance is the main link in the pathogenesis of type 2 diabetes mellitus,and its most obvious pathophysiological characteristic was disorder of hepatic glucose and lipid metabolism,leading to increased hepatic glucose output and hepatic fat accumulation,among which,gluconeogenesis and fat synthesis play an important role.Therefore,effectively inhibiting the excessive gluconeogenesis and fat synthesis of liver is one of the important targets for the treatment of insulin resistance and type 2 diabetes.In-depth exploration of the molecular mechanism of hepatic glucose and lipid metabolism regulation has become a current research hotspot.PTG,also known as glycogen targeted protein,is a protein phosphatase of protein phosphatase 1(PP1)family,which can play a role by catalyzing dephosphorylation of protein molecules that have been phosphorylated.Previous studies have shown that it can affect glycogen metabolism of liver and skeletal muscle by regulating dephosphorylation of ribosomal S6 protein kinase,glycogen synthase and glycogen phosphorylase.However,in addition to its regulation of liver glycogen metabolism,whether PTG was involved in the regulation of liver gluconeogenesis and lipid metabolism has not been fully elucidated.This study intends to investigate the effect of PTG on liver glucose and lipid metabolism and its molecular mechanism via in vivo and in vitro experiments.?Methods?In vivo experiment: Established type 2 diabetic model mice by high-fat feeding combined with low-dose streptozotocin.Intervened with PTG gene overexpression or knockdown adenovirus,adopted glucose tolerance test,insulin stimulation test,pyruvate tolerance test,liver glycogen PAS staining,glycogen content determination,liver triglyceride(TG)content determination,liver HE staining,blood lipid analysis and other methods to explore the influence of PTG on gluconeogenesis,glycogen and lipid metabolism.Fluorescence quantitative PCR and Western blot assay were used to observe the effects of it on the expression of key genes in liver glucolipid metabolism.The nuclear translocation of forkhead transcription factor 1(FOXO1)was detected by immunofluorescence.In vitro experiment: The AML-12 mouse hepatocyte gluconeogenesis model constructed by c AMP was treated with PTG knockdown or overexpression of adenovirus respectively.The effects of PTG knockdown or overexpression adenovirus on the expression of key genes in liver glycolipid metabolism were observed by fluorescence quantitative PCR and Western blot.The oil red O staining was used to observe the formation of lipid droplets in AML-12 cells.A luciferase reporter plasmid containing the phosphoenolpyruvate carboxykinase(PEPCK)promoter was constructed(PEPCK-Luc),and the activity of luciferase was detected by double fluorescent reporter gene detection system.?Results?(1)The results showed that PTG was mainly expressed in liver and muscle tissue.In starvation state,the expression of PTG,PEPCK and glucose-6-phosphatase(G6Pase)in liver tissue of mice were significantly increased.After feeding,the expression of PTG,PEPCK and G6 Pase in C57BL/6J mice were remarkably decreased.PTG was significantly increased in the liver of db/db diabetic mice,and the expression of gluconeogenic related genes such as PEPCK and G6 Pase were also dramatically increased.(2)After PTG gene specific knockdown in the liver of diabetic model mice,the results showed that the blood glucose level decreased gradually,insulin sensitivity improved,glucose tolerance and pyruvate tolerance significantly improved,and the expression levels of key enzymes of gluconeogenesis(PEPCK,G6Pase)dramatically decreased.Similarly,PTG knockdown adenovirus intervention could remarkably inhibit the expression of PEPCK and G6 Pase in AML-12 cells.(3)After overexpression of PTG gene in the liver of diabetic model mice,the results showed that the blood glucose level was increased,the glucose tolerance was significantly impaired,and the expression levels of PEPCK and G6 Pase were remarkably increased.Similarly,PTG overexpression could substantially promote the expression of PEPCK and G6 Pase in AML-12 cells.(4)Mechanism studies showed that PTG knockdown could significantly increase the phosphorylation level of FOXO1 and remarkably restricted FOXO1 nuclear entry.Overexpression of PTG decreased the phosphorylation level of FOXO1 and promoted the FOXO1 nuclear translocation.The results of luciferase reporter gene assay further showed that PTG could substantially promote FOXO1 to up-regulate the luciferase activity of PEPCK,a key gene in gluconeogenesis.In addition,we found that the promotion effect of PTG overexpression on gluconeogenesis was blocked by the FOXO1 inhibitor AS1842856,while the inhibition effect of PTG knockdown on gluconeogenesis was eliminated by FOXO1 overexpression(5)PTG knockdown significantly reduced circulating and liver triglyceride levels,reduced hepatic steatosis in diabetic mice.At the same time,the expression levels of key genes of fatty acid synthesis cholesterol regulatory element binding protein1(SREBP1)and key enzyme of triglyceride synthesis diacylglycerol acyltransferase 2(DGAT2)were significantly decreased.In addition,PTG knockdown significantly reduced lipid droplet formation and inhibited SREBP1,DGAT2,Fatty acid synthase(FAS)and Acetyl Co A carboxylase(ACC)gene expression in AML12 cells.(6)PTG overexpression remarkably increased the circulating and liver triglyceride levels,aggravated hepatic steatosis,and increased the SREBP1 and DGAT2 expression levels.Furthermore,overexpression of PTG significantly increased the expression levels of SREBP1,DGAT2,ACC and FAS in AML-12 cells.?Conclusion?In the present study,we found for the first time that PTG increases hepatic gluconeogenesis by regulating the dephosphorylation of FOXO1,and promotes the accumulation of liver fat by regulating the DGAT2 and SREBP1 mediated de novo synthesis of fat.These results suggest the close relationship between PTG and abnormal hepatic glucolipid metabolism,which may be an important protein leading to the interaction of abnormal hepatic glucose and lipid metabolism,providing experimental and theoretical basis for the research and development of anti-type 2 diabetes drugs.