Research On Cyclocarya Paliurus Triterpenic Acids Against Hepatic Gluconeogenesis-based On Glucagon/AMPK

Diabetes mellitus(DM)is a chronic non-communicable disease threatening human health,and type 2 diabetes mellitus(T2DM)accounts for more than 90%of all cases of DM.One of the pathogenesis of T2DM is the abnormal enhancement of liver gluconeogenesis.Therefore,the discovery of drugs that inhibit liver gluconeogenesis may provide new ideas for the treatment of diabetes.Cyclocarya(Batal.)Iljinskaja(CP),a specific species of China,has been reported to have anti-hyperglycemia,anti-hyperlipidemia,and anti-inflammation activities.Our previous studies found that Cyclocarya paliurus triterpenic acids(CPT)could effectively reduce hyperglycemia in diabetic mice,but its effect on liver gluconeogenesis is still unclarified.Therefore,the effect and mechanism of CPT against hepatic gluconeogenesis and the active ingredient of CPT on gluconeogenesis in vitro were evaluated in this paper.Chapter 1:Effect and preliminary mechanism of CPT on glucose and lipid metabolism disorders in diabetic mice in vivo was evaluated.High-fat feeding combined with low-dose streptozotocin(STZ)multiple injections to construct a T2DM mouse model,dabetic mice were treated with CPT(50,200 mg/kg/day)and metformin(150 mg/kg/day)by gavage for 4 weeks,then the changes in food intake,body weight and glucose and lipid metabolism was monitored.H&E staining to observe liver lipid deposition,Real-time PCR was performed to detect glucose-6-phosphate dehydrogenase(G6Pase)mRNA expression,as well as phosphoenolpyruvate carboxykinase(PEPCK).Western blotting was conducted to determine AMP-dependent protein kinase(AMPK),hepatic gluconeogenesis and glucagon signaling pathway protein expression.Results showed that CPT could significantly reduce fasting blood glucose as well as enhance glucose tolerance,and insulin resistance,meanwhile it could reduce an increase in total cholesterol(TC)and triglyceride(TG)levels induced by high-fat feeding.In addition,CPT could also inhibit the expression of G6Pase and PEPCK,which were key enzymes that regulated liver gluconeogenesis.What’s more,it increased liver PDE4B level and AMPK phosphorylation,yet reduced cAMP content,protein kinase A(PKA)and cAMP-response element binding protein(CREB)phosphorylation.These above results suggested that CPT might be a potential agent against hepatic gluconeogenesis by inhibiting cAMP/PKA/CREB signaling pathway.Chapter 2:To investigate the effect and mechanism of CPT on liver gluconeogenesis,glucagon was used to construct an in vitro gluconeogenesis export model in the primary hepatocytes.Chemical kits were used to determine glucose content in the cell supernatant.Enzyme linked immunosorbent assay(ELISA)was used to detect cAMP content.PCR and WB were used to detect the expression of related signal pathway,and AMPK inhibitor,Compound C,was applied to clarify the mechanism of CPT on hepatic gluconeogenesis.The results showed that CPT could significantly inhibit gluconeogenesis,decrease protein expression of G6Pase and PEPCK,meanwhile it could reduce cAMP content as well as PKA and CREB phosphorylation levels.In addition,CPT could significantly increase phosphorylation level of AMPK and activate PDE4B,and Compound C intervention could reverse the inhibitory effect of CPT on cAMP/CREB signaling pathway and gluconeogenesis.These above data indicated that CPT inhibited hepatic gluconeogenesis through promoting AMPK phosphorylation and inhibiting cAMP/PKA/CREB signaling pathway.Chapter 3:The main active compounds of CPT on regulating hepatic gluconeogenesis was screened in the gluconeogenesis output model.MTT method was used for evaluation index of the cytotoxic activity of the triterpene acid compounds of Cyclocarya paliurus,and chemical kit method was used to detect the content of glucose in the cell supernatant.PCR and WB were used to detect the levels of G6Pase and PEPCK in mice primary hepatocytes.The results showed that rjunolic acid,3β,23-dihydroxy-12-ene-28-ursolic acid,2α,3α,23-trihydroxyurs-12-en-28-oica could significantly reduce hepatic glucose production(HGP)induced by glucagon and decrease G6Pase and PEPCK genes and proteins levels.These results suggested that rjunolic acid,3p,23-dihydroxy-12-ene-28-ursolic acid,and 2α,3α,23-trihydroxyurs-12-en-28-oica are active components of CPT to inhibit hepatic gluconeogenesis.In summary,it was found that CPT could down-regulate cAMP signaling pathways by activating AMPK and resulting in inhibiting hepatic gluconeogenesis and alleviating T2DM.Meanwhile,active compounds of CPT inhibiting gluconeogenesis were screened out in vitro.Therefore,this study improved the material basis of CPT on diabetes and provided new strategies and scientific theoretical basis for diabetes treatment.