The Mechanism Of Huanglian-Renshen-Decoction And Its Effective Component-Oxyberberine On Inhibiting Hepatic Gluconeogenesis To Ameliorate T2DM

Section 1 The mechanisms of Huanglian-Renshen-Decoction on inhibiting hepatic gluconeogenesis to ameliorate T2DM in db/db miceObjective:To explore the potential effects and underlying mechanisms of Huanglian-Renshen-Decoction(HRD)on excessive hepatic gluconeogenesis of T2DM mice.Methods:The db/db mice were randomly divided into three groups:model group,low-dose HRD group and high-dose HRD group,while db/m mice were assigned as normal control.The anti-diabetic effects of HRD was evaluated by detecting mice body weight,fasting blood glucose(FBG),insulin level,glucose tolerance test(GTT)and insulin tolerance test(ITT)after 4 weeks intervention.To determine the potential effects of HRD on excessive hepatic gluconeogenesis of db/db mice,we examined pyruvate tolerance test(PTT),glucagon stimulation test(GST),liver positron emission computed tomography(PET)and the expression of gluconeogenic key enzymes,such as PEPCK,G6Pase.Meanwhile,liver and serum lipid profiles were also detected.After confirming the inhibitory role of HRD on hepatic gluconeogenesis,network pharmacology and transcriptomics approaches were used to clarify the underlying mechanisms.Finally,molecular biology experiments including Western Blot,Immunohistochemistry staining and Immunoprecipitation were applied for final mechanism verification.Results:HRD administration can decrease weight gain,FBG and homeostasis model assessment insulin resistance index,increase fasting insulin level,glucose clearance and insulin sensitivity in T2DM mice.The results of PTT,GST,liver PET imaging and PEPCK,G6Pase examination indicated that HRD inhibited the excessive hepatic gluconeogenesis of T2DM mice.Meanwhile,dysregulated liver and serum lipid profiles(total cholesterol,triglyceride,low-density lipoprotein cholesterol)were also corrected by HRD,which exerted a protective effect on liver steatosis.Then,network pharmacology and transcriptomics approaches illustrated that PI3K/Akt/FoxO1 signaling pathway may be responsible for the inhibitory effect of HRD on hepatic gluconeogenesis.As expected,compared with control group,the levels of p-PI3K,p-Akt,p-FoxO1 in model group were significantly decreased.HRD administration recovered the impaired PI3K/Akt/FoxO1 signaling by upregulating p-PI3K,p-Akt,p-FoxO1 levels and promoted the binding of Akt and FoxO1,which contributed to FoxO1 nuclear exclusion,eventually inhibited FoxO1-mediated gluconeogenic activity.Conclusion:Our data demonstrated that HRD can activate PI3K/Akt/FoxO1 signaling pathway,thereby promoting FoxO1 nuclear exclusion,and then inhibiting excessive hepatic gluconeogenesis,eventually ameliorating T2DM.Section 2 The mechanisms of oxyberberine on inhibiting gluconeogenesis via AMPK signaling pathwayObjective:To explore the potential effects and underlying mechanisms of oxyberberine(OBB)on gluconeogenesis.Methods:HepG2 cell insulin resistance model was established by using palmitic acid(PA)induction.Firstly,CCK-8 was performed to screen the OBB concentration used in experiments.Then,the effects of OBB on HepG2 gluconeogenesis were evaluated by detecting supernatant glucose level and the expressions of PEPCK and G6Pase.After confirming the inhibitory effect of OBB on abnormal gluconeogenesis,transcriptomics analysis was used to clarify the underlying mechanisms.Finally,molecular biology experiments were applied for mechanism verification.Meanwhile,chemical inhibitor-and targeted siRNA-based experiments were also performed according to the potential signaling pathway.Results:Compared with control group,PA-induced HepG2 cell showed an obvious increase in gluconeogenesis,while OBB significantly decreased supernatant glucose level and the protein and mRNA levels of PEPCK and G6Pase,inhibited abnormal gluconeogenesis.Transcriptomics analysis revealed that the anti-gluconeogenesis effect of OBB was closely associated with AMPK/Akt/FoxO1 signaling transduction and AMPK/CRTC2 signaling transduction.Further studies indicated that OBB administration upregulated p-AMPK,p-Akt,p-FoxO1 levels,thereby promoting FoxO1 nuclear exclusion and preventing the binding of FoxO1 and PGC-1α,and then inhibiting FoxO1-mediated gluconeogenic activity.Similarly,OBB also upregulated p-CRTC2 level,thereby promoting CRTC2 nuclear exclusion and preventing the binding of CRTC2 and CREB,and then inhibiting CRTC2-mediated gluconeogenic activity.After inhibiting AMPK signaling transduction in advance by using AMPK inhibitor Compound C and specific siRNA,OBB no longer exerted the inhibitory effects of FoxO1 and CRTC2 nuclear translocation,and the supernatant glucose level and the expressions of PEPCK and G6Pase rebounded.Conclusion:OBB can inhibit the abnormal gluconeogenesis in PA-induced HepG2 cell insulin resistance model and the underlying mechanisms are related to activating AMPK signaling pathway.OBB activates AMPK/Akt/FoxO1 signaling and AMPK/CRTC2 signaling,thereby promoting FoxO1 and CRTC2 nuclear exclusion,and eventually inhibiting FoxO1-and CRTC2-mediated gluconeogenic activity.