Study On The Mechanism Of Xinxiaoke Formula In The Treatment Of Hepatic Insulin Resistance In Type 2 Diabetes Through Acting On AMPK Pathway

BackgroundChina has the world's largest diabetes epidemic.In 2013,the estimated overall prevalence of adult diabetes in China is 10.4%,and approximately 114.4 million people have diabetes.420 million people globally have diabetes,with a projected global prevalence of 642 million by 2040.Long-term metabolic disorders in diabetes can cause damage to multiple systems and seriously threaten human health and quality of life.Type 2 diabetes mellitus(T2DM),which more than 90%of diabetes patients suffer from,is characterized by the resistance of target tissues to insulin stimulation and reduced insulin secretion with a functional defect of beta cells of the pancreas.Insulin resistance(IR)is an important pathological mechanism of both the onset and development of T2DM.Currently,alleviating IR is still one of the key avenues to treating type 2 diabetes.Hypoglycemic drugs including Metformin and thiazolidinediones both have the effect of increasing the sensitivity of peripheral tissues to insulin,but they have the adverse reactions of gastrointestinal discomfort,fracture and increased risk of heart failure respectively.Traditional Chinese medicine(TCM)has been in use for diabetes and its complications for more than 2000 years,with remarkable curative effect.Based on literature research,we find that liver-kidney Yin deficiency and dryness-heat is an important pathological mechanism of IR in T2DM,therefore nourishing Yin and clearing heat is the key therapeutic method for T2DM and IR.My supervisor has been engaged in clinical and basic research on the treatment of diabetes and its complications with traditional Chinese medicine for a long time.On the basis of Xiaoke Formulas from Danxi' Experiential Therapy written by Zhu Danxi and Case Records as a Guide to Clinical Practice written by Ye Gui respectively,Xinxiaoke Formula is constructed,consisted of Rhizoma Coptidis 10g,Radix Rehmanniae 30g,Rhizoma Anemarrhenae 10g,Gypsum Fibrosum 30g,Radix Ophiopogonis l Og,Cortex Lycii 30g,Radix Paeoniae Alba 10g,and Radix Glycyrrhiza 6g,and is usually used for T2DM patients with the syndrome of Yin deficiency and effulgent fire,aquiring a significant effect.Modern pharmacological studies on the Chinese herbal medicines from Xinxiaoke Formula show that it plays an important role in regulating target proteins associated with insulin pathways,such as AMP-activated protein kinase(AMPK),insulin receptor(InsR),insulin receptor substrate(IRS),phosphatidylinositol 3-kinase(PI3K)/AKT,glucose transporter type 4(GLUT4),silent information regulator 1(SIRT1),peroxisome proliferator-activated response-? coactivator-1?(PGC-1?),nuclear factor kappa B(NF-?B),NADPH oxidase 4(NOX4)and peroxisome proliferator-activated response-y(PPARy)in liver,skeletal muscle and adipose tissue,and can significantly improve inflammatory reaction and oxidative stress.Liver is the crucial organ which plays a central role in the maintenance of glucose homeostasis by balancing gluconeogenesis and glycogen synthesis.Hepatic IR can lead to elevated gluconeogenesis and reduced glycogen synthesis in the liver,and then causes hyperglycemia.Therefore,hepatic IR contributes to the onset and development of T2DM.Xinxiaoke Formula is established for the treatment of T2DM people with liver-kidney Yin deficiency and dryness-heat,which may have a significant effect of regulating hepatic IR.Oxidative stress has been considered as a major hallmark for the pathogenesis and development of T2DM.More and more evidence show that hyperlipidemia,hyperglycemia,and proinflammatory factors in T2DM,usually contribute to overproduction of reactive oxygen species(ROS)from NADPH oxidase,which through acting on stress-signaling pathways,such as c-jun N-terminal kinase(JNK),NF-?B,p53 mitogen-activated protein kinase(p53MAPK),can lead to increasing serine/threonine-phosphorylated IRS proteins and reduced tyrosine-phosphorylated IRS proteins,and impair the PI3K/AKT signaling pathway as well.Increased production of ROS and oxidative stress have been involved in the pathogenesis and development of T2DM.AMPK can regulate hepatic gluconeogenesis and hepatic glycogen synthesis to maintain blood glucose homeostasis.Thus,AMPK dysregulation contributes to the onset and development of T2DM.PI3K/AKT pathway plays an important role in insulin signaling pathway,which is considered as the key regulator relevant to gluconeogenesis and glycogen synthesis.Previous studies have shown that activation of AMPK can activate hepatic PI3K/AKT signaling pathway and increase hepatic insulin sensitivity.The expression of SIRT1 in liver can improve hepatic steatosis,steatohepatitis,and increase insulin sensitivity,thus SIRT1 dysfunction may be an important pathological mechanism of the occurrence of hepatic IR in T2DM.SIRT1 can activate AMPK by deacetylating the upstream liver kinase B1(LKB1),which promotes LKB1 translocation from the nucleus to the cytosol,where it is activated and in turn phosphorylates and activates AMPK.Likewise,AMPK can activate SIRT1 by increasing the NAD/NADH ratio.Collectively,these findings suggest the existence of an AMPK-SIRT1 cycle that links the cell's energy and redox states,which can prevent diabetes in experimental animals.Recent studies have shown that the AMPK/SIRT1 cycle not only plays an important role in regulating hepatic glucolipid metabolism,but inhibits excessive production of NADPH oxidase-derived ROS to improve oxidative stress.Functional disturbance of a single molecule,NOX4 as a source of selective IR and responsiveness,is sufficient to induce the key harmful features of deranged insulin signaling in T2DM.Based on the modern pharmacological research,Xinxiaoke Formula has shown to have an important effect of improving oxidative stress as well as AMPK/SIRT1 signaling pathway.It is suggested that Xinxiaoke Formula may not only improve the impairment of hepatic IRS/AKT signaling pathway in diabetes and activate hepatic AMPK/PI3K/AKT signing pathway to ameliorate hepatic insulin resistance in T2DM,but also through the activation of AMPK/SIRT1/NOX4 signing pathway,may inhibit NOX4-mediated oxidative stress and overproduction of ROS to improve the impairment of hepatic IRS1/PI3K/AKT signaling pathway in T2DM,which plays a significant role in treating hepatic IR in T2DM.ObjectiveTo clarify the effects of Xinxiaoke Formula in improving hepatic IR in type 2 diabetic rat induced by combined high-fat diet(HFD)and streptozotocin(STZ)injection and IR-HepG2 cell model induced by glucosamine administration by performing vivo and vitro experiments,and to explore the underlying molecular mechanisms of Xinxiaoke Formula activating AMPK/PI3K/AKT signaling pathway to ameliorate hepatic IR in T2DM,and activating AMPK/SIRT1/NOX4 signaling pathway to inhibit the excessive production of NOX4-mediated ROS in the liver to prevent oxidative stress and overproduction of ROS from disturbing IRS-1/AKT insulin signaling pathway to improve hepatic IR.Methods1.In vivo part:type 2 diabetes in vivo was induced by combined high-fat diet(HFD)and streptozotocin(STZ)injection in male SD rats.The control group had 10 male SD rats.Forty-eight type 2 diabetic rats were randomly divided into 6 groups(n=8 for each group):type 2 diabetic rats treated with vehicle only,type 2 diabetic rats treated with high dose of Xinxiaoke Formula,medium dose of Xinxiaoke Formula,low dose of Xinxiaoke Formula,mixture component of four hypoglycemic monomers from Xinxiaoke Formula(berberine,catalpa alcohol,mangiferin,and glycyrrhizic acid),and metformin respectively.The intervention time was seven weeks.Weight changes of SD rats in all groups were monitored before intervention and every week after intervention.Fasting blood glucose(FBG)was measured before intervention,and at week 4 and week 7 after intervention.Seven weeks later,rats were administrated with oral glucose tolerance test(OGTT)and insulin tolerance test(ITT)assay,and area under glycemic curve(AUC)was assessed respectively.After that,rats were anesthetized and euthanized using pentobarbital(30 mg/kg,i.p.),the blood samples were exsanguinated via the abdominal aorta to be used for detecting and measuring Hemoglobin alc(HbAlc),fasting blood insulin(FINS)along with the assessment of HOMA-IR,total cholesterol(TC),triglycerides(TG),low density lipoprotein(LDL),high density lipoprotein(HDL),superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),malondialdehyde(MDA),alanine aminotransferase(ALT),and aspartate aminotransferase(AST).The whole liver was harvested and weight of liver was measured.Hematoxylin-ensin(HE)staining was used for pancreas and liver histological analysis.Periodic acid schiff(PAS)staining was used to detect liver glycogen deposits,and glycogen quantification was performed by the glycogen content assay kit.By measuring these indicators,we were aimed at investigating the effects of Xinxiaoke Formula in regulating glycolipid metabolism,alleviating IR and oxidative stress,protecting liver function,lowering the weight of liver organ,improving hepatic steatosis and steatohepatitis,increasing glycogen content in T2DM rat.To further clarify the molecular mechanism of the Xinxiaoke Formula ameliorating hepatic IR,western-blot was used to detect the relative expression levels of target proteins relevant to hepatic AMPK signaling pathway,including AMPK,p-AMPK(Thrl 72),SIRT1,NOX4,IRS-1,p-IRS-1(Ser307),AKT,p-AKT(Ser473),GSK3B,p-GSK3?(Ser9)and PEPCK.The expression levels of mRNA related to hepatic AMPK signaling pathway,including AMPK,SIRT1,NOX4,IRS-1 and AKT were detected by RT-PCR respectively.2.In vitro part:IR in vitro was induced by glucosamine administration in HepG2 cells.Both cell morphology and CCK-8 assay used to estimate cell viability were applied to determine the intervention concentration range of the Xinxiaoke Formula,as well as the appropriate intervention concentrations of the mixture component of four hypoglycemic monomers from Xinxiaoke Formula,and metformin.Glucose production and glycogen content in all groups were respectively tested to verify the IR in IR-HepG2 cell model,and to assess the effects of the Xinxiaoke Formula in inhibiting glucose production and increasing glycogen content.The content of ROS and the levels of SOD,GSH-PX and MDA in each group were detected to evaluate oxidative stress injury in IR-HepG2 cell model and the effects of the Xinxiaoke Formula in improving oxidative stress respectively.In order to further clarify the molecular mechanism of Xinxiaoke Formula in improving IR in IR-HepG2 cell model and to verify the results of animal experiments,the expression levels of proteins related to AMPK signaling pathway,including AMPK,p-AMPK(Thr172),SIRT1,NOX4,IRS-1,p-IRS-1(Ser307),AKT,p-AKT(Ser473),GSK3p,p-GSK3?(Ser9),FOXO1,p-FOXO1(Ser256)and PEPCK,were respectively detected by western-blot in each group,and the expression levels of mRNA associated with AMPK signing pathway,including AMPK,SIRT1,NOX4,IRS-1,AKT,and GSK3?,were respectively detected by qRT-PCR.Results1.In vivo part:(1)Xinxiaoke Formula and mixture component of four hypoglycemic monomers from Xinxiaoke Formula had no effect on body weight of type 2 diabetic rats(P>0.05),but all showed significant effects that could lower FBG,HbAlc,FINS,HOMA-IR(P<0.05),improve the glucose tolerance and increase the insulin sensitivity(P<0.05),lower the levels of serum TC,TG(P<0.05),increase the level of serum SOD and decrease the level of serum MDA(P<0.05),and significantly decrease the level of serum ALT(P<0.05).The above effects was most outstanding in high concentration group of Xinxiaoke Formula.(2)Xinxiaoke Formula and mixture component of four hypoglycemic monomers from Xinxiaoke Formula could significantly improve pancreatic and hepatic histomorphology in type 2 diabetic rat,lower the weight of liver organ,improve hepatic steatosis and steatohepatitis,and increase hepatic glycogen content.Likewise,high dose of Xinxiaoke Formula may have a better effect of improving the above indexes.(3)the results of western-blot showed that the expression levels of hepatic proteins related to AMPK signing pathway,including p-AMPK(Thr172),SIRT1,NOX4,IRS-1,p-IRS-1(Ser307),p-AKT(Ser473),p-GSK3 ?(Ser9)and PEPCK in type 2 diabetic rat were well improved treated with different doses of Xinxiaoke Formula,and mixture component of four hypoglycemic monomers from Xinxiaoke Formula respectively.Among them,high-dose Xinxiaoke Formula could significantly up-regulate the relative expression levels of p-AMPK(Thrl72),SIRT1,IRS-1,p-AKT(Ser473),p-GSK3 ?(Ser9),and down-regulate the relative expression levels of NOX4,p-IRS-1(Ser307)and PEPCK in the liver tissues(P<0.05).(4)the results showed that the Xinxiaoke Formula,especially the high-dose Xinxiaoke Formula,could significantly up-regulate the expression levels of SIRT1 and IRS-1 mRNA,and down-regulate the expression level of NOX4 mRNA(P<0.05).The mixture component of four hypoglycemic monomers from Xinxiaoke Formula can significantly up-regulate the expression level of IRS-1 mRNA,and down-regulate the expression level of NOX4 mRNA(P<0.05).2.In vitro part:(1)when HepG2 cells were treated with 18mM glucosamine for 18h,glucose generation was significantly increased while glycogen content was significantly decreased(P<0.05),which suggested the successful establishment of IR-HepG2 cell model.Glucose production in IR-HepG2 cells all could be obviously reduced treated with different concentrations of Xinxiaoke Formula,and mixture component of four hypoglycemic monomers from Xinxiaoke Formula respectively(P<0.05).The glycogen content in IR-HepG2 cells was significantly increased treated with medium or high concentrations of Xinxiaoke Formula(P<0.05).(2)the production of ROS could be reduced in a dose-dependent way with the Xinxiaoke Formula.The content of ROS could be significantly reduced in the medium and high concentrations of the Xinxiaoke Formula,and the mixture component of four hypoglycemic monomers from Xinxiaoke Formula(P<0.05).The levels of SOD and GSH-PX were significantly increased and the level of MDA was decreased in the Xinxiaoke Formula groups and mixture component of four hypoglycemic monomers from Xinxiaoke Formula(P<0.05).(3)the medium concentration of the Xinxiaoke Formula had a significant effect in improving IR in IR-HepG2 cell model,which was used to treat IR-HepG2 cells,and to investigate the molecular mechanism.The results of western-blot showed that the expression levels of proteins associated with AMPK signing pathway,including p-AMPK(Thr172),SIRT1,IRS-1,p-AKT(Ser473),p-FOXO1(Ser256)all were significantly up-regulated in both medium concentration of the Xinxiaoke Formula group and mixture component group(P<0.05),and the expression levels of target proteins,including NOX4,p-IRS-1(Ser307)and PEPCK,were down-regulated in both medium concentration of the Xinxiaoke Formula group and mixture component group(P<0.05).In addition,the expression level of p-GSK3 ?(Ser9)also was significantly up-regulated in the medium concentration of the Xinxiaoke Formula group(P<0.05).(4)the results of qRT-PCR showed that the expression level of IRS-1 mRNA was significantly up-regulated,and NOX4 mRNA was significantly down-regulated in the medium concentration of the Xinxiaoke Formula group and mixture component group(P<0.05).In addition,medium concentration of the Xinxiaoke Formula had a better effect of down-regulating the expression level of GSK3? mRNAConclusion1.The Xinxiaoke Formula significantly improved IR and oxidative stress in HFD/STZ rat and in glucosamine-induced HepG2 cells.2.The Xinxiaoke Formula regulated hepatic gluconeogenesis and hepatic glycogen synthesis through acting on AMPK and PI3K/AKT signing pathway in the liver of HFD/STZ rat and in glucosamine-induced HepG2 cells to improve hepatic IR.3.The Xinxiaoke Formula ameliorated the impairment of IRS/AKT signing pathway in the liver of HFD/STZ rat and in glucosamine-induced HepG2 cells.The Xinxiaoke Formula might activate AMPK/SIRT1/NOX4 signing pathway to prevent NOX4-mediated oxidative stress and overproduction of ROS from disturbing IRS-1/AKT insulin signaling pathway,thus to improve hepatic IR.Further research is needed.