| IntroductionType 2 diabetes mellitus(T2DM)is a group of systemic metabolic disease.Its clinical characteristics mainly include two aspects:(1)hyperglycemia;(2)various tissues and organs damages caused by hyperglycemia.Clinical trials found that almost all T2DM patients have insulin resistance(decreased insulin sensitivity/reactivity),which is a clinical manifestation of reduced insulin physiological effects,namely that the target cells of insulin cannot use insulin effectively,eventually leading to glucose disorders and elevated blood glucose in the whole body level.At present,studies on insulin resistance mainly focus on tissues closely related to material metabolism such as liver and adipose cells,among which liver,as the largest metabolic organ in the body,plays a key role in blood glucose control.Glucose transported to the liver is mainly involved in energy metabolism through the following ways:(1)decomposition for energy supply;(2)conversion to glycogen;(3)conversion to non-sugar storage and utilization such as fat and protein.Therefore,hepatocyte glucose metabolism disorder plays an important role in the occurrence and development of insulin resistanceCurrently,the exact pathophysiological mechanisms of insulin resistance have not been elucidated,and the occurrence of insulin resistance is closely related to the impairment of insulin signaling pathways.The imbalance of this pathway is mainly related to the expression or activation(phosphorylation)of some key proteins,such as insulin receptor(InsR),insulin receptor substratel(IRS1),phosphatidylinositol 3-kinase(PI3K),glucose transporter 4(GLUT4)and glycogen synthase kinase 3 beta(GSK3β),leading to reduced glucose utilization and increased blood glucose through inhibiting glucose uptake and glycogen synthesis.G protein coupled receptor kinase 2(GRK2)is a serine/threonine kinase.GRK2 is a comprehensive node of the signal network.Its functions mainly have two aspects:(1)mediates the desensitization and resensitization of G protein-coupled receptors;(2)inhibits or activates proteins.It have reported that GRK2 could inhibit InsR expression and tyrosine phosphorylation of IRS1,resulting in the impairment of InsR/IRS1/PI3K signaling pathway,which leads to impaired insulin action and insulin resistance.Importantly,inhibition of GRK2 could improve glycogen synthesis in mouse liver FL83B cells and glucose metabolism in db/db mice.Therefore,GRK2 may be a new target for insulin resistance regulation by drugsPaeoniflorin-6’-O-benzene sulfonic acid ester(CP-25)is an ester compound produced by our group through the reaction of paeoniflorin(Pae)with benzenesulfonyl chloride.Studies have shown that CP-25 can significantly improve the inflammation in animal models of adjuvant arthritis and collagen-induced arthritis by inhibiting GRK2 to regulate inflammatory signaling pathways transmission,inhibit the production of pro-inflammatory factors and exert immunomodulatory effects.Inflammatory response plays an important role in the progression of T2DM.Inflammation can extensively destroy the structure and function of β cells and various target cells,leading to reduced insulin secretion and physiological effects of insulin and causing insulin resistance.However,whether CP-25 can improve insulin resistance by regulating GRK2 is unclear.Therefore,this study aims to explore the effects of CP-25 on improving hepatic insulin resistance in T2DM mice,and its effects on insulin signaling pathway by regulating GRK2,providing a new strategy for insulin insulin resistance treatmentObjectiveClarify the effect of CP-25 on hepatic insulin resistance in T2DM mice,and to reveal the regulatory effect of CP-25 on insulin signaling pathway through GRK2 and its molecular mechanismsMethodsWe established the T2DM animal model by high fat diet combined with intraperitoneal injection of streptozotocin.The C57BL/6 mice were divided into normal group,T2DM group,CP-25(70mg/kg)group,gliclazide(20mg/kg)group,metformin(200mg/kg)and CP-25(70mg/kg)-combined gliclazide(200mg/kg)group.Each group has 12 mice.After successful modeling,mice were given the drugs by gavage for 5 weeks.We measured the body weight and fasting blood glucose once a week.After administration,we detected the glucose tolerance,serum insulin level and insulin sensitivity.Meanwhile,we observed the effect of CP-25 on liver pathology by hematoxylin-eosin staining.we measured the effect of CP-25 on GRK2 and insulin signaling pathway proteins(InsR,p-IRS/IRS,PI3K,p-GSK3β/GSK3β)expression in liver by western blot and immunofluorescence.In vitro,we induced the insulin resistant HepG2 cell model to detect the role of CP-25 on glucose consumption,glycogen content and the expression of GRK2 and insulin signaling pathway proteins(InsR,p-IRS/IRS,PI3K,p-GSK3β/GSK3β,GLUT4)we examined the interaction between GRK2 and InsR or IRS1 by co-immunoprecipitation and confocal microscopy.Furthermore,GRK2 siRNA and GRK2 inhibitor GSK180736A inhibited GRK2 expression and activity to verity the effect of inhibiting GRK2 on insulin signaling pathwayResults1.The effects of CP-25 on body weight,glucose tolerance and insulin sensitivity in T2DM mice.CP-25 could improve the body weight of T2DM mice.Compared with T2DM group,the body weight was significantly increased in CP-25 and CP-25-combined gliclazide groups(P<0.05).CP-25-combined gliclazide group obviously reduced fasting blood glucose and serum insulin level(P<0.01,P<0.01);CP-25 and CP-25-combined gliclazide groups improved glucose tolerance and insulin sensitivity(P<0.05,P<0.01)Especially,the effects of CP-25-combined gliclazide group was the most obvious2.The effects of CP-25 on liver pathology in T2DM mice.Hematoxylin-eosin staining showed that CP-25 and CP-25-combined gliclazide groups significantly improved liver damage in T2DM mice,reducing lipid vacuoles and different degrees of hepatocyte necrosis along with hepatocytes retain intact cell structure.3.The effects of CP-25 on glucose consumption and glycogen content in insulin resistant HepG2 cells.1μM insulin stimulated HepG2 cells for 48 hours to induce insulin resistant HepG2 cell model.CP-25 treatment at different concentrations(1μM,100nM,10nM and 1nM)increased glucose consumption and glycogen content in insulin resistant HepG2 cells4.The effects of CP-25 on insulin signaling pathwayWestern blot and immunofluorescence results showed that CP-25 and CP-25-combined gliclazide groups significantly increased the expression of InsR,p-IRS/IRS and PI3K,and inhibited GRK2 expression and GSK3β phosphorylation in the liver of T2DM.Meanwhile,CP-25(luM,100nM,lOnM,1nM)significantly increased the expression of InsR,p-IRS/IRS,PI3K and GLUT4,and inhibited GRK2 expression and GSK3β phosphorylation in insulin resistant HepG2 cells.Furthermore,co-immunoprecipitation and confocal microscopy results showed that CP-25 could attenuate the interaction between GRK2 and InsR or IRS1.5.The effects of down-regulation of GRK2 expression and function on glucose consumption,glycogen content and insulin signaling pathway in insulin resistant HepG2 cellsSilencing GRK2 gene expression and inhibiting GRK2 activity can increase glucose consumption,glycogen content,and insulin signaling pathway key proteins(InsR,p-IRS/IRS,PI3K,GLUT4)expression,and inhibit GSK3β phosphorylation in insulin resistant HepG2 cellsConclusion1.CP-25 improved glucose metabolism disorder and insulin sensitivity,and relieved liver damage in T2DM mice.CP-25 increased glucose consumption and glycogen content.2.The interaction between GRK2 and InsR,GRK2 and IRS1 inhibited the expression of InsR and tyrosine phosphorylation of IRS1 to prevent InsR/IRS1/PI3K insulin signaling pathway transduction.The molecular mechanism of CP-25 improving hepatic insulin resistance may be related the up-regulation of insulin signaling pathway by inhibiting GRK2 expression and activity.3.CP-25-combined gliclazide not only improved the hypoglycemic effect of gliclazide,but also better protect liver damage in T2DM mice.Therefore,CP-25-combined hypoglycemic drugs may have important prospects for T2DM hepatic insulin resistance. |
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