Study On The Mechanism Of Arginyl-fructosyl-glucose Improving Insulin Resistance In Diabetic Mice

Objective:To investigate the effect of Arginyl-fructosyl-glucose(AFG)on improving insulin resistance in diabetic mice,and to identify AFG targets based on network pharmacology and molecular docking technology.To elucidate the molecular mechanism of AFG to improve insulin resistance by inhibiting mTORCl and then regulating phosphorylation balance of IRS1 at Ser636/Tyr612.Methods:Part 1:Systematic review and meta-analysis were conducted to explore the effects of ginseng amino sugars on insulin resistance in type 2 diabetes mellitus.PubMed,Embase,Web of Science,CNKI,VIP Chinese Journal Service platform(VIP)and Wanfang Data Knowledge Service platform(Wanfang)were searched for relevant studies on ginseng non-saponin amino carbohydrate in the treatment of insulin resistance in type 2 diabetes mellitus and its mechanism of action.The retrieval time was from the establishment of the database to January 31,2023.A systematic review and meta-analysis of the included studies were conducted according to the PRISMA reporting process.Part 2:To investigate the effect and mechanism of AFG in the treatment of insulin resistance in type 2 diabetes based on animal experiments1.C57BL/6J mice were fed with high-fat diet for 8 weeks to establish the early type 2 diabetic model,and 2-month-old db/db mice were used as the middle and late type 2 diabetic model,and AFG intervention was given for 4 weeks.Body weight,fasting blood glucose(FBG),oral glucose tolerance test(OGTT),insulin tolerance test(ITT),blood lipids,insulin secretion,insulin resistance index(HOMA-IR),the ratio of adipose tissue plastids,and HE staining of liver and islets were detected.The expression levels of insulin signaling pathway proteins p-AKT/AKT and membrane GLUT4 in skeletal muscle were determined.2.Target sets of AFG and insulin resistance diseases were searched based on network pharmacology to obtain potential targets of AFG treatment of IR,establish protein interaction network and screen core targets and signaling pathways.SailVina software was used for molecular docking,to analyze the binding force of AFG at the rapamycin-mTORC1 molecular docking site,and to simulate the dynamics of AFG-mTORC1 docking.3.Western blotting was used to detect the changes of p-mTOR/mTOR levels in skeletal muscle of mice with early and middle and late T2DM,and to detect the expression level of IRS1 phosphorylated protein at Ser636/Tyr612.Results:Part 1:Systematic review and meta-analysis were conducted to explore the effects of ginseng amino sugars on insulin resistance in type 2 diabetes mellitus.The results showed that the fasting blood glucose(P<0.00001),insulin resistance index(P=0.02),total cholesterol(P=0.008),triglyceride(P=0.007),malondialdehyde(P=0.004),tumor necrosis factor-α(P=0.004)in type 2 diabetic insulin resistance model animals treated with ginseng amino carbohydrate substance were significantly decreased,while the levels of superoxide dismutase(P=0.003)and glutathione(P=0.006)were significantly increased.It is suggested that ginseng amino carbohydrate has the potential to improve insulin resistance in type 2 diabetes mellitus.Part 2:To investigate the effect and mechanism of AFG in the treatment of insulin resistance in type 2 diabetes based on animal experiments.1.AFG treatment significantly decreased the levels of fasting blood glucose(FBG)(P<0.05),total cholesterol TC(P<0.01),triglyceride(TG)(P<0.05)and low-density lipoprotein(LDL-C)(P<0.05)in early type 2 diabetes model mice.Increased the level of high-density lipoprotein HDL-C(P<0.01),decreased the area under OGTT(P<0.05)and ITT(P<0.01)curves,decreased insulin secretion(P<0.001)and HOMA-IR(P<0.0001),but had no significant effect on the lipid body ratio;HE staining showed that the model group had no obvious organic changes compared with the control group.In the model group,HE staining showed edema,and AFG could significantly improve liver edema.AFG significantly increased the expression level of skeletal muscle insulin signaling pathway protein pAKT/AKT(P<0.001)and membrane GLUT4(P<0.01).The changes of blood biochemical and pathological indexes in db/db mice in the middle and late diabetic model were basically the same as those in the early diabetic model mice.AFG treatment significantly decreased the levels of FBG(P<0.001),TC(P<0.01),TG(P<0.001)and LDL-C(P<0.05),and increased the levels of HDL-C(P<0.001)in db/db diabetic model mice.The area under OGTT(P<0.05)and ITT(P<0.01)curves decreased,the insulin secretion(P<0.01)and HOMA-IR(P<0.01)were significantly decreased,and the lipid body ratio was not significantly changed,but HE staining showed obvious damage in the islet and liver tissue in db/db mice.AFG can improve islet and liver damage in db/db mice.AFG also increased the expression level of skeletal muscle insulin signaling pathway protein p-AKT/AKT(P<0.001)and membrane GLUT4(P<0.0001).2.Network pharmacological screening revealed 237 potential binding sites for insulin resistance and AFG.Molecular docking analysis showed that AFG could bind the binding site of rapamycin and mTORC1,and molecular dynamics simulation results showed that AFG and mTORCl had stable binding,suggesting that AFG might play a role through mTORCl.3.Compared with the control group,the protein expressions of p-mTOR/mTOR(P<0.001)and p-IRS1(Ser636)/IRS1(P<0.05)in skeletal muscle of mice in the early diabetic model group were significantly increased,while p-IRS1(Tyr612)/IRS1 protein expression was significantly decreased(P<0.0001)in the western blotting.Compared with the model group,after AFG treatment,p-mTOR/mTOR(P<0.01)and p-IRS1(Ser636)/IRS1(P<0.001)protein expression levels were significantly decreased,while p-IRS1(Tyr612)/IRS1 protein expression levels were increased(P<0.0001).In db/db mice in the middle and late diabetic model,compared with db/m mice in the control group,the protein expression levels of p-mTOR/mTOR(P<0.0001)and pIRS1(Ser636)/IRS1(P<0.0001)in db/db mice were significantly increased.PIRS1(Tyr612)/IRS 1(P<0.001)protein expression was significantly decreased.Compared with the model group,protein expression levels of p-mTOR/mTOR(P<0.0001)and p-IRS1(Ser636)/IRS1(P<0.001)were decreased after AFG treatment,while protein expression levels of p-IRS1(Tyr612)/IRS1(P<0.0001)were increased.The change trend of mTOR-IRS pathway protein was basically consistent with that of early diabetic model mice.Conclusions:1.Arginyl-fructosyl-glucose(AFG)could improve insulin resistance in early and late type 2 diabetic mice;2.AFG may improve the insulin signaling pathway by inhibiting mTORCl and regulating the balance of IRS1 phosphorylation at Ser636/Tyr612,thereby improving the early and late insulin resistance in type 2 diabetic mice.