Puerarin Modulates Insulin Sensitivity And Fatty Acids Metabolism In Skeletal Muscle

Skeletal muscle is the major organ and important peripheral tissue dependent on insulin to uptake and utilize glucose and fatty acids.Muscular insulin-resistance is considered an important event in pathophysiology of type 2 diabetes mellitus(T2DM).It is a current research focus to find out new pathway independent on insulin for stimulating skeletal muscle to uptake and utilize glucose.Muscular insulin-resistance is closely related to metabolic disorders of glucose and fatty acids and mitochondrial dysfunction.Mitochondria is the key place for oxidize glucose and fatty acids.It is an important reason of muscular insulin-resistance for mitochondrial dysfunction and/or mitochondrial quantity decrease leading to lipid intermediate metabolites such as longchain acyl-CoA,DAG,ceramide and metabolic byproducts ROS accumulating in skeletal muscle cells.Puerarin is a natural isoflavone compounds.Puerarin has been reported to lower plasma glucose and lipids and to improve glucose homeostasis for treatment of T2DM patients in clinical.However,the cellular and molecular mechanisms involved in its antidiabetic actions remain unclear.Especially whether puerarin can improve muscular insulin-resistance has not been reported.We conducted the present study to determine the effects of puerarin on muscular insulin-resistance as well as the mechanism by looking into its effects on fatty acids metabolism and mitochondrial function in skeletal muscle of T2DM rats and in palmitate(PA)-induced insulin-resistant L6 myotubes.Objective:We adopted skeletal muscle tissue of T2DM rats and PA-induced insulinresistant L6 myotubes to explore the effects of puerarin on insulin sensitivity,fatty acids catabolism and mitochondrial function in order to elaborate the mechanisms of puerarin on improving insulin-resistance and regulating glucose and fatty acids metabolism in skeletal muscle.Methods:1.L6 myotubes were treated with 0.75 mM PA for 24 hours to establish insulin-resistant model.In puerarin-treatment group,puerarin was pre-incubated with L6 myotubes for 24 hours then co-incubated with 0.75 mM PA for 24 hours in presence of or absence of insulin(100nM)during the last 30 minutes of incubation with the PA.In some experiments,1.0 uM of wortmannin were added one hour before the puerarin treatment.Key proteins involved in insulin signaling pathway such as Akt,AS 160 were detected by western blot analysis.Glucose uptake by myotubes was performed by flow cytometric analysis of 2-NBDG.Cell membrane were separated and membrane GLUT4 contents were determined by western blot.2.60 male Sprague-Dawley rats were divided into two groups,10 were used as normal control,the other 50 were used to develop T2DM by long time high-fat diets combined with low dose streptozotocin(STZ,35mg/kg)injection intraperitoneally.After diabetic model established,the diabetic rats were randomly divided into diabetic group,puerarin-treatment group,naloxone-treatment group,naloxone combined with puerarin-treatment group.4 weeks later,the rats were sacrificed.Fasting blood glucose and serum insulin levels were measured.The contents of POMC and ?-endorphin in hypothalamus,adrenal glands and pancreas were determined by ELISA and real time RT-PCR.In addition,the expressions of MOR and related proteins involved in insulin signaling pathway in skeletal muscle were determined by Western blot.Interactions between MOR and IRS1 was detected by Co-immunoprecipitation(Co-IP).3.Established T2DM rats model and PA-induced insulin-resistant L6 myotubes model according to above methods.The expressions of related proteins regulating fatty acids catabolism as well as mitochondrial biogenesis and function were detected by real time RT-PCR and western blot.Mitochondrial structures of skeletal muscle and L6 myotubes were observed under transmission electron microscope.Results:1.Puerarin activated key molecules such as Akt,AS 160 in insulin signaling pathway and significantly enhanced acute insulin-mediated GLUT4 translocation to cell membrane and 2-NBDG uptake in PA-induced insulin-resistant L6 myotubes.These results indicated that puerarin could ameliorate insulin signal transduction in insulin-resistant states.2.The blood glucose concentration was decreased and glucose tolerance was increased after T2DM rats were treated with puerarin for 4 weeks.The MOR expression and phosphorylation of skeletal muscle were enhanced under the treatment of puerarin.Puerarin ameliorated impaired insulin signaling of skeletal muscle in T2DM rats.In addition,puerarin up-regulated the expression and phosphorylation of MOR and enhanced glucose uptake in PA-induced insulin-resistant L6 myotubes.All those effects of puerarin in T2DM rats and PA-induced insulin-resistant L6 myotubes could be abolished by MOR antagonist naloxone.MOR possibly interacts directly with IRS1 in skeletal muscle.3.Puerarin significantly up-regulated the contents of phosphorylated AMPK and ACC as well as the expressions of CPT1b and LCAD leading to increase fatty acids oxidation of skeletal muscle,subsequently improved lipids disorders and reduced serum TG and FFA contents in T2DM rats.The content of p-p66Shc/Shc increased but the expressions of UCP2,FOXO3a and SOD2 decreased,mtDNA content decreased,and the expressions of proteins involved in mitochondrial fusion and fission such as Mfn2,OPA1,DRP1,and respiratory chain complexes such as NDUFS3,UQCRFS1,MTCO1 also decreased in skeletal muscle resulted in decreasing amounts of mitochondria and impaired mitochondrial function in diabetic model group,compared with those in normal control group.In puerarin-treatment group,the above changes were reversed,significant differences of those were found as compared to those in diabetic model group.Puerarin decreased ROS content but increased mitochondrial membrane potential and complex I activity thus enhanced ATP level of PA-treated L6 myotubes.The effects of puerarin on fatty acids catabolism and mitochondrial function in PAinduced insulin-resistant L6 myotubes were consistent with those in skeletal muscle of diabetic rats.Conclusions:Puerarin can improve the insulin signaling pathway via activation of MOR in skeletal muscle and increase muscular insulin sensitivity.In addition,puerarin can enhance mitochondrial function and increase fatty acids oxidation.Therefore,puerarin can coordinate the utilization of fatty acids and glucose in skeletal muscle cells.These results provide new insight on the molecular mechanism of puerarin on regulating the metabolic steady of glucose and fatty acids in insulin-resistant states.