Intervention Of Daizongfang On Insulin Resistance In Skeletal Muscle Cells And Its Mechanism

Metabolic syndrome(MS)is characterized by a cluster of metabolic abnormalities gathered in the same individual.It can directly increase the risk of cardiovascular disease,type 2 diabetes and all-cause death.The rapid increase in its prevalence has become a major public health problem affecting the world.In the United States,the prevalence of MS among adults over 20-year-old has risen from 34%to 44.2%.The China Health and Nutrition Survey in 2009 found that the prevalence of MS among adult has reached 18.2%,25.5%among the elderly population.Insulin resistance is a condition whereby response of metabolic organs or tissues to insulin is impaired.It is the common pathology of metabolic disorders such as abdominal obesity,hyperglycemia,high blood pressure and dyslipidemia.Skeletal muscle is a major site of glucose uptake and utilization,and it is also an important site of insulin resistance.Skeletal muscle insulin resistance is mainly manifested as a decrease in insulin-stimulated glucose uptake and utilization.It is one of the causes of systemic insulin resistance and metabolic diseases such as MS.Therefore,it is of great significance to regulate glucose metabolism and alleviate insulin resistance in skeletal muscle in order to prevent the occurrence and development of MS.My professor believes that abdominal obesity is the core component of MS,which is caused by excessive accumulation of lipid in the abdomen,site of the disease is spleen and stomach.According to the spleen governs the distribution of essence,spleen governs muscles and extremities theory,lipid accumulation in the abdomen,overflowing limbs is an important cause of skeletal muscle insulin resistance.On the basis of more than twenty years of clinical experience and modern clinical pharmacology research,professor Liu Ximing created Daizongfang(DZF)to treat MS.DZF is composed of Rhizoma Coptidis,Jiang Banxia,pericarpium trichosanthis and other herbs.In the national 12th Five-Year new drug research project "preclinical pharmacology study of Daizongfang intervening glucose and lipid metabolism disorders in early stage of Metabolic syndrome",efficacy has been carried out in the comprehensive study,the toxicology,clinical and experimental study,we have carried out the study of pharmacy,pharmacodynamics,toxicology,clinical and basic research of DZF.We confirmed that DZF can decrease the body weight of MS patients,alleviate the glucose and lipid metabolism disorders;promote glucose consumption and intracellular triglyceride decomposition in adipocyte,regulate the homeostasis of lipid synthesis and decomposition;down-regulate C/EBP?,PPAR-?,FAS,ACC gene expression to inhibit 3T3-L1 pre-adipocyte differentiation;inhibit oleic acid-induced lipid accumulation in the liver cells.This study combines TCM theory with skeletal muscle insulin resistance to provide further evidence for DZF treatment of MS.Objective1.To study the effects of DZF serum on the glucose metabolism of C2C12 skeletal muscle cells,and to explore the mechanisms based on GLUT1 and GLUT4 membrane protein expression.2.To study the effects of DZF serum on palmitate-induced insulin resistance in C2C12 skeletal muscle cells,and to explore its mechanisms from PI3K/AKT signaling pathway,AMPK pathway and inflammation.3.To study the effects of DZF on glucose and lipid metabolism disorders in type 2 diabetic KKAy mice.Methods1.Preparation of DZF serum:Prepare the DZF serum and blank serum.The activity of C2C12 skeletal muscle cells intervened by 1%,5%,10%and 20%concentration of DZF serum respectively after 24h was detected to establish the proper concentration of DZF serum.2.C2C12 skeletal muscle cells were intervened by 10%concentration of DZF serum(DZFh),5%concentration of DZF serum(DZFm)and 2.5%concentration of DZF serum(DZF1)respectively for 8h,16h and 24h and the glucose consumption was detected by Glucose Oxidase.The glucose uptake of C2C12 skeletal muscle cells in base state and insulin stimulated state was detected by 2-NBDG after the intervention of high concentration of DZF serum for 24h.3.The expressions of GLUT1 and GLUT4 membrane protein of C2C12 skeletal muscle cells in basal state and insulin stimulated state were detected by Western-blot after the intervention of high concentration of DZF serum for 24h.4.C2C12 skeletal muscle cells were incubated by 0.5 mM palmitate for 24h to induce the model of palmitate-induced IR.The glucose uptake of IR C2C12 skeletal muscle cells was detected by 2-NBDG.The concentration of triglyceride of IR C2C12 skeletal muscle cells was detected by Triglyceride Enzyme.5.The mRNA expressions of key molecule IRS-1,PI3K,AKT and GLUT4 in the PI3K/AKT pathway of IR C2C12 skeletal muscle cells were detected by RT-PCR.The mRNA expressions of inflammatory factors IL-6,TNF-?,NF-?B were also detected by RT-PCR.6.The expression of AKT,GSK-3? phosphorylated protein and GLUT4 membrane protein in the PI3K/AKT pathway of IR C2C12 skeletal muscle cells were detected by Western-blot.The expressions of AMPK and ACC phosphorylated protein in the AMPK pathway of IR C2C12 skeletal muscle cells were also detected by Western-blot.7.IR C2C12 skeletal muscle cells were incubated by DCFH-DA for 20mins and the fluorescence intensity was detected by fluorescence microscope and flow cytometry respectively.The level of ROS was estimated by the fluorescence intensity.8.The NF-?B nuclear transport of IR C2C12 skeletal muscle cells was detected by NF-?B activated nuclear transport assay kit.9.8-week-old KKAy mice were fed with high fat diet and 60 mice whose fasting blood-glucose higher than 13.9mmol/L were selected.All the mice were randomly divided into 5 groups.They are control group(Con),Metformin group(Met,200mg/kg/day),high dose DZF group(DZFh,2000mg/kg/day),Medium dose DZF group(DZFm,1500mg/kg/day)and low dose DZF group(DZF1,1000mg/kg/day).Another eight 8-week-old C57BL/6J mice were chosen as normal group(Nor).All the mice were oral gavaged once a day for 8 weeks with their drugs.Mice from Con and Nor were also oral gavaged with sterile water.Mice food-intake,body weight,fasting blood-glucose were detected every week and mice sugar tolerance was tested at the 8th week.After the execution,mice blood and muscle were obtained,and the blood glucose,TG,TC,HDL-C,LDL-C,serum insulin were detected and insulin sensitivity index(QUICKI)were calculated.10.Gastrocnemius of 4 mice selected randomly in every group were obtained,fixed in formalin,dehydrated,embedded in paraffin sections.The expression of GLUT4 protein of mice gastrocnemius was detected by immunohistochemistry.Results1.Compared with normal control group,there was no significant difference in cell survival rate of blank serum group and DZF serum groups.2.Intervened by DZF serum for 8h,compared with blank serum group,cellular glucose consumption in DZFh and DZFm group increased significantly(P<0.05),by 18.75%,22.54%respectively;after 16h and 24h intervention of DZF serum,cellular glucose consumption in DZFh,DZFm and DZF1 group increased significantly(P<0.05),among the three groups,DZFh had the best effect.Intervened by high dose of DZF serum for 24h,cellular glucose uptake were conducted,in basal state,the fluorescence intensity of DZFh group increased by 19.88%,in insulin-stimulated state,the fluorescence intensity increased by 9.54%,the differences were statistically significant(P=0.041,P=0.030).3.Intervened by high dose of DZF serum for 24h,in basal state,the cellular GLUT1,GLUT4 membrane protein expression significantly increased by 30.98%and 30.98%respectively(P=0.008,P=0.008)compared with the blank serum group,in insulin-stimulated state,GLUT1 membrane protein expression significantly increased by 42.26%.(P=0.000)compared with the blank serum group.4.Incubation of C2C12 myotubes with palmitate(24h)prior to insulin challenge inhibited insulin-stimulated glucose uptake by 30.72%,indicating the induction of insulin resistant state.After 24h intervention of DZF serum,insulin-stimulated glucose uptake increased,especially in DZFh and DZFm group,increased by 13.27%and 20.62%respectively(P=0.039,P=0.001).Incubation with palmitate for 24 h,intracellular triglyceride concentrations increased significantly(P=0.000),the presence of DZF serum decreased its concentrations significantly(P=0.004).5.Intervened by high dose of DZF serum for 24h,the AKT,GLUT4 gene expression significantly increased(P=0.042,P=0.041),DZF serum greatly enhanced respectively the phosphorylation of AKT at ser473,GSK-3? at ser9(P=0.002,P=0.002),GLUT4 translocation(P=0.001).6.Intervened by high dose of DZF serum for 24h,the phosphorylation of AMPK and ACC prominently increased(P=0.006,P=0.006).7.Intervened by high dose of DZF serum for 24h,the IL-6,TNF-? gene expression significantly increased(P=0.003,P=0.003),ROS production was abolished(P<0.05),gene expression and nuclear transport were significantly increased(P =0.002)8.Though body weight of KKAy mice in all DZF groups have no significant difference compared with control group,their growth has a downtrend.The fasting blood-glucose of KKAy mice in all DZF groups has a downtrend compared with control group.The fasting blood-glucose in DZFh decreased 15.19%(P<0.05)compared with control group at the 6th week.The fasting blood-glucose in DZFh and DZFm decreased 17.46%(P<0.05)and 15.29%(P<0.05)respectively compared with control group at the 8th week.The sugar tolerance in DZFh is improved significantly compared with control group(P<0.05).The TC level in DZFh decreased 11.33%compared with control group(P<0.05).The TG level in DZFh and DZFm decreased 28.78%(P<0.05)and 18.80%(P<0.05)respectively compared with control group,The HDL-C levels in all DZF groups have no significant difference compared with control group(P>0.05).The LDL-C level in DZFh is decreased 25.20%compared with control group(P<0.05).The fasting insulin levels and QUICKI in DZFh and DZFm decreased significantly(P<0.05).9.The GLUT4 protein expression of skeletal muscle cells in DZFh increased significantly compared with control group(P=0.003).Conclusion1.There is no significant effect on the activity of C2C12 skeletal muscle cells when the concentration of DZF serum is lower than 20%.The glucose consumption and uptake of skeletal muscle cells can be increased by DZF serum.The increased expression of GLUT1 and GLUT4 membrane protein of skeletal muscle cells may be its mechanism.2.The IR of C2C12 skeletal muscle cells can be improved by DZF serum.The possible mechanism is the mRNA expression and protein phosphorylation of AKT,the key molecule in PI3K/AKT pathway,can be up-regulated by DZF serum,leading to the promotion of the expression of GLUT4 mRNA and membrane translocation and the activity reduction of Glycogen synthase kinase GSK-3.AMPK pathway may also be activated by DZF serum,leading to the inflammation inhibition of skeletal muscle cells.3.The fasting blood-glucose of KKAy mice can be improved by DZF serum.The TC,TG,LDL-C and fasting insulin level of KKAy mice can be down-regulated by DZF serum.The insulin sensitivity and the GLUT4 protein expression of skeletal muscle cells of KKAy mice can be increased by DZF serum.