Experimental Study On Effects And The Impact Molecular Mechanism Of Musclin On Rat Differentiated Myoblasts Glucose Uptake

ObjectiveMusclin were first identified and reported as a novel skeletal muscle-derived secretory factor by signal sequence trap of mouse skeletal muscle cDNAs in 2004. Functionally,recombinant musclin significantly attenuated insulin-stimulated glucose uptake and glycogen synthesis in C₂C₁₂myocytes.Therefore,musclin likely to be involved in insulin resistance(IR).IR is widespread in human.Particularly the most patients with type 2 diabetes and obesity are accompanying with insulin resistance.IR was also reported exist in 25%of normal glucose tolerance and 25%of the elderly.In addition,even in the normal physiological circumstances,IR will occur,such as pregnancy.Therefore,the mechanism of IR will be the fundamental method to the ultimate control and improve the insulin sensitivity.In preliminary studis,mostly around the insulin target organs and the insulin receptor;in recent years,with gradually recognized that the adipose tissue is an endocrine organ,it was found that adipose tissue is the important bridge connecting the endocrine dysfunction,such as obesity,diabetes and IR.The abnormal expression of fat-derived factors,such as leptin,resistin, adiponectin and so on,are participat in and increase IR,injuryβ-cell function,and induce diabetes as an important molecular mechanism.As the largest insulin sensitivity tissue,skeletal muscle is participat in at least 90%of the insulin-stimulated glucose disposal in healthy.Therefore,the discovery of musclin is bound to lead the people to have a more in-depth understanding on insulin resistance. IR is the performance of insulin-mediated glucose metabolism not sensitive in its target organs,such as the liver,muscle,adipose tissue,and so on.Mostly study on the molecular mechanism of glucose uptake is focus on glucose transporter and the insulin signal transduction pathway.Among them,the largest study of glucose transporter in skeletal muscle is glucose transporter 4(GLUT₄),which mainly storaged within cell vesicles in the absence of insulin-stimulated.When insulin receptor binding insulin,a series of cascading effect are inspired,leading to the vesicles which rich with abound of GLUT₄,move to the cell membrane,with the cell membrane and vesicle membrane fusion,GLUT₄ translocation to the cell membrane and increased its activity,and combinate with glucose,then its structural changes,transport glucose to the cells,and then revert to its original structure.An important feature of Type 2 diabetes is the resistance of insulin-stimulated glucose uptake.As a result of this process which is mediated by GLUT₄,GLUT₄ was considered to play a key role in insulin resistance;the phosphatidylinositol 3-kinase(PI3K)pathway has the largest studies of insulin signal transduction pathway.PI3K is a peptide substance which function of cell signal transduction and molecular switches,protein kinase B(PKB/AKT)is its downstream effector molecule.Phosphorylation of PI3K activats activated PKB,which starts in the skeletal muscle of insulin signal transduction pathway,thus impacts GLUT₄ mRNA synthesis,cytoplasmic protein synthesis in the Golgi,transport and cytoplasmic membrane translocation,thereby affects glucose uptake.PPARγis involved in glucose metabolism as a nuclear factor receptor,and linked its downstream factor LXRαby subunit.Dalen,et,al,confirmed GLUT₄ regulated by the nuclear factor of liver X receptor(liver X receptors,LXRs)α(LXRα);LXRαgene regulars GLUT₄ by a conservative equence of the promoter region-DR-4 response element(GLUT₄ LXRE), and to improve the glucose uptake of peripheral organizations.It was not clear whether these cytokines will participate in musclin induced rat differentiation myoblast glucose uptake,and how to participate.It has not been reported in domestic,in particular.This study was a series of experiments on rats to explore musclin of glucose uptake in myocytes and the impact of molecular mechanism;and using rosiglitazone,one members of the family-PPARγagonist Thiazolidinediones (TZD)drugs,to observe the effect of PPARγon musclin induced insulin resistance,and the effect of musclin on glucose uptake in myocytes and molecular mechanism link of molecular biology.Methods1.Rat myocyte isolated,culture and differentiation.(1)Rat myocyte isolatedOn the experimental day,rats were anesthetized with chloral hydrate(0.3ml/100g body wt,i.p.)The mtocytes were gained as described by Pei,X.T.Single myofibers were isolated from the hindlimb EDL and soleus muscles after collagenase digestion and cultured individually in Matrigel-coated wells.After initial adherence,myofibers were either fixed(time 0)or supplemented with our standard myogenic growth medium (DMEM[high glucose,with L-glutamine,110mg/L sodium pyruvate,and piridoxine hydrochloride supplemented with 100μg/ml penicillin and 100μg/ml streptomycin GIBCO-Invitrogen)containing 20%fetal bovine serum(Sigma-Aldrich),10%horse serum(HyClone),1%chicken embryo extract prepared from whole 10-day old embryos])for the indicated periods before fixation.This growth medium supports both proliferation and spontaneous differentiation of myoblasts.(2)Rat myocytes primary cultureThe cells were maintained in DMEM supplemented with 20%(v/v)FCS,2mM glutamate,15 mM Hepes,PH 7.5,100μg/ml penicillin and 100μg/ml streptomycin (here termed 20%FCS-DMEM),All incubations were at 37℃in medium continuouly gassed with 95%O₂/5%CO₂.Clones of satellite cells were established from isolated myofibers.The cells were cultured after 24-hour,replacement inoculated medium, joined 0.25%trypsin digestion digestive juice,gently shaking,the mixed cells were Suspended.Centrifugation(1000 g,10 minutes),then absorbed the supernatant,added 10 ml containing 10%fetal calf serum DMEM medium with pipette,and then selected single cell to 96-well plates.When cells grow to be more than 50 clones,Cells digestived by 0.25%trypsin digestion juice,rinse,after centrifugation,added containing 10%fetal calf serum DMEM medium with pipettes.Cells then inoculated in 24-well plates,replaced medium every 2-3 days for a solution,passaged cells with 0.25%trypsin digestion digestive fluid when cells were grown to 90%confluency. Blood cell counts plate count cells.Seed cells in culture bottles,and culture cells in the humid incubator at 37℃and 5%CO₂.Passaged cells with 0.25%trypsin digestion digestive fluid;replaced differentiation medium when cells were grown to 90% confluency.(3)Rat myocytes immunohistochemical identificationRat myocytes immunohistochemical identification was fixed in acetone for 5 min and preincubated with phosphate buffered saline(PBS)containing 2%bovine serum albumin(BSA).The sections were then incubated overnight at 4℃the polyclonal anti-MyoD antibody(Wuhan Boster Biological Technology Ltd,Wuhan,China.) diluted at 1:100.Immunoreactivity was detected using goat anti-mouse IgG(Wuhan Boster Biological Technology Ltd,Wuhan,China.)at a dilution of 1:500 for 1 h.(4)Rat myocytes differentiationTo obtain fully differentiated mytocytes,cells were grown to 90%confluency and incubated for 1 day in the above medium containing only 1%(v/v)FCS(here termed 1%FCS-DMEM).Cells were then seeded at a density of 1×10⁶ cell/cm² into 10cm dishes or 6-well plates precoated with 1%(v/v)gelation,in 1%FCS-DMEM containing 10μM cytosine-1β-D-arabinofuranoside.Medium was replaced at 1 and 2 days,and after 4 days mtocytes were return to 1%FCS-DMEM alone and used for experiments after a further 24 h.(5)Rat myocytes Identification by iron-hematoxylin stainingP₃ myocytes were grown to 90%confluency continue incubating in medium containing 1%FBS and 100μg/ml penicillin,and 100μg/ml streptomycin in DMEM medium at 37℃,5%CO₂ under humid conditions for 7 days,then abandoned the medium,washed with distilled water 40℃,fixed with 10%formalin for 30 minutes, washed three times with distilled water,2.5%iron alum disseminated at room temperature for 30 minutes;2.5%iron alum +10%hematoxylin disseminated 40 minutes,rinsed three times by distilled water,2.5%iron alum disseminated for two minutes,rinsed three times with distilled water again.And then added 4 ml distilled water,the stained cells were placed under phase contrast microscope camera records staining situation.2.Experimental groups(1)The effects of musclin on rat differentiated myocytes glucose uptake in the different concentrations of glucoseA,5mmol/l Glucose +10mU/ml Insulin;B,25mmol/l Glucose +10mU/ml Insulin;C,5mmol/l Glucose +10mU/ml Insulin +0.5μg/ml Musclin;D,25mmol/l Glucose +10mU/ml Insulin +0.5μg/ml Musclin.(2)Experimental study on the effects and the molecular mechanism of musclin on rat differentiated myocytes glucose uptake in 25mmol/l glucoseA,25mmol/l Glucose;B,25mmol/l Glucose + 0.5μg/ml Musclin;C,25mmol/l Glucose+10mU/ml Insulin;D,25mmol/l Glucose + 0.5μg/ml Musclin + 10mU/ml Insulin.(3)Experimental study on the effects and the molecular mechanism of rosiglitazone on rat differentiated myocytes glucose uptake treated by musclinA,(Control)25mmol/l Glucose;B,25mmol/l Glucose + 0.5μg/ml Musclin;C,25mmol/l Glucose + 0.5μg/ml Musclin + 0.4μg/ml Rosiglitazone;D,25mmol/l Glucose + 10mU/ml Insulin.E,25mmol/l Glucose + 0.5μg/ml Musclin + 10mU/ml Insulin;F,25mmol/l Glucose + 10mU/ml Insulin + 0.5μg/ml Musclin + 0.4μg/ml Rosiglitazone + 10mU/ml insulin.3.Detection of indicators and methods(1)Myocytes identified by immunohistochemistry method;(2)Identified myocytes differentiat to skeletal muscle cells(myoblasts)by iron hematoxylin staining;(3)Glucose uptake by isotope tracer method;(4)PI3K,AKT,PPARγ,LXRαand GLUT₄ mRNA expression level detected by RT-PCR;(5)Protein expression of measured AKT and pAKT by Western blot.4.Statistical calculationsResults are presented as mean±SEM.Significance of a difference between two groups was determined by applying ANOVA and paired two-tailed t tests to the data. The linear regression and multivariate analyses(MINOVA and discrimination function tests)were carried out by Minitab statistical software.P values＜0.05 were considered to be statistically significant.Results1.The effects of musclin on rat differentiated myocytes Glucose Uptake in different concentrations of glucose(1)Myocytes culture,differentiation and identification Muscle cells function as a syncitium,with several nucleated cells fusing to form the muscle fibre-the key cellular activator of this process being the satellite cell.In this experiment,satellite cells divide to produce satellite cell-derived myoblasts that further proliferate,before committing to differentiation and fusing to form myotubes,which then mature into myofibers.(2)The effects of musclin on rat differentiated myocytes glucose uptake in the different concentrations of glucoseCompare with the control group(group A),glucose uptake of the musclin intervention group(group B)decreased significantly(P＜0.05);compared with group B,glucose uptake of insulin and musclin intervention group(Group D)was significantly increased(P＜0.05). 2.Experimental study on the effects and the molecular mechanism of musclin on rat differentiated myocytes glucose uptake in 25mmol/l glucose(1)Myocytes culture,differentiation and identification The results of this part were similar to the results of experimental one.(2)Experimental study on the effects of musclin on rat differentiated myocytes glucose uptake in 25mmol/l glucoseCompare with the control group(group A),glucose uptake of the musclin intervention group(group B)decreased significantly(P＜0.05);compared with group B,glucose uptake of insulin and musclin intervention group(Group D)was significantly increased(P＜0.05).(3)Semi-quantitative RT-PCR of PI3K,AKT,PPARγ,LXRα,and GLUT₄ mRNAs expression levelsCompare with the control group(group A),PI3K,AKT,PPARγand LXRαmRNA levels of the musclin intervention group(group B)decreased significantly(P＜0.05); compared with group B,PI3K,AKT,PPARγand LXRαmRNA levels of insulin and musclin intervention group(Group D)were significantly increased(P＜0.05),but the expression levels of GLUT₄ did not alter(data were not shown)which is similar to Nishizawa et al.(4)Musclin attenuated the protein expression levels of AKT and pAKT in insulin-stimulated glucose uptake of rat myocytes treated by musclinCompare with the control group(group A),AKT and pAKT protein levels of the musclin intervention group(group B)decreased significantly(P＜0.05);compared with group B,AKT and pAKT protein levels of insulin and musclin intervention group (Group D)were significantly increased(P＜0.05). 3.Experimental study on the effects and the molecular mechanism of rosiglitazone on rat myocytes glucose uptake treated by musclin(1)Myocytes culture,differentiation and identification The results of this part were similar to the experimental one.(2)Rosiglitazone improve rat differentiated myocytes glucose uptake treated by musclin in 25mmol/l glucoseCompare with the control group(group A),glucose uptake of the musclin intervention group(group B)decreased significantly(P＜0.05).Compared with group B,glucose uptake of insulin and musclin intervention group(Group C)was significantly increased(P＜0.05);glucose uptake of musclin,rosiglitazone and insulin total intervention group(group F)was significantly increased(P＜0.01).(3)Rosiglitazone improve PI3K,AKT,PPARγ,LXRα,and GLUT₄ mRNAs expression levels of rat differentiated myocytes treated by musclin in 25mmol/l glucoseCompare with the control group(group A),PI3K,AKT,PPARγand LXRαmRNA levels of the musclin intervention group(group B)decreased significantly(P＜0.05); compared with group B,PI3K,AKT,PPARγand LXRαmRNA levels of insulin and musclin intervention group(Group D)were increased(P＜0.05);PI3K,AKT,PPARγand LXRαmRNA levels of musclin,rosiglitazone and insulin total intervention group (group F)were significantly increased(P＜0.05).But the expression levels of GLUT₄ did not alter(data were not shown).(4)Rosiglitazone improve the protein expression levels of AKT and pAKT of rat differentiated myocytes treated by musclin in 25mmol/l glucoseCompare with the control group(group A),AKT and pAKT protein levels of the musclin intervention group(group B)decreased significantly(P＜0.05);compared with group B,AKT and pAKT protein levels of insulin and musclin intervention group (Group D)were increased(P＜0.05);AKT and pAKT protein levels of musclin, rosiglitazone and insulin total intervention group(group F)were significantly increased (P＜0.05). Conclusion1.Musclin attenuated insulin-stimulated glucose uptake in rat differentiated myocytes in 25mmol/l glucose.2.Musclin attenuated insulin-stimulated glucose uptake in rat differentiated myocytes in 25mmol/l glucose is related to factors belong to insulin signal transduction pathway-PI3K/AKT and the nuclear factors-PPARγand LXRα,which is not linked to GLUT₄ mRNA alter.3.Rosiglitazone improve rat differentiated myocytes glucose uptake treated by musclin in 25mmol/l glucose;and rosiglitazone improves PI3K/AKT mRNAs expression levels and the AKT activity of rat differentiated myocytes treated by musclin in 25mmol/l glucose,which is related to the nuclear factors-PPARγ,LXRα.