Effects Of Myostatin On Insulin Resistance And Insulin Signaling Pathway In Skeletal Muscle Of Mice With Type 2 Diabetes Mellitus

Background:Myostatin(MSTN)is a member of the transforming growth factor-β(TGF-β)superfamily and is a secreted protein highly produced by skeletal muscle that negatively regulates its growth and development.MSTN also plays a role in regulating glucose and lipid metabolism.Previous studies have shown that people with type 2 diabetes mellitus(T2DM)and insulin resistance have elevated serum and skeletal muscle MSTN levels.The expression of MSTN in skeletal muscle of T2DM mice increased.After receiving MSTN antibody injection,the fasting and postprandial blood glucose levels improved.The available evidences show that MSTN can affect the body’s glucose uptake and utilization and participate in the occurrence of insulin resistance.However,the molecular mechanism of MSTN involved in regulating T2DM insulin resistance is not clear.In this study,based on the MSTN knockout(KO)mice constructed by CRISPR/Cas9 gene editing,a high-fat diet combined with low-dose streptozotocin(STZ)was used to construct a T2DM animal model.The body composition,muscle strength,fatigue resistance,glucose regulation and histopathological changes in skeletal muscle were observed,and the expression levels of proteins on insulin signaling pathway were measured to explore the effect of MSTN on skeletal muscle glucose metabolism and insulin resistance in T2DM.Objective:To analyze effects of MSTN on insulin resistance and insulin signaling pathway in skeletal muscle of mice with T2DM.Methods: MSTN knockout heterozygous C57BL/6N mice were constructed by CRISPR/Cas9 gene editing techniques.We acquired finally male mice consisting of 12 wild type mice,12 heterozygous mice and 12 homozygous mice through breeding and PCR.All animals were randomly assigned to 2 groups including 6 groups in total and 6 in each group: WT group,MSTN(+/-)group,MSTN(-/-)group,WT+DM group,MSTN(+/-)+DM group and MSTN(-/-)+DM group.The first three groups were fed 6-week standard chow and the rest were fed 6-week high-fat diet for combined with small-dose streptozocin(STZ)injection to structure models of T2DM.After measuring fasting plasma glucose(FPG)to evaluate animal models,all mice were measured body weight(BW),body length(BL),abdominal circumference(AC),gastrocnemius muscle weight(BMW),abdominal adipose weight(VAW)and calculated ratios(GMW/BW,VAW/BW)to evaluate the changes of body composition.All animals were measured gripping force and rotating time to exhaustion to evaluate muscle strength and fatigue resistance.All groups were measured fasting serum insulin(FIns),tolerance test(GTT),insulin tolerance test(ITT)and calculated insulin sensitivity index(ISI),homeostasis model assessment-insulin resistance index(HOMA-IR)and area under curve(AUC)to evaluate directly and indirectly insulin sensitivity,insulin resistance and regulation of blood sugar.HE staining analyzed the muscle fiber morphology and cross-sectional area in gastrocnemius muscles.Western blot(WB)quantified expressions of MSTN,Ins R,IRS1,p-IRS1,PI3 K,p-PI3 K,Akt,p-Akt,GLUT4,GSK3β and p-GSK3β on the insulin signaling pathway in gastrocnemius muscles.Measurement data among multiple groups were conducted using one-way analysis of variance(ANOVA)by SPSS software.Images of HE staining and WB were analyzed semiquantitatively by Image J software.Graphs were produced using Graph Pad Prism software.Results:1.The WT+DM group showed obvious decreases in BW,AC,GMW/BW,gripping force,rotating time to exhaustion and the cross-sectional area of gastrocnemius muscle fibres,obvious increases in VAW/BW and MSTN of gastrocnemius muscle(P<0.05)while no statistical difference in BL compared with WT group(P>0.05).The WT+DM group showed obvious increases in FPG,HOMA-IR,GTT-AUC and ITT-AUC,and obvious decreases in FIns,ISI,Ins R,p-IRS1/IRS1,p-PI3K/PI3 K,p-Akt/Akt,GLUT4 and p-GSK3β/GSK3β of gastrocnemius muscle compared with WT group(P<0.05).2.There were no statistical differences in VAW/BW and rotating time to exhaustion among WT group,MSTN(+/-)group and MSTN(-/-)group(P>0.05).The MSTN(-/-)group showed obvious increases in BW,BL,AC,GMW/BW,gripping force and cross-sectional area,while obvious decreases in MSTN compared with WT group(P<0.05).There were significant statistical differences in above indicators except BW and gripping force between MSTN(-/-)group and MSTN(+/-)group(P<0.05).MSTN(+/-)group showed obvious statistical differences in cross-sectional area and MSTN compared with WT group(P<0.05).3.There were no statistical differences in FPG,FIns,ISI and HOMA-IR among WT group,MSTN(+/-)group and MSTN(-/-)group(P>0.05).MSTN(-/-)group showed obvious decrease in GTT-AUC,while obvious increases in ITT-AUC,p-PI3K/PI3 K,p-Akt/Akt,GLUT4 and p-GSK3β/GSK3β compared with WT group(P<0.05).4.There were no statistical differences in rotating time to exhaustion among WT+DM group,MSTN(+/-)+DM group and MSTN(-/-)+DM group(P>0.05).The MSTN(-/-)+DM group showed obvious increases in BW,BL,AC,GMW/BW,gripping force and cross-sectional area,while obvious decreases in VAW/BW and MSTN compared with WT+DM group(P<0.05).There were significant statistical differences in AC,GMW/BW,cross-sectional area and MSTN between MSTN(-/-)+DM group and MSTN(+/-)+DM group(P<0.05).There were significant statistical differences in AC,GMW/BW,cross-sectional area,VAW/BW and MSTN between MSTN(+/-)+DM group and WT+DM group(P<0.05).5.There was no statistical difference in FPG and FIns among WT+DM group,MSTN(+/-)+DM group and MSTN(-/-)+DM group(P>0.05).MSTN(-/-)+DM group showed obvious decreases in absolute value of ISI,HOMA-IR,GTT-AUC,ITT-AUC,while obvious increases in Ins R,p-IRS1/IRS1,p-PI3K/PI3 K,p-Akt/Akt,GLUT4 and p-GSK3β/GSK3β compared with WT+DM group(P<0.05).Conclusion:1.T2DM leads to decreases in muscle mass,muscle strength and fatigue resistance,and accumulation in the visceral fat,probably associated with atrophy of muscle cells due to increased MSTN.T2DM also decreases expressions of insulin receptor and insulin receptor substrate,downregulates insulin signaling pathway,reduces glucose transport and glycogen synthesis which leads to insulin resistance.2.MSTN-deficiency can lead to increases in body weight,body size,muscle mass,muscle strength and myocyte hypertrophy,and the latter is related to gene expression level of MSTN.Loss-of-function MSTN can increase expressions of insulin receptor and insulin receptor substrate,upregulate insulin signaling pathway,promote glucose transport and glycogen synthesis,which improve regulation of blood glucose and insulin sensitivity.3.Deficiency of MSTN can prevent decreases in muscle mass and strength,accumulation in the visceral fat and atrophy of muscle cells,furthermore improve impaired glucose tolerance and insulin resistance due to T2DM through upregulating insulin signaling pathway,promoting glucose transport and glycogen synthesis to some extent.