Mechanism Of GLP-1 Agonist In Alleviating Muscle Loss In Type 2

Part One Relationship between muscle mass and GLP-1 level in type 2 diabetic patientsObjective:Low muscle mass aggravates the disorder of glucose metabolism in type 2 diabetes patients.GLP-1 analogues are novel hypogly-cemic drugs,which have multi-organ protective effect in addition to hypogly-cemic effect.Therefore,whether it has an effect on muscle mass is discussed in this experiment.Method:Fifty-one non-obese male patients with type 2 diabetes admitted to our hospital from March 2020 to February 2021 were retrospectively selected.Age and diabetes history were collected,blood pressure,height and weight of all subjects were measured,and body mass index（BMI）was calculated.After fasting for 8 h,venous blood was collected from the next morning to detect fasting blood glucose（FBG）,lipid levels（TC,TG,HDL-c,LDL-c）and glycosylated hemoglobin（Hb A1c）.GLP-1 and DPP-4 levels were determined by ELISA.Skeletal muscle mass of all subjects was measured by dual-energy X-ray absorptiometry（DXA）to calculate skeletal muscle index（SMI）.SMI was divided into groups A（n=25,SMI<7 kg/m2）and B（n=26,SMI≥7 kg/m2）.Results:1.There was no statistical significance in the disease duration,age,fasting blood glucose,triglyceride,total cholesterol,low density lipo-protein cholesterol,high density lipoprotein cholesterol and DPP4 levels between group A and group B（P>0.05）.GLP-1 level in group A was signifi-cantly lower than that in group B[（0.3±0.22）ng/ml vs（0.5±0.20）ng/ml,P<0.01].2.Univariate analysis showed a positive correlation between SMI and GLP-1（r=0.526,P<0.05）.3.Multiple linear regression analysis showed that age（β=-0.299,95%CI:-0.054～-0.009,P=0.015）and GLP-1（β=0.435,95%CI:0.18～1.612,P=0.001）were independent risk factors for SMI.4.Binary Logistic regression analysis showed that with SMI as the independent variable and age,disease progression,fasting blood glucose,glycated hemoglobin,and GLP-1 level as the dependent variable,the risk of low SMI occurrence at GLP-1 level below P_50th was 10.55 times higher than that above P_50th（95%CI:2.894-38.504,P=0.001）.Conclusions:GLP-1 levels are decreased in patients with type 2 diabetes and low SMI.SMI is independently and positively correlated with GLP-1levels in type 2 diabetes,and the risk of low SMI increases significantly as GLP-1 levels decline.Part Two Mechanism of GLP-1 receptor agonist alleviating muscle loss in type 2 diabetic miceObjective:To study the effect of Liraglutide,a GLP-1 receptor agonist,on muscle loss in diabetic mice by directly inhibiting the expression of MAFbx and Mu RF1.Methods:1.Cell part1)C2C12 cells cultured in vitro were stimulated with liraglutide at different concentrations（100n M,200n M,500n M）,and cell activity was detected by MTT assay 48 h later.2)Cells are added to drugs according to random groups:Control group（untreated group）,negative control group（mannitol 25mmol/L）,high glucose group（glucose 25mmol/L）,high glucose+liraglutide group（glucose 25mmol/L+liraglutide 200nmol/L）,The skeletal muscle specific ubiquitin protease E3（MAFbx and Mu RF1）and AMPKαprotein expression levels were determined by Western blot.3-MH was detected by ELISA.2.Animal part:KK-Ay mice were divided into two groups,liraglutide treatment group（250ug/kg/d）and the same amount of normal saline treatment group.Food intake was recorded every 3 days,and the intervention lasted for8 weeks.Fat and skeletal muscle mass（SMM）were measured by bone densitometer.Before the mice were sacrificed,blood was taken from the eyeballs of the mice,glucose oxidase method was used to determine glucose concentration.After the mice were sacrificed with neck broken,skeletal muscle tissues of the limbs were quickly dissected,washed with PBS buffer solution,the surface residual liquid was dried,and wet weight was obtained by weighing with electronic balance.The m RNA and protein levels of mice skeletal muscle MAFbx and Mu RF1 were detected by Real-time RT-PCR and Western blot.Results:1.Compared with mannitol group and control group,AMPKαlevel in high glucose group was significantly decreased（P<0.01）,and after liraglutide-200 intervention,AMPKαlevel was significantly increased（P<0.05）.2.The levels of MAFbx and Mu RF1 in high glucose group were significantly higher than those in blank control group and mannitol group（P<0.01）,and the levels of MAFbx and Mu RF1 in high glucose group were significantly lower after liraglutide-200 intervention（P<0.01）.3.There was no difference in 3-MH level between the mannitol group and the blank control group.The 3-MH level in the high glucose group was significantly higher than that in the blank control group and the mannitol group（P<0.01）.4.Compared with the saline treatment group,liraglutide group of KK-Ay mice had less food intake,weight loss and blood glucose decrease（P<0.05）.The wet weight of skeletal muscle in liraglutide treatment group was significantly higher than that in normal saline group（P<0.01）.5.mRNA and protein expression levels of Mu RF1 and MAFbx in skeletal muscle of KK-Ay mice in liraglutide treatment group were signifi-cantly decreased compared with saline group（P<0.05）.Conclusions:In cell experiments,AMPKαexpression was inhibited at high glucose concentration,m RNA and protein levels of MAFbx and Mu RF1were increased in high glucose environment,and 3-MH expression was increased,which was the increase of skeletal muscle protein decomposition.liraglutide intervention could alleviate this pathological process.In animal experiments,liraglutide can reduce the blood sugar and weight of diabetic mice,and increase the skeletal muscle mass.It was also observed that liraglu-tide can inhibit the m RNA and protein expression levels of Mu RF1 and MAFbx in the skeletal muscle tissues of diabetic mice.Part Three To explore the Mechanism of GLP-1 receptor agonists to relieve muscle lossObjective:Dexamethasone was used to create a model of muscle loss,and GLP-1 receptor agonist（Liraglutide）was used to intervene and observe its mechanism of alleviating muscle loss.Method:1.Cultured C2C12 muscle cells were stimulated with different concen-trations of liraglutide（10n M,100n M,1000n M）and divided into blank control group,liraglutide 10n M group,liraglutide 100n M group and liraglutide1000n M group.The C2C12 cells were fluorescically stained and the nuclei were stained by DAPI.The efficiency of muscle tube differentiation was evaluated by MHC staining.2.Cultured C2C12 cells were treated with 10u M dexamethasone to create a muscle-reducing model.Different concentrations of liraglutide（10n M,1000n M）were used for intervention,which were divided into blank control group,dexamethasone group,dexamethasone+liraglutide group,10n M dexamethasone+liraglutide group,1000n M dexamethasone+liraglutide group.Fluorescence staining was also performed to evaluate the efficiency of muscle tube differentiation by MHC staining.The m RNA expression levels of MAFbx and Mu RF1 were detected by Real-time RT-PCR.3.MAFbx and Mu RF1 overexpressed recombinant adenovirus with GFP fluorescence were transfected.Adenovirus only expressing GFP fluorescence was used as the negative control.The m RNA expression levels of MAFbx and Mu RF1 were detected by Real-time RT-PCR.Results:1.The fluorescence intensity of MHC was enhanced with the increase of liraglutide concentration,and the fluorescence intensity of MHC was the highest in liraglutide 1000n M intervention group.Real-time RT-PCR results showed that the m RNA expression levels of MAFbx and Mu RF1 decreased gradually with the increase of liraglutide concentration,and the decrease was most significant in liraglutide 1000n M intervention group,with statistical significance（P<0.05）.2.The fluorescence intensity of MHC decreased significantly after dexamethasone treatment,and gradually increased after liraglutide treatment at 10n M,100n M and 1000n M,and the increase was most obvious in Lirag-lutide-1000n M intervention group,with statistical significance（P<0.05）.3.The fluorescence intensity of MHC in Ad-GFP group was significantly reduced after dexamethasone treatment,and increased after liraglutide treat-ment at 1000n M,with statistical significance.Ad-MAFbx and Ad-Mu RF1significantly antagonized the effect of liraglutide on increasing MHC fluores-cence intensity,and the difference was statistically significant（P<0.05）.Conclusions:GLP-1 receptor agonist liraglutide promotes myoblast differentiation in a dose-dependent manner and inhibits the transcriptional expression of MAFbx and Mu RF1 m RNA in a dose-dependent manner.In the dexamethasone treatment group,muscle duct formation was significantly reduced and the intracellular expression levels of MAFbx and Mu RF1 were increased.Liraglutide reduced the induced high expressions of MAFbx and Mu RF1 in a dose-dependent manner,thereby improving muscle duct loss.Liraglutide promotes muscle differentiation and alleviates dexamethasone-induced muscle loss by inhibiting the expression of MAFbx and Mu RF1.