The Effect And Molecular Mechanism Of Weight-bearing Treadmill Exercise And Low-intensity Pulsed Ultrasound On The Gastrocnemius Muscle Atrophy In Type I Diabetic Rats

Objective:Type 1 diabetes mellitus(T1DM)is characterized by autoimmunity against pancreatic B cells,resulting in their destruction and the patients' subsequent dependency on lifelong insulin replacement.T1DM patients have many complications such as cardiovascular,retina and retina disorders.Among them,the skeletal muscle is a major target tissue of diabetic damage,type 1 diabetic subjects without insulin treatment display a dramatic loss of muscle,skeletal muscle atrophy is characterized by a decrease in protein content,fiber diameter,force production,and fatigue resistance,which affecting the normal life and activities seriously.Weight-bearing training is one of the effective ways to improve muscle atrophy,however,the effect of different exercise on muscle atrophy did not have detailed report,and its molecular mechanism have not been studied yet.Low-intensity pulsed ultrasound(LIPUS)is a common treatment for skeletal muscle injury.In muscle tissues,LIPUS can stimulate the proliferation of myogenic precursor cells and myogenic cells.Moreover,accumulating evidence suggests that LIPUS therapy can increase protein synthesis.However,whether LIPUS could improve skeletal muscle atrophy in type 1 diabetes rats has not been previously investigated.Myostatin is a negative regulator of skeletal muscle development,which binds to cell surface receptors causing muscle loss.Active myostatin binds to its receptor ActR ?B with high affinity and regulates the expression of target genes through a TGF-? signaling pathway.Exercise can inhibit MSTN effectively which can promote skeletal muscle hypertrophy.Furthermore,myostatin signaling is able to inhibit Akt phosphorylation therefore down-regulating the PI3K/Akt hypertrophy pathway.In addition to the effects of myostatin on muscle mass and protein accretion,this protein also has an important role in tissue glucose uptake.Earlier studies showed the protein level of mTOR by Akt lead to the activation of pathways promoting protein synthesis and inactivating glycogen synthase.Akt also inactivates the FoxO1 to inhibit muscle atrophy.However,how weight-bearing training and LIPUS improve muscle atrophy have not been reported.The study used the methods of weight-bearing training,low-intensity pulsed ultrasound(LIPUS)and the combination of the two ways,explore the influence of different interventions on the gastrocnemius muscle of type 1 diabetes rats.Discussing the mechanisms of improving muscle atrophy induced by type 1 diabetesby testing physiological and biochemical indexs and different levels research of proteins and genes.It could help us find atraumatic and meaningful adjuvant therapies on the gastrocnemius muscle atrophy induced by type 1 diabetes.Method:Eighty male Sprague-Dawley rats(8 weeks,200-240 g)were obtained from the Laboratory Animal Breeding and Research Center of Xi' an Jiaotong University(Xi'an,China)and were housed in a controlled room(22 ± 2 ?,60%± 5%humidity,and 12 hours light/dark cycle).After 5 days of acclimation,rats were randomly assigned to either the normal control group(NC,n = 10)or the T1DM model group(T1DM,n = 70).Diabetes was induced by a single intraperitoneal(i.p)injection of STZ at 60 mg/kg body weight.After STZ administration,STZ diabetic group was confrmed by measuring blood glucose levels in a tail vein blood samples in first,third,seventh and tenth days.Rats with a blood glucose concentration greater than or equal to 16.7 mmol/L(300 mg/dL),and who were given oral glucose tolerance test(OGTT)were considered diabetic.Once diabetes was induced,diabetic rats were randomly allocated into five groups:diabetic control group(DC),diabetic insulin treated group(DI)as the positive control,diabetic LIPUS therapy group(DU),diabetic weight-bearing training group(DWT),diabetic with low-intensity pulsed ultrasound and weight-bearing training group(D+U+WT).The study test the change of indexes,body weight,food intake,water consumed,muscle strength and blood glucose.Through the OGTT were considered modeling susscess.Using Elisa kit and normal biochenmical kits test insulin,glycosylated hemoglobin,creatine kinase and lactic dehydrogenase levels.HE staining of rat gastrocnemius test morphological changes in muscle fiber cross-sectional area.Western Blot and Quantitative Real-time PCR were used in signaling pathways MSTN/Akt and various signaling molecules downstream signaling pathways from protein levels and gene expression.Results:1.Rats in T1DM group was significantly lose weight throughout the duration of modeling(P<0.01),while after modeling NC group was not significantly different from those before.The T1DM group was significantly loss weight throughout the study when compared with NC group(P<0.01).After STZ injected,rats in metabolic cages amount of food intake and water consumed for 6 days.Compared with NC rats,the T1 DM rats exhibited significantly higher amount of food intake and water consumed(P<0.01).Whilst,all diabetic groups had significantly lower glucose clearance rates compared with NC group(P<0.01).These results obviously showed that modeling succeed by STZ injection.2.The body weights of the NC group continued to be remarkably higher than those in the other five diabetic groups during the whole experimental period(P<0.01).6 weeks of weight-bearing training and LIPUS could increase body weight of rats(P<0.01),the average peak force(P<0.01),and muscle mass and strength(P<0.01).The combination of two intervention ways is better than a single intervention.3.Compared with NC group,HbAlc level in DC group was extremely significant(P<0.01),HbA1c level in the groups of DI,DWT and D+U+WT rats were significantly increased(P<0.05).After 6 weeks of intervention,HbAlc levels significantly reduced compared with DC group(P<0.01).4.After 6 weeks of intervention,the serum creatine kinase activity and CK activity in the gastrocnemius muscle of T1DM group rats were significantly different from the NC group(P<0.01).While,the serum CK activity of DI,DU,DWT and D+U+WT rats were significantly decreased compared with the DC group(P<0.01),however,the CK activity in the gastrocnemius muscle cell of those four groups were significantlyincreased(P<0.01,P<0.05,P<0.05,P<0.05 for DI,DU,DWT and D+U+WT rats,respectively).The LDH level in the gastrocnemius muscle was extremely decreased compared with the NC group(P<0.01),and DI,DWT and D+U+WT group rats were significantly increased compared with the DC group(P<0.01).These results showed that T1DM rats would decreased CK activity in gastrocnemius muscle,lead to muscle atrophy.The three different interventions would improve CK activity in gastrocnemius muscle and LDH level,thus enhance energy metabolism and the improvement of muscle atrophy.5.Western Blot and RT-qPCR methods showed that 6 weeks of weight-bearing training,LIPUS and the combination of two ways can significantly reduce MSTN mRNA levels and protein expression(P<0.01),but there was no significant difference between insulin level and insulin receptor protein expression compared with that of the DC group(P>0.05).Weight-bearing training and LIPUS significantly increased the expression of Akt mRNA(P<0.01),and the protein level of Akt and its phosphorylated(P<0.05).The expression of FoxO1 mRNA and protein level in DU,DWT,and D+U+WT groups was significantly lower than that in DC group,and the expression of mTOR mRNA and protein was significantly higher than that in DC group.Type 1 diabetes significantly decreased GLUT4 mRNA and protein expression(P<0.01),lead to decreased glucose transport capacity and decreased glucose metabolism.Compared with the DC group,weight-bearing training,LIPUS and the combination of two ways significantly increased GLUT4 mRNA and protein expression(P<0.01).These results indicated that different methods can improve muscle atrophy induced by type 1 diabetes,and thus enhance the glucose transport capacity of skeletal muscle.Exercise training and the the combination of the two ways have a better improvement in muscle atrophy than LIPUS.Conclusion:1.According to our results of MSTN/Akt signaling pathways,we found that decreases in MSTN protein levels and the mRNA expression by weight-bearing training and LIPUS in diabetes rats may contribute to improvements of skeletal muscle atrophy.2.Weight-bearing training and LIPUS can markedly up-regulate the expression of Akt,mTOR and down-regulate the expression of FoxO1.It is suggested that MSTN play an important role in improved muscle atrophy by weight-bearing treadmill running and LIPUS via the Akt/mTOR and Akt/FoxO1 signaling pathway.