Relationship Between Vitamin D And Sarcopenia In Older Adults And Preliminary Study On Its Underlying Mechanism

Purpose Muscle stem cells could differentiate into myoblasts,proliferate,differentiate,and fuse with each other to form multinucleated muscle fibers.Decreased differentiation and fusion of myoblasts will lead to thinning or reduction of muscle fibers,which is one of the key mechanisms for inducing sarcopenia.Though there were a series of studies assessed the effect of Vitamin D（VD）on the proliferation of myoblasts,the potential regulatory mechanisms of action on VD-induced cell fusion still unclear.Our previous study found that the deficiency of VD in older population was closely related to low muscle mass,further studies based on muscle stem cell model indicated that the lack of VD inhibited the proliferation and differentiation of myoblasts.In the present study,the association between VD and sarcopenia,as well as the effects of VD on cell fusion and sarcopenia phenotype were studied from three aspects:population-based cohort study,in vitro and in vivo studies and randomized controlled trial.This study is helpful to clarify the mechanism of muscle mass reduction and progression to sarcopenia caused by VD deficiency and provide a new approach for the prevention and control of sarcopenia and related drug development.Method 1)Population research.The participants were selected from the Tianjin Chronic Low-grade Systemic Inflammation and Health Cohort Study from 2015 to2018 in Tianjin,China.Serum 25-（OH）D concentrations,which is the gold standard for measuring the body’s VD level,were detected by enzyme-linked immunoassay.Grip strength（GS）,one of the main indicators of sarcopenia,was evaluated by using electronic grip meter.Analysis of covariance was used to analyze the association between baseline serum VD and the annual change of GS.2)In vitro experiments.Cell proliferation of mouse skeletal muscle cell precursor cells-myoblast C2C12 were detected after administrated with VD（1,25-dihydroxyvitamin D₃,1,25-（OH）₂D₃for 24,48,and 72 h.Immunofluorescence was used to observe the effect of myoblasts fused with each other to form multinucleated muscle fibers after VD treatment and differentiation for 5 days.In addition,the potential role of VD on cells fusion and protein synthesis as well as muscle fiber formation via VD receptor（VDR）/Dock180/Rac1 axis or Akt/mTOR/p70s6k pathway were further investigated.3）In vivo experiments.21-month-old natural aging muscle failure mice were randomly divided into 4 groups,fed with deficiency（150IU/kg feed）,normal（1000IU/kg feed）,low-dose（2500IU/kg feed）and high-dose（5000IU/kg feed）of Vitamin D₃（VD₃）.After feeding elderly mice for 3 months,the effect of VD₃intervention on the phenotype of sarcopenia（muscle mass,muscle strength,motor function,muscle fiber tissue morphology,etc.）were clarified.The serum VD,blood calcium and phosphorus,and tissue ATPase changes in activity and glycogen content were analyzed.4)Randomized controlled trial:we recruited 195 elderly sarcopenic adults with vitamin D insufficiency.The participants were randomly divided into three groups according to intervention:placebo group;low dose group（600IU/d vitamin D3）and high dose group（1000IU/d vitamin D3）.The individual participation period comprised 12 months.We gather information as follows:physical examination,blood measurement and sarcopenia component assessments.The main outcome measures were 6m gait speed,grip strength and muscle mass.Kruskal-Wallis test was used to compare the change of outcome variables before and after the intervention for the three groups.Results 1)Population research.A total of 3,625 participants（2,111 men and 1,514women）aged over 40 years were enrolled in the cohort study（3-year follow-up period;mean,2 years）.The mean value of all participants for the annual change of GS was-0.197kg（men,-0.039kg;women,-0.443kg）.The result of the cohort study showed that after adjusting for multiple confounding factors,a positive correlation between 25-（OH）D level and annual change of GS was found.The annual change of GS in 25-（OH）D level at<30 nmol/L,30-50 nmol/L and>50 nmol/L was-0.33（-0.62,-0.03）,-0.24（-0.54,-0.05）,and-0.23（-0.53,0.07）（P<0.01）,respectively.By Bonferroni multiple comparison,compared with the annual change of GS with 25-（OH）D<30nmol/L,the annual change of GS with 25-（OH）D at 30-50 and>50 nmol/L was statistically significant.25-（OH）D had no interaction with all covariates（P≥0.06）.2)In vitro experiments.In vitro studies showed that different concentrations of1,25（OH）₂D₃ interfered cell viability of C2C12 cells,and the effect of cell proliferation was best at 48 h.After C2C12 cells differentiating for 5 days,the embryonic myosin heavy chain（eMyHc）protein of muscle fibers was significantly overexpressed.The supplement of 1,25（OH）₂D₃promoted the mutual fusion of myoblasts and formed more multinucleated muscle fibers.In addition,after 1,25（OH）₂D₃ intervention,the expression of VDR was significantly up-regulated,while the expression of Dock180/Rac1 decreased.Furthermore,the expression levels of p-Akt/mTOR proteins increased significantly after 1,25（OH）₂D₃administration.However,the p70S6K,which is a downstream molecule,did not change significantly.3)In vivo experiments.For the in vivo study,the body weight and food intake of mice in each group did not change significantly after different doses of VD₃ formula administration.Interestingly,the serum 25-（OH）D,1,25（OH）₂D₃ and blood calcium levels were significantly increased,but no significant increase in phosphorus was observed.In the determination of body composition of mice,the lean mass of the 2500IU-dose and 5000IU-dose groups increased significantly,but no statistical change was observed in bone density.In the sarcopenia phenotypic experiment,the GS of mice in VD₃intervention group was significantly higher than that of the lacking group,but the tendency of the declined GS with the increase of months could not be reversed,and the ability to rotate exercise was significantly enhanced,similar with GS.The ATPase activity and glycogen content in muscle tissue elevated significantly in VD₃intervention group than that of the lacking group,and the morphological staining of tibial anterior muscle indicated that VD₃supplementation could promote muscle fiber area to become larger,tightly arranged,tissue interstitial small,and regular shape,suggesting that VD₃ supplementation could improve aged mice muscle atrophy.4)Randomized controlled trial.After 12 months intervention,the average annual changes（median（interquartile range））of gait speed in placebo group,low dose group（600UI/d）and high dose group（1000UI/d）were-0.09（-0.23,0.05）m/s（within the groups:P<0.0001）,-0.04（-0.16,0.09）m/s（within the groups:P=0.09）and 0.01（-0.13,0.13）m/s（within the groups:P=0.72）（among groups:P<0.01）,respectively.By Bonferroni multiple comparison,there was significant difference in gait speed change between high dose group and placebo group（P<0.05）.No significant intervention effects were found for GS and muscle mass.After 12months intervention,the serum25-（OH）D（P=0.02）and 25-（OH）D₃（P=0.03）concentration significantly increased in placebo group.the serum 25-（OH）D,25-（OH）D2,and 25-（OH）D3 concentration significantly increased in low dose group and high dose group（all P<0.0001）.The changes of serum 25-（OH）D,25-（OH）D2,and 25-（OH）D3 concentration in low dose group and high dose group was significantly higher than those in placebo group（P<0.0001）.Meanwhile,the changes of serum 25-（OH）D,25-（OH）D2,and 25-（OH）D3 concentration in high dose group was significantly higher than those in low dose group（P<0.0001）.Conclusions In middle-aged and elderly people over 40 years old,higher serum 25-（OH）D levels were correlated with the increased change of GS.1,25（OH）₂D₃supplementation promoted the proliferation of C2C12 cells,and the formation of muscle fibers via p-Akt/p-mTOR axis.The increased expression of VDR receptor down-regulated the Dock180/Rac1 pathway,and increased the formation of multinucleated muscle fibers.In addition,VD₃ supplementation improved the phenotype,blood biochemical indexes,and tissue morphology of elderly mice with sarcopenia.Moreover,a randomized controlled trial demonstrated that daily 1,000 IU vitamin D supplementation had beneficial effect on the maintenance of gait speed in sarcopenic old adults.The results of this study provided novel clues and new approaches to prevent and manage the occurrence and development of sarcopenia.