| BackgroundThe prevalence of type 2 diabetes mellitus(T2DM)is increasing.There is a largest number of T2DM patients of the world in China.T2DM-related health expenditures were increasing.Sarcopenia is defined as the progressive loss of muscle mass and strength associated with aging that increases the risk of falls,disability,hospitalization,and death.T2DM is one of the risk factors for sarcopenia.Generally,T2DM and sarcopenia coexist and promote each other,with the loss of muscle mass and strength accelerated,resulting in poor quality of life of the patients.Studies have confirmed that T2DM-related sarcopenia is an independent disease entity.Hence it is of great significance to explore the pathogenesis of T2DM-related sarcopenia,as well as the effective interventions.Systemic chronic inflammation(SCI)plays an important role in the pathogenesis of both T2DM and sarcopenia,and may act as a bridge between the two conditons.Group 2 innate lymphoid cells(ILC2s)are the first-line responders to tissue damage and participate in the development of allergic and metabolic diseases.Several studies have shown that the proportion of ILC2s in mice with high risk factors for T2DM such as obesity or high-fat diet was reduced,and that ILC2s might exert its function in the pathogenesis of T2DM.ILC2s were involved in the repair of acute skeletal muscle injury,as well as in the development of Duchenne muscular dystrophy.Whether ILC2s play a role in T2DM-related sarcopenia has not been reported.Studies have demonstrated decreased serum and fecal butyrate levels in T2DM patients.The sodium butyrate(NaB)could increase the proportion of slow muscle fibers and improve the insulin resistance in mice treated with high-fat diet,and inhibit oxidative stress and autophagy through PI3K/AKT/mTOR signaling pathway with the atrophy of skeletal muscle cells alleviated in diabetic nephropathy db/db mice.These results indicated that NaB had a protective effect on T2DM-related skeletal myopathy.Our previous experiments demonstrated that NaB could up-regulate the ILC2s in db/db mice.In this study,we established an animal model of T2DM-related sarcopenia with db/db mice.By administering ILC2s regulators such as NaB,IL-2/Jes6(stimulator of ILC2),anti-CD90.2(inhibitor of ILC2s),etc.,ILC2s were up-regulated and downregulated in vivo.Hence we would investigate the role of ILC2s and related mechanisms in T2DM-related sarcopenia,and explore the effect of NaB treatment on the T2DM-related sarcopenia.Metabolic syndrome(MetS)is a high risk factor for T2DM.The joint report of the American Diabetes Association and the European Diabetes Association published in 2005 proposed to treat MetS as a pre-disease state of T2DM.Metabolic diseases such as T2DM,MetS,and obesity are closely related to sarcopenia.Therefore,it is helpful to investigate the relationship between Mets and sarcopenia for the early diagnosis and prevention of T2DM-related sarcopenia.Moreover,MetS patients,as well as T2DM patients,are at high risk of atherosclerotic cardiovascular disease(ASCVD).Whether the muscle mass reduction associated with MetS has an impact on ASCVD is worth to be elucidated.In this study,we would retrospectively investigate the relationship between the reduced muscle mass and carotid artery atherosclerosis in a MetS population.Section I The role and mechanism of ILC2s in type 2 diabetesrelated sarcopeniaObjectives1.To investigate whether ILC2s are involved in T2DM-related sarcopenia.2.To clarify the mechanism of ILC2s in the pathogenesis of T2DM-related sarcopenia.3.To explore whether NaB exerts a protective effect on T2DM-related sarcopenia by influencing ILC2s.Methods1.Establishment of animal model of T2DM-related sarcopeniaAfter 1 week of adaptive feeding,the 7-week-old male C57BL/6J-db/db mice and wild-type male C57BL/6J mice were were grouped as C57BL/6J group(n=5)and db/db group(n=5).(1)Mesurement of body weight and blood glucose regularly(2)Performent of intraperitoneal glucose tolerance test(IPGTT)(3)Measurement of skeletal muscle mass by dual energy X-ray(DXA)and gastrocnemius/body weight ratio(4)Measurement of skeletal muscle mass strength by hanging grid test and forelimb grip strength test(5)Measurement of exercise ability by treadmill exhaustive running test 2.Groups of experimental animalsThe 7-week-old male C57BL/6J-db/db mice and wild-type male C57BL/6J micewere grouped randomly as follows with a time of different treatments for 9 weeks:(1)For the mechanism of ILC2s and effect of NaB:C57BL/6J+water group(n=12),C57BL/6J+NaB group(n=12),db/db+water group(n=12),db/db+NaB group(n=12)(2)For the ILC2s expansion experiments:db/db+PBS group(n=10),db/db+IL2/Jes6 group(n=10)(3)For the ILC2s suppression experiments:db/db+NaB+PBS group(n=10),db/db+NaB+anti-CD90.2 group(n=10)3.Monitor mouse body weight,blood sugar,muscle mass,muscle strength and exercise capacity as in step 14.Detection of serum IL-33 and IL-13 by ELISA5.Detection of Treg cells and ILC2s cells by flow cytometry6.Histopathological examinationHematoxylin-eosin(HE)staining of gastrocnemius muscle tissue was performed to observe morphological changes and calculate the cross-sectional area(CSA)of muscle fibers.Immunofluorescence staining of slow and fast MyHC was performed to calculate the ratio of slow muscle fibers to fast muscle fibers.7.Molecular examinationWestern Blotting was performed to detect Atrogin 1,MuRF 1,Fast MyHC,Slow MyHC,PGC-lα,GATA-3,P-STAT3 and STAT3 in gastrocnemius muscle tissue.8.StatisticsStatistical analysis and graphing were performed using SPSS 20.0.The means of 2×2 factorial design were compared using the factorial variance analysis method to analyze the two main influencing factors "T2DM" and "NaB".Comparisons between two groups were performed using independent samples t-test.Comparisons between multiple groups were performed using One-way ANOVA and LSD post-hoc analysis.P<0.05 was considered statistically significant.Results1.Establishment of T2DM-related sarcopenia animal modelCompared with C57BL/6J group,the area under the curve(AUC)of IPGTT and body weight of db/db group were significantly increased(both P<0.05).The muscle mass index measured by DXA,gastrocnemius muscle/body weight ratio,forelimb grip strength,time of hanging grip test,treadmill exhaustive running time and distance were significantly reduced in db/db group than those in C57BL/6J group(all P<0.05).These results suggested that T2DM-related sarcopenia animal model was successfully established in db/db mice.2.Effects of NaB and modulating ILC2s on ILC2s in spleen and skeletal muscle of db/db mice(1)Compared with the C57BL/6J+water group,the proportion of ILC2s in the spleen and skeletal muscle of db/db+water group were significantly reduced(both P<0.05).Compared with the db/db+water group,the proportion of ILC2s in the spleen and skeletal muscle in the db/db+NaB group was significantly increased(both P<0.05).These results suggested that NaB could up-regulate the proportion of ILC2s both in spleen and skeletal muscle of db/db mice.(2)Compared with db/db+PBS group,the proportion of ILC2s in spleen and skeletal muscle of db/db+IL-2/Jes6 group was significantly increased(both P<0.05).Compared with db/db+NaB+PBS group,the proportion of ILC2s in spleen and skeletal muscle in db/db+NaB+anti-CD90.2 group was significantly decreased(both P<0.05).These results suggested that the ILC2s stimulator IL-2/Jes6 and inhibitor anti-CD90.2 were effective.3.Effects of NaB and modulating ILC2s on glucose metabolism in db/db mice(1)Compared with the db/db+water group,AUC of IPGTT was significantly decreased in the db/db+NaB group(P<0.05).This result suggested that NaB could improve the glucose tolerance of db/db mice.(2)Compared with the db/db+PBS group,AUC of IPGTT was significantly decreased in the db/db+IL-2/Jes6 group(P<0.05).Compared with the db/db+NaB+PBS group,there was no significant difference in the AUC of IPGTT in the db/db+NaB+anti-CD.90.2 group(both P>0.05).These results suggested that expansion of ILC2s could improve the glucose tolerance of db/db mice.4.Effects of NaB and modulating ILC2s on muscle mass,muscle strength and exercise capacity in db/db mice(1)Compared with the db/db+water group,db/db+NaB group had a significant improvement in the treadmill running time and distance(both P<0.05),indicating that NaB could improve the running capacity of db/db mice.(2)Compared with the db/db+PBS group,the treadmill running time and distance of the db/db+IL-2/Jes6 group were significantly increased(both P<0.05).Compared with db/db+NaB+PBS group,the grid hanging time,treadmill running time and exercise distance of the db/db+NaB+anti-CD.90.2 group were significantly reduced(all P<0.05).These results suggested that expansion of ILC2s could improve the running capacity whereas supression of ILC2s could inhibit the running capacity of db/db mice.5.Effects of NaB and modulating ILC2s on myofiber atrophy of db/db mice(1)Compared with the C57BL/6J+water group,the CSA of myofibers in the db/db+water group was significantly decreased(P<0.05),and the expressions of Atrogin 1 and MuRF 1 were significantly increased(both P<0.05).Compared with the db/db+water group,the CSA of myofibers in the db/db+NaB group was significantly increased(P<0.05),and the expression of MuRF 1 was significantly decreased(P<0.05).These results suggested that NaB could improve myofiber atrophy in db/db mice.(2)Compared with the db/db+PBS group,the CSA of myofibers in the db/db+IL2/Jes6 group was significantly increased(P<0.05),and the expressions of Atrogin 1 and MuRF 1 were significantly decreased(both P<0.05).Compared with the db/db+NaB+PBS group,the CSA of myofibers in db/db+NaB+anti-CD90.2 group was significantly decreased(P<0.05),and the expression of MuRF 1 was significantly increased(P<0.05).These results suggested that ILC2s could improve myofiber atrophy in db/db mice.6.Effects of NaB and modulating ILC2s on skeletal muscle fiber type switching of db/db mice(1)Compared with the C57BL/6J+water group,the db/db+water group had significantly decreased proportion of slow muscle fibers and ratio of Slow MyHC/Fast MyHC(both P<0.05).Compared with the db/db+water group,the db/db+NaB group had significantly increased proportion of slow muscle fibers and ratio of Slow MyHC/Fast MyHC(both P<0.05).These results suggested that NaB could increase the proportion of slow muscle fibers in gastrocnemius muscle of db/db mice.(2)Compared with the db/db+PBS group,the db/db+IL-2/Jes6 group had significantly increased proportion of slow muscle fibers and ratio of Slow MyHC/Fast MyHC(both P<0.05).Compared with the db/db+NaB+PBS group,the db/db+NaB+anti-CD90.2 group had significantly decreased proportion of slow muscle fibers and ratio of Slow MyHC/Fast MyHC(both P<0.05).These results suggested that ILC2s could increase the proportion of slow muscle fibers in gastrocnemius muscle of db/db mice.7.Effects of NaB and modulating ILC2s on inflammatory status in db/db mice(1)Compared with the C57BL/6J+water group,the db/db+water group had a significantly decreased proportion of Treg(P<0.05).Compared with the db/db+water group,the db/db+NaB group had a significantly increased proportion of Treg(P<0.05).These results suggested that NaB could improve the inflammatory status in db/db mice.(2)Compared with the db/db+PBS group,the db/db+IL-2/Jes6 group had a significantly increased proportion of Treg(P<0.05).There was no significant difference in the proportion of Treg between db/db+NaB+PBS group and db/db+NaB+antiCD90.2 group(P>0.05).These results suggested that ILC2s could improve the inflammatory status in db/db mice.8.Effects of NaB and modulating ILC2s on serum IL-33 and IL-13 in db/db mice(1)Compared with the C57BL/6J+water group,the db/db+water group had a significantly increased ILC2s upstream factor IL-33 level and a significantly decreased ILC2s downstream factor IL-13(both P<0.05).Compared with db/db+water group,the db/db+NaB group had a significantly decreased IL-33 level and a significantly increased IL-13(both P<0.05).These results suggested that NaB could up-regulate the IL-13 with a down-regulation of IL-33 in db/db mice.(2)Compared with db/db+PBS group,the db/db+IL-2/Jes6 group had a significantly increased IL-13 level(P<0.05),and there was no significant difference in the IL-33 level between the two groups(P>0.05).Compared with db/db+NaB+PBS group,the db/db+NaB+anti-CD90.2 group had a significantly decreased IL-13 level(P<0.05),and there was no significant difference in the IL-33 level between the two groups(P>0.05).These results suggested that ILC2s could up-regulate the IL-13 level,with modulating ILC2s unaffected by IL-33 in db/db mice.9.Effects of NaB and modulating ILC2s on the ILC2s/IL-13/STAT3 pathway in db/db mice(1)Compared with the C57BL/6J+water group,the db/db+water group had significantly decreased ratio of P-STAT3/STAT3,as well as expression of PGC-1α and GATA-3 in the skeletal muscle by Western blotting(all P<0.05).Compared with the db/db+water group,the db/db+NaB group had significantly increased ratio of PSTAT3/STAT3,as well as expression of PGC-1α and GATA-3 in the skeletal muscle by Western blotting(all P<0.05).These results suggested that NaB could improve the running capacity by up-regulating the expression of slow myofibers via activation of ILC2s/IL-13/STAT3 pathway.(2)Compared with db/db+PBS group,the db/db+IL-2/Jes6 group had significantly increased ratio of P-STAT3/STAT3,as well as expression of PGC-1α and GATA-3 in the skeletal muscle by Western blotting(all P<0.05).Compared with db/db+NaB+PBS group,the db/db+NaB+anti-CD90.2 group had significantly decreased ratio of P-STAT3/STAT3,as well as expression of PGC-1α and GATA-3 in the skeletal muscle by Western blotting(all P<0.05).In combination with above description,i.e.the modulating ILC2s were not influenced by IL-33,these results suggested that IL-33 independent ILC2s/IL-13/STAT3 pathway was responsible for the improvement of running capacity in db/db mice.Conclusions1.Diabetes and sarcopenia coexist in db/db mice aged more than 7 weeks,which can be used as an animal model of T2DM-related sarcopenia.2.Decreased proportion of ILC2s in db/db mice can lead to muscle atrophy,reduced proportion of slow-twitch muscle fibers,and decreased endurance running capacity,ultimately promoting the pathogenesis of T2DM-related sarcopenia.3.IL-33-independent activation of ILC2s/IL-13/STAT3 pathway ameliorates T2DM-related sarcopenia in db/db mice.4.NaB may be an effective intervention for T2DM-related sarcopenia by activating ILC2s.Section II Effects of low skeletal muscle mass on carotid atherosclerosis in the metabolic syndrome population in Shandong province of ChinaObjectives1.To explore the relationship between low skeletal muscle mass and carotid atherosclerosis in MetS population in Shandong province of China.2.To assess the predictive value of low skeletal muscle mass for carotid atherosclerosis in MetS population in Shandong province of China.Methods1.Subjects1.1This was a retrospective study.Gender-and age-matched subjects(n=376)aged 24-85 years were selected from the MetS database established by the Department of Cardiology in Qilu Hospital of Shandong University.The diagnosis of MetS was based on the 2005 International Diabetes Federation(IDF)definition.1.2Groups Subjects were divided into MetS group(n=192)and non-MetS group(n=184)according to whether they met the MetS criteria,and then the non-MetS group was further divided into 2 groups according to waist circumference-to-height ratio(WHTR):(1)Non-MetS obesity group(WHTR>0.5),a total of 118 cases,including 50 males and 68 females;⑵ Healthy control group(WHTR<0.5),a total of 66 cases,including 18 males and 48 females.The non-MetS obesity group and the MetS group were further stratified according to whether the skeletal muscle mass was decreased.The non-MetS obesity group was divided into ?non-MetS obesity without muscle mass loss group,?non-MetS obesity with muscle mass loss group.The MetS group was divided into ?MetS without muscle mass loss group,(2)MetS with muscle mass loss group.2.Data Collection The anthropometric data were collected.Standing height(Ht)and body weight(BW)were measured and BMI was calculated.Waist circumference(WC)and hip circumference(HC)were measured and waist-hip ratio(WHR)and WHTR were calculated.Systolic blood pressure(SBP)and diastolic blood pressure(DBP)were also measured with the difference value being pulse pressure(PP).The hematological data were also collected.Total cholesterol(TC),density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C),and triglyceride(TG),fasting blood glucose(FBG)? insulin concentrations,hemoglobin Ale(HbAlc)and uric acid(UA)were measured.HOMA-IR was calculated as fasting blood glucose multipled by insulin and then divided by 22.5.The cut-off value of HOMA-IR was 2.5 to identify individuals with insulin resistance(IR).3.Appendicular skeletal muscle mass index The appendicular skeletal muscle mass(ASM)was acquired by anthropometric skeletal muscle mass prediction formulas derived from magnetic resonance imaging:ASM(kg)= 0.244><BW + 7.80><Ht-0.098xage+6.6xsex + race-3.3? here sex=l for male and 0 for female,race=-1.2 for Asian.The appendicular skeletal muscle mass index(ASMI)was calculated as follows:ASMI(%)= total ASM(kg)/body weight(kg)x 100%.Muscle mass loss was defined as 1 standard deviations(SD)below the sex-specific average value of skeletal muscle mass of the control group,which were <39.04% for men and <28.43% for women in this study.4.Carotid artery ultrasonography Carotid intima-media thickness(IMT)was measured by ultrasound in all subjects.IMT> 1.0 mm was defined as carotid atherosclerosis.5.Statistical analysis Statistical analyses were performed using SPSS 20.0 software.For comparison between groups,ANOVA was used for normally distributed data,Kruskal-Wallis test was used for non-normally distributed data,and %2 test for trend was used for prevalence comparison.The relationship between MetS,muscle mass loss and IMT was analyzed by binary logistic regression model,and the predictive effect of ASMI on carotid atherosclerosis in MetS patients was evaluated.AUC was calculated using receiver operating characteristic(ROC)curves to assess the diagnostic value of muscle mass loss for carotid atherosclerosis.F<0.05 was considered statistically significant.Results1.Clinical data.In both males and females,there was no statistical difference in age among the three groups: healthy control group,non-MetS obese group and MetS group(all>0,05).For both males and females,anthropometric parameters(BW? BMI,WC,HC and WHR)showed an increasing trend with WHTR and MetS(all P <0.05).The metabolic parameters(SBP,DBP,PP,TC? LDL-C,TG,UA,Glu,insulin,HbAlc,HOMA-IR)and IMT in the MetS group were significantly higher than those in healthy control group and the non-Mets obese group(all P<0.05).On the other hand,SMI and HDL-C showed a decreasing trend both in males and females(all P<0.05).2.Prevalence of carotid atherosclerosis among groups In both males and females,the prevalence of carotid atherosclerosis showed an increasing trend from the healthy control group,the non-MetS obesity group to the MetS group,and the trend difference was statistically significant(P for trend=0.001 for females,P for trend=0.009 for males).3.The effects of muscle mass loss on carotid atherosclerosis In males,there was significant difference in IMT between MetS with muscle mass loss group and non-MetS obesity with muscle mass loss group(P>0.020).There were no significant differences in IMT between MetS with muscle mass loss group and MetS without muscle mass loss group(P>0.05)5 as well as between non-MetS obesity with muscle mass loss group and non-MetS obesity without muscle mass loss group(P>0.05).In females,there was significant difference in IMT between MetS with muscle mass loss group and MetS without muscle mass loss group(P=0.009),as well as between MetS with muscle mass loss group and non-MetS obesity with muscle mass loss group(P=0.009).There were no significant differences in IMT between MetS without muscle mass loss group and non-MetS obesity without muscle mass loss group(P>0.05)? as well as between non-MetS obesity with muscle mass loss group and nonMetS obesity without muscle mass loss group(P>0.05).4.The predictive effect of muscle mass loss on carotid atherosclerosis All parameters that differed significantly among the MetS groups,non-MetS obesity groups and control groups were introduced into the binary logistic regression model for univariate analyses.The SBP,DBP,PP,WC,TC,TG,LDL-C,IR and ASMI were left in the regression model.These parameters were further used for multivariate analyses.SBP and ASMI were left in the regression model.These results suggested that SBP and ASMI had good predictive value for carotid atherosclerosis with their OR values and 95% confidence intervals 1.026(1.010-1.033)and 2.788(1.559-4.985)respectively.5.The diagnostic value of muscle mass loss on carotid atherosclerosis The ROC curves were depicted for SBP and ASMI respectively,and showed an AUC of 0.641 for ASMI,and 0.692 for SBP(both P<0.001).These results suggested that muscle mass loss had certain diagnostic value for carotid atherosclerosis.Conclusions1.The prevalence of carotid atherosclerosis is higher in MetS patients.2.Low skeletal muscle mass is closely associated with carotid atherosclerosis in the MetS population in Shandong province of China,and the association is more obvious in women.3.In the MetS population in Shandong province of China,low skeletal muscle mass is an independent risk factor for carotid atherosclerosis.4.Low skeletal muscle mass(ASMI male <39.04%,female <28.43%)has good predictive effect and certain diagnostic value on carotid atherosclerosis. |
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