| Sarcopenia is the age-associated loss of skeletal muscle mass, strength and function, which increases the costs of the edged health care and results in great economic burdens to the society and families. It has been reported that the skeletal muscle mass control processes involve in complicated pathology of sarcopenia, including protein synthesis pathway, autophagy, proteasome system, and apoptosis pathway, and it is related to muscle mass control disturbance. AMPK and Sirtl act as prime sensors of energy metabolic in muscle, and the AMPK/Sirtl signaling axis (pathway) involves in the regulation of energy metabolic, protein synthesis, protein degradation and apoptosis, affects the progress of sarcopenia. It is studied that physical activity or regular exercise has beneficial effect on the body health and sarcopenia prevention, while it is not clear whether there is an inner relation between AMPK/Sirtl signaling axis and sarcopenia. And it needs to be found out if different exercise scheme affects the skeletal muscle mass reformation. In this paper we focused on the relationship between AMPK/Sirtl signaling axis and its down-signaling pathways involved in protein turnover, apoptosis and the physiological relevance of these signal pathways and exercise control. Hence, understanding the effect of AMPK/Sirtl signaling axis on the quality control of muscle mass induced by exercise could guide us to identify and improve preventive and therapeutic strategies for SarcopeniaOBJECTIVETo explore the effect of different intensity endurance training program on AMPK/Sirtl signaling axis and the related downstream of skeletal muscle quality control mechanism of the senescence accelerated mouse prone 6 (SAMP6) mice. Firstly, comparing with the SAMR1 mice, this paper discussed the changes of the SAMP6 mice AMPK/Sirtl signaling axis and related downstream of skeletal muscle quality control mechanisms (including protein synthesis, autophagy, ubiquitin-proteasome system, apoptosis and so on) during aging process and observed changes containing the gender characteristics of skeletal muscle of SAMP6 mice in order to provide new ideas for reveal the molecular mechanism of sarcopeinia aging that occurs in the process; secondly, designed the treadmill endurance training schemes in different period, different intensity of exercise intervention on the adaptability of SAMP6 mice, observed the changes of skeletal muscle AMPK/Sirtl signaling axis and its related downstream of skeletal muscle quality control mechanism to provide the data support for the rational design of the exercise prescription for prevention of skeletal muscle attenuation; in addition, in view of the important role of autophagy in skeletal muscle in quality control, this study adopted AMPK and Sirtl’s corresponding agonist AICAR and RSV so as to verify the relation between AMPK/Sirtl signaling axis and autophagy by using cell model.METHOD88 SAMP6 (P)and 20 SAMRl(R) male (m)and female (f) mice were randomly assigned to two groups:group land group 2, each group consisted of five subgroup: young control group (R1m, n=5; Rlf, n=5; R2m, n=5; R2f, n=5), old control group (P1m, n=5; P1f, n=5; P2m, n=5; P2f, n=5), high intensity endurance exercise group (H1m, n=6; H1f, n=6; H2m, n=6; H2f, n=6), moderate intensity endurance exercise group (M1m, n=5;M1f, n=5; M2m, n=6; M2f, n=6) and low intensity endurance exercise group (L1m, n=5; L1f, n=5; L2m, n=6; L2f, n=6). Food and water were provided ad libitum. According to the training program, mice of exercise groups undertook different intensity(28m/min,18m/min and 8m/min,5°inclined) treadmill endurance training,6 days/week. The intervention period lasted for 4 weeks in group 1 and 8weeks for group 2. Mices were killed 12h after the end of exercise training protocol. The gastrocnemius muscle was rapidly excised, immediately frozen in liquid nitrogen. Samples were subsequently stored at-80 ℃ until analysis.(1) The effect of aging and endurance training on body weight and skeletal muscle mass of mice:body weight and gastrocnemius muscle wet weight.(2) The effect of aging and endurance training on the genes expression related to AMPK/Sirtl signaling axis in skeletal muscle:AMPK alpha, AMPK alpha 2, NAMPT, SIRT mRNA expression levels were measured by Real-time PCR (RT-PCR); AMPK, Sirtl total protein contents were measured by Western blot(WB) and p-AMPK, ac-p53 protein contents refleced the AMPK/Sirtl pathway activation level; AMPK/Sirtl signaling axis upstream signaling molecules FYN, LKB1 mRNA expression and the ratio of p-LKB1/LKB1 in skeletal muscle were measured by RT-PCR and WB.(3) The effect of aging and endurance training on the protein synthesis system in skeletal muscle:PI3K, PDK1, IGF-1, AKT, mTOR, GSK3,4E-BP1, S6K1, beta MyOD, MSTN, MHC1, MHC2A, MHC2B, MHC2X, mRNA relative contents were measured by RT-PCR; PI3K, AKT, IGF-1, MSTN, MHC1, MHC2a, p-Foxos protein contents were measured by WB; HE staining was used and the muscle cross-sectional area (CSA) measured by IPP software.(4) The effect of aging and endurance training on autophagy in skeletal muscle: BECN1, ATG7, ULK1, LC3, ATG13, BNIP3, SQSMT1/P62, LAMP2 mRNA relative contents were measured by RT-PCR; p62 protein contents and the ratio of LC3II/LC3I were measured by WB; induced C2C12 cell line differentiate into myotubes and treated them with AICAR or RSV for 12h, and the LC3 expression level was measured by immunofluorescence technology.(5) The effect of aging and endurance training on proteasome system in skeletal muscle:FOXO1, FOXO3a, UBC PSMC, ATROGIN-1, MURF1 mRNA relative contents were measured by RT-PCR; Atrogin-1, MuRF1 protein contents and p-Foxos levels were measured by WB.(6) The effect of aging and endurance training on apoptosis in skeletal muscle of mice:P53, KU70, BAX BCL-2, Caspase-3, Caspase-9 mRNA relative contents were measured by RT-PCR; Bax, Bcl-2, Caspase-3, p53 protein contents and the ratio of Bcl-2/Bax were measured by WB.RESULTS(1) Comparing with corresponding group R, the skeletal muscle mass index of mice in group P2m was decreased; the body weight of group P1f and P2f was significantly increased (P<0.01), however, the skeletal muscle mass index of these groups was significantly decreased (P<0.05). Comparing with corresponding group P, the body weight of mice was significantly decreased (P<0.01) and skeletal muscle mass index tended to be increasing in group H2m, M2m and L2m, but there was no change in group H2f, M2f and L2f; the skeletal muscle mass index of group H2f, M2f and L2f was significantly increased (P<0.05)(2) Comparing with corresponding group R, the AMPK and Sirtl protein cotents in skeletal muscle of mice in group P were not changed, but the ratio of p-AMPK/AMPK in skeletal muscle of group P1f and P2m/f was significantly decreased (P<0.05); the ac-p53 protein content in skeletal muscle of group Plm and P2m/f was significantly increased (P< 0.05). Comparing with corresponding group P, the AMPK protein and the ratio of p-AMPK/AMPK in skeletal muscle of group H2m were significantly increased (P<0.01), but the ratio of p-AMPK/AMPK in skeletal muscle of group H2f, M2f and L2f was significantly increased (P<0.05); the Sirtl protein content in skeletal muscle of group H2f was significantly increased(P<0.05); the ac-p53 protein content in skeletal muscle of group H2m/f, M2m/f and L2m/f was significantly decreased (P<0.05)(3) Comparing with corresponding group R, the CSA of group Plf and P2f was significantly decreased (P<0.01); the IGF-1 and AKT protein contents in skeletal muscle of group P2m and P2f were significantly decreased (P<0.05); the PI3K protein content in skeletal muscle of group P2m was significantly decreased (P< 0.05); the MHC1 protein content of group P1f and P2m/f was significantly decreased (P<0.01).Comparing with corresponding group P, the CSA of group H2f and M2f was significantly increased (P<0.05); the IGF-1 protein contents in skeletal muscle of group M2f and AKT protein content of group H2f were significantly increased (P<0.05), the IGF-1 and AKT protein contents in skeletal muscle of group H2m, M2m and L2m were significantly increased (P<0.05); the MSTN protein content in skeletal muscle of group H2f, M2f and L2f was significantly decreased (P <0.05); the MHC1 protein content of group H2m/f, M2m/f and L2m/f was significantly increased (P<0.05)(4) Comparing with corresponding group R, the ratio of LC3Ⅱ/LC3Ⅰ in skeletal muscle of group Plm and P2m was significantly decreased(P<0.05); the p62 protein content in skeletal muscle of group P2m was significantly increased (P<0.01) Comparing with corresponding group P, the ratio of LC3Ⅱ/LC3 Ⅰ in skeletal muscle of group H2m/f, M2m/f and L2m was significantly increased (P<0.05); the p62 protein content in skeletal muscle of group H2m/f, M2m/f and L2f was significantly decreased (P<0.05)(5) Comparing with corresponding group R, the p-Foxol, p-Foxo3a and Atrogin-1 protein levels in skeletal muscle of group P2m and P2f were significantly increased (P <0.05); the MuRF protein content in skeletal muscle of group P2f was significantly increased (P<0.05).Comparing with corresponding group P, the p-Foxoland p-Foxo3a protein levels in skeletal muscle of group H1f, M1f, L1f, H2m/f, M2m/f and L2m/f were significantly decreased (P<0.05); the Atrogin-1 protein content in skeletal muscle of group H2f, M2f, and L2f was significantly decreased (P<0.01)(6) Comparing with corresponding group R, the p53 protein level in skeletal muscle of group P1 m/f and P2m/f was significantly increased (P< 0.05); the Bax and Caspase-3 protein levels in skeletal muscle of group P2m and P2f were significantly increased (P<0.05), however, their Bcl-2 protein content and the ratio of Bcl-2/Bax was significantly decreased (P<0.05).Comparing with corresponding group P, the p53 protein level in skeletal muscle of group H2m was significantly increased (P< 0.05); the Bax protein content in skeletal muscle of group H2m, M2m and L2m was significantly decreased (P<0.01); the Bcl-2 protein content in skeletal muscle of group H2m/f, M2m and L2m was significantly increased (P<0.05); the ratio of Bcl-2/Bax in skeletal muscle of group H2m/f and M2m was significantly increased (P <0.05); the Caspase-3 protein content in skeletal muscle of H2m/f, M2m/f and L2m was significantly decreased (P<0.05)CONCLUSION(1) The skeletal muscle mass index of SAMP6 female mice was significantly decreased and below the index of 2SD of SAMR1 mice, which suggested that SAMP6 female mice could be used as animal model for sarcopenia study; the skeletal muscle mass index of 5 months old male SAMP6 mice was below 1SD of SAMR1, which suggested that the SAMP6 male mice would be with risk of sarcopenia. Endurance exercise especially high intensity endurance exercise training for two months could improve skeletal muscle mass to prevent the occurrence of sarcopenia induced by aging.(2) The ratio of p-AMPK/AMPK in skeletal muscle of SAMP6 mice was significantly decreased, however ac-p53 protein content was increased during aging, which suggested that activity of AMPK/Sirtl signaling axis in skeletal muscle was inhibited by aging through inhibited AMPK and Sirtl activities. Endurance training especially high intensity endurance training 2 months could reverse these conditions to improve the activity of AMPK/Sirtl signaling axis.(3) The function of IGF-1/PI3K/AKT pathway was inhibited during aging, and it involved in the decreased of MHC protein depression which caused occurrence of sarcopenia. The endurance exercise training promoted IGF-1, PI3K and AKT protein content and inhibited MSTN protein depression in skeletal muscle of SAMP6mice, consequently, it improved MHC1 and MHC2A protein synthesis to prevent the sarcopenia.(4) The level of autophagy was inhibited by decreased LC3Ⅱ/LC3 I ratio and increased p62 protein content in skeletal muscle of 5 month old SAMP6 mice during aging, which suggested that autophagy decline may be involved in the sarcopenia in the aging process. Endurance training of 2 months of high intensity and moderate intensity promoted autophagy level and maybe involved in the prevention of sarcopenia through protected skeletal muscle against harmful stimulation.(5) The protein levels of Atrogin-1 and MuRF1 in skeletal muscle of 5 month old SAMP6 mice were increased during aging which may promote activity of ubiquitin proteasome system involving in the occurrence of sarcopenia. This deleterious effect was eliminated by 2 months of endurance training to prevent the occurrence of sarcopenia(6) The increase protein level of p53 in skeletal muscle of SAMP6 mice was to promote apoptosis through improve Bax expression and inhibit Bcl-2 expression during aging.2 months of endurance training activated the AMPK/Sirtl axis to inhibit the transcription activity of the p53 which induced apoptosis and had an important significance for the prevention and treatment of sarcopenia. |
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