The Effects Of AMPK On The Functional Activity Of Myoblasts And Its Metabolic Regulation

Background and Objective:Muscle satellite cells are the most important stem cells in skeletal muscle tissue.It is responsible for the postnatal growth and regeneration of skeletal muscle,constituting the major cellular basis for regeneration and plasticity of skeletal muscle.In recent years,studies have shown that cell energy metabolism plays an important role in the self-renewal,proliferation and differentiation of stem cells.In the process of proliferation and differentiation,muscle satellite cells also showed significant changes in energy metabolism.AMP activated protein kinase(AMPK)is a key regulator of cellular energy metabolism,activated AMPK can inhibit the metabolic pathway of ATP production on the one hand,and simultaneously start-up the intracellular catabolism of ATP,so as to restore or maintain the balance of cell energy metabolism.In this study,focusing on AMPK,we firstly observed the effects of activation of AMPK on the proliferation and differentiation of C2C12 myoblast,as well as the functional properties of cellular mitochondria.Then we observed the effects of caloric restriction on the microenvironment of skeletal muscle and the regulation of AMPK signaling pathway in muscle satellite cells based on the previous reports.Our aim is to elucidate the possible mechanism of metabolic factors involved in the regulation of stem cell function,and to explore the role of AMPK signaling pathway in regulation of the proliferation and differentiation potential of skeletal muscle satellite stem cells,and to explore the potential molecular targets for promoting the regeneration of skeletal muscle injury in aged animals.Methods:1.Cell cultureC2C12 cell growth medium is a high glucose DMEM medium containing 10% fetal bovine serum.The differentiation medium was low glucose DMEM medium containing 2% horse serum.The incubator temperature was 37℃ and the CO2 concentration was 5%.2.Detection of AMPK signaling pathway proteins expression in C2C12 cells after metformin treatmentThe expression of p-AMPKα,AMPKα and SIRT1 protein were examined by WB at 12 h,24h,36 h and 48 h after culture of C2C12 cells in growth medium containing metformin(final concentration of 1 mmol/L).3.Measuring the proliferation of C2C12 cells after induction of AMPK activationC2C12 cells were cultured in growth medium containing metformin(final concentration 1 mmol/L).CCK-8 was used to detect the cell proliferation status at 0h,24 h and 48 h after culture.Flow cytometry was used to detect the changes of cell cycle at 24 h and 48 h after culture.DNA synthesis assay was analysed by using ethynyl-deoxyuridine(Edu)incorporation method.4.Detection of myogenic transcription factors expressionin and myotube formation after induction of AMPK activation in C2C12 cells(1)C2C12 cells were cultured in growth medium containing metformin(final concentration of 1 mmol/L).The expression of Pax7 protein was examined by WB for 12 h,24h,36 h and 48 h respectively.Real-time PCR was used to detect the expression of Myo D mRNA at 24 h and 48 h after culture.(2)C2C12 cells were pretreated in growth medium containing metformin(final concentration of 1 mmol/L)for 48 h,then induced differentiation with differentiation medium.The expression of Myogenin m RNA was detected by Real-Time PCR after induction of 24 h,48h and 72 h respectively.The myotube formation was checked by Giemsa staining after 72 h,and the fusion index was calculated(the proportion of myotube containing more than two nuclei).5.Detection of mitochondrial membrane potential in C2C12 cells after induction of AMPK activationC2C12 cells were cultured in growth medium containing metformin(final concentration of 1 mmol/L).The mitochondrial membrane potential of the cells was measured by flow cytometry with JC-1 mitochondrial membrane potential probe at 24 h and 48 h,respectively.At the same time,the mitochondrial membrane potential of the cells was detected by laser confocal microscopy with TMRE mitochondrial membrane potential probe at 24 h.6.Detection of mitochondria biogenesis,distribution and PGC1α expression in C2C12 cells after induction of AMPK activationC2C12 cells were cultured in growth medium containing metformin(final concentration of 1 mmol/L).Mitochondria were labeled by Mito-Tracker Green probe to detect the mitochondria biogenesis and distribution.At the same time,the expression of PGC1α protein was examined by WB for 12 h,24h,36 h and 48 h respectively.The expression of PGC1α m RNA was detected by Real-Time PCR at 24 h and 48 h.7.Establishment of calorie restriction animal modelThirteen month male C57BL/6 mice(n = 30)were divided into two groups according to simple random sampling: free eating group(AL)and 40% calorie restriction group(CR).All single cage fed,free drinking water.The calorie restriction mice were fed daily for 60% of the free-eating group for 12 weeks.8.Detection of AMPK signaling pathway proteins expression in mouse skeletal muscle after calorie restrictionChanges of p-AMPKα,AMPKα and SIRT1 protein expression in the tibialis anterior muscle of mice after calorie restriction were examined by WB.9.Observation of mitochondrial morphology and detection of PGC1α protein expression in skeletal muscle of mice after calorie restrictionThe mitochondrial morphological changes of tibialis anterior muscle were observed by transmission electron microscopy.The density,length and width of mitochondria were calculated respectively.The expression of PGC1α,the key mitochondria biogenesis-related protein in the tibialis anterior muscle was detected by WB.10.Detection of AMPK signaling pathway proteins expression in mouse muscle satellite cells after caloric restrictionIsolation of muscle satellite cells is accomplished by collagenase and neutral protease digestion.And then subjected to flow cytometry by multiple fluorescent labeling,wherein CD45-/ CD11b-/ Sca-1-/ CXCR4 + / CD29 + cells were defined as muscle satellite cells.The total protein was extracted from the cells and then the expression of p-AMPKα,SIRT1 and PGC1α were detected by ELISA.Results:1.The phosphorylation level of AMPKα was significantly increased after metformin treatment(P<0.05).The expression of SIRT1 protein was also up-regulated,and the difference was significant(p<0.05)between the treatment and control group.2.CCK-8 experiment showed that C2C12 cell proliferation rate became slow after metformin treatment.OD450 nm decreased significantly at 48h(p<0.01)compared with the control group.The proportion of S phase cells increased in the treatment group,and the difference was significant(p<0.05)after 48 h,while the proportion of G2/M phase cells decreased.DNA synthesis test showed that the proportion of Edu positive cells increased significantly(p<0.01)comparing with the control group after 48 h of metformin treatment.3.After 24 hours of metformin treatment,the expression of Pax7 protein in C2C12 cells was significantly higher than that in control group(p<0.01),while Myo D mRNA expression decreased significantly(p<0.01)compared with the control group after 48 h treatment.After differentiation induction,the expression of Myogenin mRNA in the metformin pretreatment group was lower than that in control group at thedetection time points.The myofibers fusion index were significantly decreased after 72 hours of induction in the pretreatment group(both p<0.01).4.The mitochondrial membrane potential of C2C12 cells was significantly lower than that of the control group(both p<0.01)after 24 hours of metformin treatment by two different mitochondrial membrane potential probes.5.After 48 h of metformin treatment,the mitochondria of C2C12 cells were closely distributed around the nucleus through detected by the Mito-Tracker Green probe.The average fluorescence intensity of mitochondria was significantly higher than that of the control group(p<0.01).Comparing with the control group,the expression of PGC1α protein and m RNA expression in C2C12 cells treated with metformin were significantly higher(p<0.01 and p<0.05,respectively).6.Compared with the AL group,the phosphorylation level of skeletal muscle AMPKα was significantly up-regulated(p<0.01),and the expression of SIRT1 protein was also increased(p<0.01)in the CR group.7.Compared with the AL group,the mitochondrial density of skeletal muscle was significantly increased(p<0.01),and the volume of mitochondria was significantly larger(length: p<0.05,width: p<0.01)in the CR group.The expression of PGC1α protein was also up-regulated(p<0.05).8.The expression of p-AMPKα,SIRT1 and PGC1α in muscle satellite cells of CR group were significantly higher than those in AL group(p<0.01;p<0.05;p<0.05).Conclusion:1.Metformin can activate the AMPK pathway of C2C12 cells and promote the expression of related proteins.2.Metformin can inhibit the proliferation of C2C12 cells and block the cell cycle in S phase.At the same time,it can up-regulate the expression of Pax7 protein and down-regulate the transcription level of Myo D gene,thereby preserving the low differentiation state of C2C12 cells,even under differentiated medium induction conditions.These effects may be dependent on the activation of AMPK pathway and the up-regulation of its associated protein expression in C2C12 cells.3.Activation of AMPK by metformin can induce the decrease of mitochondrial membrane potential in C2C12 cells and down-regulate the level of energy metabolism.At the same time,it can up-regulate the transcription and protein expression of PGC1αand promote mitochondrialbiogenesis and re-distribution around the nucleus.It suggested that metformin might maintain the quiscent state of C2C12 cells through AMPK pathway activation.4.Calorie restriction can activate AMPK pathway in skeletal muscle,increase AMPK-related protein expression,promote skeletal muscle mitochondrial production,it also activate the AMPK signal in muscle satellite cells.Combined with previous studies,it suggest that calorie restriction may help preserve the stemness of aged muscle satellite cells by activation of the AMPK signaling pathway.