The Effect Of Cyclic Mechanical Stretch On Leptin Resistance Of C2C12 Myoblasts And Its Mechanisms

ObjectMost of obese subjects show leptin resistance,characterized by abnormal increase of serum leptin but diminished effects of leptin on inhibiting appetite,enhancing energy expenditure and decreasing blood glucose.Leptin resistance that develops in brain and also directly in peripheral tissues such as skeletal muscle,adipose tissue and liver is considered as central and peripheral leptin resistance,respectively.Skeletal muscle is one of the most important metabolic organs of body and the reduction of leptin resistance in skeletal muscle is of great significance for the treatment of obesity and obesity-related diseases.However,there are few studies on the effects of exercise on skeletal muscle leptin resistance,and its mechanism is still unclear.Signal transducer and activator of transcription 3(STAT3),an important indicator of leptin sensitivity,mediates most of the effects of leptin,and the increases in phosphorylation and expression of STAT3 enhance leptin sensitivity.While suppressor of cytokine signaling 3(SOCS3)and protein tyrosine phosphatase 1B(PTP1B)negatively regulate leptin signaling,and reducing both enhances leptin sensitivity.Moreover,the onset of leptin resistance is often accompanied by disrupted glucose metabolism,and decreased leptin resistance can be evidenced by improved glucose metabolism.The insulin-like growth factor 1(IGF-1)-insulin receptor substrate(IRS-1)-serine/threonine kinase(Akt)-glucose transport protein 4(GLUT4)signaling pathway is involved in the regulation of glucose metabolism,and the enhancement of its activity increases glucose uptake.Importantly,the action of leptin down-regulating blood glucose and up-regulating glucose uptake is related to this signaling pathway.Here,we established a leptin resistance model of C2C12 myoblasts and used mechanical stretch to simulate exercise to explore the effects and mechanism of mechanical stretch on leptin resistance in C2C12 myoblasts(regulate STAT3,PTP1 B and SOCS3 expressions),and further explore the mechanism of mechanical stretch on increasing glucose uptake after reducing leptin resistance of C2C12 myoblasts(stimulate IGF-1/IGF-1R/IRS-1/Akt/GLUT4),thus providing a new insight into the mechanisms of exercise-induced decrease in peripheral leptin resistance and the relationship between leptin signal and insulin signal.Methods1.C2C12 myoblasts were cultured in medium containing different glucose concentrations at 25 mmol/L(control group,C),55,65,75 mmol/L for 24 h,which is used to induce leptin resistance.The concentration of glucose in medium supernatant was determined by Glucose detection kit;the mRNA levels of leptin,Lep R and GLUT4 determined by RT-PCR.The criteria for successful leptin resistance model of C2C12 myoblasts: increased leptin mRNA expression,decreased leptin receptor(Lep R)mRNA expression,decreased GLUT4 mRNA expression,and decreased glucose uptake.2.The C2C12 myoblasts were divided into control group,leptin resistance group and leptin resistance + mechanical stretch group that was subjected for 15% cyclic mechanical stretch at 0.5 Hz for 3 h or 6 h by Flexcell-5000 tension system,which could help to investigate the effect of mechanical stretch on leptin resistance and its mechanisms.The concentration of glucose in medium supernatant was determined by Glucose detection kit;the mRNA levels of leptin,Lep R,STAT3,SOCS3,PTP1 B of leptin signaling determined by RT-PCR;the mRNA levels of IGF-1R,IRS-1,Akt,GLUT4 of insulin signaling determined by RT-PCR;the protein expressions of SOCS3,IGF-1 and GLUT4 determined by Western blot.Results1.Establish a leptin resistance model of C2C12 myoblasts Both high glucose(65 mmol/L)and 75 mmol/L could induce leptin resistance of C2C12 myoblasts.However,compared with 75 mmol/L glucose,65 mmol/L glucose triggered leptin resistance as better as expected.Therefore,the concentration at 65 mmol/L was selected for subsequent experiments.2.Cyclic mechanical stretch improves leptin resistance in C2C12 myoblasts Compared with the control group,leptin resistance group displayed a significant increase in leptin,a decrease in Lep R mRNA expressions,and reduces in glucose uptake and GLUT4 mRNA and its protein expressions,while 3 h and 6 h stretch decreased leptin mRNA,increased Lep R mRNA in the stretch group;increased glucose uptake and elevated GLUT4 mRNA level,and 6 h stretch also increased the protein expression of GLUT4 where the stretch for 6 h increased more STAT3 mRNA expressions than that of the stretch for 3 h.3.The mechanism of improved leptin resistance in C2C12 myoblasts by cyclic mechanical stretch-increase STAT3,reduce PTPT1 B and SOCS3 Compared with the control group,leptin resistance group displayed a decrease in STAT3 mRNA level and increases in PTP1 B and SOCS3 mRNA levels,while 3 h and6 h stretch increased STAT3 mRNA level in the stretch group,where the stretch for 6h increased more STAT3 mRNA expressions than that of the stretch for 3 h,and reduced PTP1 B and SOCS3 mRNA levels where the stretch for 6 h decreased more SOCS3 mRNA expressions than that of the stretch for 3 h;3 h and 6 h stretch also reduced SOCS3 protein level of the stretch group.These finding have shown that improved leptin resistance in C2C12 myoblasts by stretch is possibly due to increased STAT3 and reduced PTP1 B and SOCS3.4.The mechanism of improved glucose uptake after reducing leptin resistance of C2C12 myoblasts by cyclic mechanical stretch-activate IGF-1/IGF-1R/IRS-1/Akt signal pathway Compared with the control group,leptin resistance group displayed decreases in IGF-1 protein expressions(without statistical significance),while 6 h stretch increased its IGF-1 protein expressions.Compared with the control group,leptin resistance group displayed decreases in IGF-1R and Akt mRNA expressions,while 3h and 6 h stretch increased IGF-1R and Akt mRNA expressions in the stretch group,where the stretch for 6 h increased more IGF-1R mRNA expressions than that of the stretch for 3 h.Besides,15% stretch for 6 h also increased IRS-1 mRNA expression while stretch for 3 h showed no effects.These finding have shown that increased glucose uptake in C2C12 myoblasts by stretch is possibly due to the stimulation of IGF-1/IGF-1R/IRS-1/Akt signal pathway.Conclusion1.High glucose at 65 mmol/L for 24 h resulted in leptin resistance of C2C12 myoblasts,associated with increased leptin mRNA,decreased Lep R mRNA level,and decreased glucose uptake.2.15% mechanical stretch for 3 h or 6 h significantly improved leptin resistance(most significant at 6 h stretch),which was related to increased STAT3 and decreased PTP1 B and SOCS3 expressions.3.15% mechanical stretch significantly increased glucose uptake after improving leptin resistance of C2C12 myoblasts,which was related to the activation of IGF-1/IGF-1R/IRS-1/Akt/GLUT4 signaling pathway.