Mechanisms Of The Effects Of Lcariin On Irisin/FNDC5 Expression In Skeletal Muscle

ObjectiveAs a cause of many chronic diseases,obesity has been listed as one of the six risk factors by the world health organization.Each year at least 3.4 million people worldwide died from obesity-related diseases.It is urgent to control such situation.Obesity is closely related to the function of skeletal muscle.Skeletal muscle cells can oxidize free fatty acids to reduce the size of adipose tissue so as to protect against obesity.This effect is associated with regulating the function of mitochondria in skeletal muscle cells and the endocrine functions of skeletsl muscle cells.Irisin is a cytokine secreted by skeletal muscle cells.After sheared by Type Ⅲ domain-containing protein5(FNDC5)in skeletal muscle,irisin was released into circulatory system.Irisin has anti-obesity effects through increasing the expression of adipose tissue related thermogenic protein Uncoupling protein 1(UCP-1)in white adipose tissue(WAT).As one the upstream regulatory proteins of FNDC5,Peroxisome proliferator-activated receptor gamma coactivator 1-alpha(PGC-1α)promotes the expression of Irisin/FNDC5 in skeletal muscle.Our previous study has found that Erxian Decoction(a prescription for tonifying kidney)could improve the endocrine function of WAT in menopausal rats,as well as increase the heat-production related proteins PGC-1α and UCP-1 expression,which ultimately reduced the ovariectomized rat weight gain.We also found that icariin,the main component of herba epimedii(Chinese herb),could increase the expression of heat-producing protein in Brown adipose tissue(BAT).Adenosine 5’-monophosphate(AMP)-activated protein kinase(AMPK)is an AMP-dependent protein kinase and a key factor in energy metabolism regulation.AMPK is closely related to the body’ s glucose and lipid metabolism.Previous study has found that Erxian Decoction could promote the expression of BAT related thermogenic proteins and activate the AMPK pathway.Based on the previous studies,this study investigates the effect and mechanism of icariin on the expression of Irisin/FNDC5 in skeletal muscle and the possible role of AMPK pathway.Promoting the secretion of irisin is conducive to providing new ideas for the treatment of obesity-related diseases in clinical practice.Methods1.To investigate the effect of icariin on irisin and the phosphorylation of AMPK in C2C12 myotubes at the cellular levelC2C12 myoblasts were differentiated into myotubes by horse serum,and divided into different groups according to concentration gradient:the blank control group(CON,0 μg/mL),the low-dose icariin group(ICAL,20 μg/mL),the medium-dose icariin group(ICAM,40 μg/mL),and the high-dose icariin group(ICAH,80 μg/mL).Time gradient grouping:icariin at a concentration of 40 μg/mL acting on myotubes for 0h,12h,24h and 48h respectively.CCK-8 assay was used to detect the viability rate of cells in different concentration and time gradient groups.The contents of irisin in the supernatant of myotubes treated with icariin at different concentration gradients were determined by ELISA.Protein and mRNA levels of FNDC5 and PGC-1α in each group with different concentration gradients and time gradients were detected by Western-blot and fluorescence quantitative PCR.The protein expression of FNDC5 in myotubes was detected by immunofluorescence.The protein expression of AMPK and pAMPK was detected by Western-blot after 15min of treatment.The cells were pretreated with AMPK Inhibitors Compound C(50 μM)for lh and culture media was replaced with 40 μg/mL icariin for 24h.At the same time,AMPK agonist AICAR(0.1mM)were used for myotubes for 24h.Protein and mRNA levels of FNDC5 were detected.siRNA transfection was conducted in the cells to interfere AMPK,and the protein level of FNDC5 was detected by Western-blot.2.To investigate the effect of icariin on irisin and the phosphorylation of AMPK in skeletal muscle of C57BL/6 mice at the integral and organ levelC57BL/6 mice were randomly divided into the 3 groups:normal control group(CON,gavage with equivalent normal saline),the low-dose icariin group(ICAL,gavage with icariin at a concentration of 10mg/kg),and the high-dose icariin group(ICAH,gavage with icariin at a concentration of 40mg/kg).Corresponding solution was given to each group for 14 consecutive days.The changes of body mass of mice in each group were detected.At the organ level,the protein and mRNA levels of FNDC5 and PGC-1α were detected by Western-blot and fluorescence quantitative PCR.Expression levels of AMPK and pAMPK were detected by Western-blot.Immunohistochemical assay was used to detect the expression of thermogenic protein UCP-1 in subcutaneous adipose tissue in the groin of mice in each group.Results1.After icariin treatment on differentiated myotubes,the concentration of irisin in the cell supernatant was higher than that of CON group(P<0.05).Western-blot and fluorescence quantitative PCR results indicated that contrasting with the CON group,the protein and mRNA levels of FNDC5 and PGC-1αin myotubes were increased in icariin intervention group in a time-and concentration-dependent manner(P<0.05).Western-blot results showed that compared with CON group,the phosphorylation level of AMPK in myotubes was obviously increased in medium-dose icariin group and high-dose icariin group(P<0.05).The expression level of FNDC5 was determined by using AMPK agonist AICAR,AMPK inhibitor Compound C,as well as siRNA interference of AMPK in myotubes.Western-blot results showed that the protein expression level of FNDC5 was up-and down-regulated after the phosphorylation level of AMPK pathway was increased and decreased respectively(P<0.05).The differences were statistically significant.2.After 14 days of intragastric administration of icariin in C57BL/6 mice,the body weight gain of the high-dose group was significantly lower than that of the CON group(P<0.05).Western-blot and fluorescence quantitative PCR results showed that compared with CON group,the protein and mRNA levels of FNDC5 and PGC-1α in gastrocnemius muscle in low-dose group and high-dose group were significantly increased(P<0.05).Western-blot analysis indicated that the phosphorylation level of AMPK in the gastrocnemius muscle was higher in the icariin intervention group compared with the CON group(P<0.05).Immunohistochemical results showed that the expression of UCP-1 in subcutaneous adipose tissue in groin was significantly increased(P<0.05)in the medicine intervention group.The differences were statistically significant.Conclusion1.Icariin could increase irisin level and the protein and mRNA expression levels of FNDC5 and PGC-1α in C2C12 myotubes and this effect is associated with activation of AMPK pathway.2.Icariin could increase the secretion of irisin and phosphorylation level of AMPK pathway in the gastrocnemius muscle in C57 mice.It could promote the browning of WAT thereby reducing the body mass gain of mice and playing the role of anti-obesity.