Role And Mechanism Of FGF19 In Improving Muscle Damages Caused By High Fat Induced Obesity

Objectives:1.To investigate the effect of FGF19 on improving muscle atrophy caused by HFD-induced obesity.2.To clarify the effect of FGF19 on alleviating the lipid and glucose metabolic derangement caused by HFD-induced obesity.3.To investigate the effect of FGF19 on attenuating the decreased irisin levels caused by HFD-induced obesity.4.To investigate the protective role of FGF19 against skeletal muscle damages caused by HFD-induced obesity through AMPK/SIRT-1/PGC-1?.Methods:1.In vivo:(1)5 weeks and 15 months C57 BL / 6J male mice were randomly divided into 4 groups: control group,FGF19 group,high-fat-diet(HFD)group,and FGF19+HFD group.The HFD group and FGF19+HFD group were given a 5-month HFD to establish a mouse model of obesity and sarcopenic obesity.The control group and FGF19 group were fed with ordinary diet.After successful construction,mice in the FGF19+HFD group and FGF19 group were given intraperitoneal injection of FGF19(0.1mg / kg)once a day for 3 weeks.Meanwhile,the control group and HFD group were treated with the same amount of normal saline.(2)The body weight and gastrocnemius muscle weight of the mice in each group were measured by an electronic balance,the maximum grip of the mouse was analyzed by the electronic grip strength meter,and the changes of body composition was detected by the dual-energy X-ray bone densitometer(DXA).(3)The blood samples were collected from mice to measure glucose tolerance test and blood glucose,blood lipid,FGF19,irisin,and insulin expression in each group of mice.(4)H&E staining,transmission electron microscope(TEM),Oil red O staining,and immunohistochemistry were used to observe muscle atrophy,gastrocnemius lipid accumulation,and muscle FNDC-5 protein expression in mice of each group.(5)Western blot were used to detect the expression of muscle atrophy markers(FOXO-3,Atrogin-1,Mu RF-1),myogenic differentiation markers(MHC,Myo D,Myo G),glucose uptake related markers(IRS-1,GLUT-4),signaling pathway molecules(AMPK,SIRT-1,PGC-1?)as well as FNDC-5/ irisin.2.In vitro:(1)Cell group: control group,FGF19 group(100ng / m L),PA group(0.5m M),FGF19 + PA group(100ng / m L + 0.5m M).(2)Western blot were used to detect the expression of muscle atrophy markers,myogenic differentiation markers,glucose metabolism markers,signaling pathway molecules,and FNDC-5.(3)The irisin levels in the culture supernatant was detected by ELISA.(4)Immunofluorescence,2-NBDG,and Oil red O staining were used to detect myotube atrophy,glucose uptake,and lipid droplet accumulation.(5)The AMPK and SIRT-1 inhibitors as well as the PGC-1?-si RNA were used to investigate the effect of FGF19 on improving PA-induced muscle atrophy,lipid glucose metabolic derangement and decreased irisin levels.Results:1.In vivo:(1)The mouse models of obesity and sarcopenic obesity were constructed by HFD in young and old mice.The results showed that the body weight and fat weight of the mice in the HFD group were increased compared with the control group,while the muscle mass and grip strength were decreased significantly.In the HFD group,the blood glucose and blood lipids,and insulin were increased;the glucose tolerance was abnormal;the plasma FGF19 and irisin expression levels were decreased.FGF19 treatment could reduce the weight of mice,improve muscle mass and grip strength,restore blood glucose and lipid levels,and alleviate blood FGF19,irisin and insulin levels,suggesting that obesity and sarcopenic obesity caused by HFD could lead to inhibit muscle mass and function,lipid and glucose metabolic derangement,and decrease of irisin levels.However,FGF19 may attenuate the occurrence and development of obesity and sarcopenic obesity.(2)The analysis of H&E staining,TEM,Oil red O staining,and immunohistochemistry showed that the diameter of muscle fibers in the HFD group was smaller than that in the normal control group,and the structure of the muscle fibers was damaged.A large amount of lipid droplets accumulated in the gastrocnemius muscle,and the expression of FNDC-5 in the gastrocnemius muscle was reduced.FGF19 administration could alleviate muscle fiber atrophy,lipid droplet accumulation,and increase the expression of FNDC-5,suggesting that FGF19 could improve HFD-induced skeletal muscle atrophy,lipid deposition,and decreased irisin levels.(3)The results of western blot showed that the expression of muscle atrophy-related protein(FOXO-3,Atrogin-1,Mu RF-1)in the gastrocnemius muscle of the HFD group was higher than that of the normal control group,while the expression of myogenic differentiation markers(MHC,Myo D,Myo G),glucose uptake-related protein(IRS-1,GLUT-4)and FNDC-5/irisin were lower than that of the control group.FGF19 treatment could inhibit the increase of muscle atrophy markers,promote the expression of myogenic differentiation markers,glucose uptake-related proteins and FNDC-5/irisin,and enhance the expression of energy metabolism-related molecules in adipose tissue,suggesting that FGF19 has ability to alleviate HFD-induced obesity caused muscle atrophy,metabolic impairment,and decline in FNDC-5/irisin expression.2.In vitro:(1)The results of western blot found that the protein expression of muscle atrophy-related factors were up-regulated in the PA group when compared with the control group,while myogenic differentiation markers,glucose uptake-related factors and FNDC-5 were decreased.FGF19 treatment could significantly alleviate PA-induced abnormal expressions of these markers,suggesting that FGF19 has an ability to improve PA-induced muscle atrophy,glucose metabolic disorders and decreased irisin levels.(2)ELISA results showed that the expression of irisin increased in the cell culture supernatant of the FGF19 group,while the expression of irisin in the PA group was decreased compared with the control group.FGF19 could alleviate the decrease in irisin expression caused by PA.Analysis of the correlation between irisin expression and muscle atrophy markers and glucose metabolism factors,we found that irisin was positively correlated with the expression of IRS-1 and GLUT-4,suggesting that FGF19 may promote glucose metabolism in skeletal muscle.(3)The results of immunofluorescence,2-NBDG,and Oil red O staining showed that the diameter of myotubes and the fusion index of myotubes in PA group was decreased compared with the control group.Moreover,PA could inhibit glucose uptake of myoblasts and aggravate lipid droplets accumulation in myotubes.However,FGF19 could alleviate PA-induced myotube atrophy,abnormal glucose uptake and lipid droplets accumulation.(4)According to using AMPK/SIRT-1 inhibitors and PGC-1?-si RNA to inhibit the expression of AMPK,SIRT-1,and PGC-1?,FGF19 was unable to alleviate PA-induced the up-regulation of muscle atrophy markers expression,and the down-regulation of glucose uptake factors and FNDC-5 expression.This result suggests that the AMPK/SIRT-1/PGC-1? signaling pathway play a key role in the protective effect of FGF19.Conclusion:High fat-induced obesity could lead to muscle atrophy,inhibit muscle mass and function,metabolic derangement,and decline in FNDC-5/irisin expression.FGF19 has an ability to improve high fat-induced muscle damages through AMPK/SIRT-1/PGC-1? signaling pathway.Therefore,FGF19 may become a therapeutic target for the obesity and sarcopenic obesity in the future.