Role Of Chemerin In Lipid Deposition In Skeletal Muscle Of Mice Regulated By Diet And Exercise

PurposeObesity leads to excessive deposition of lipids in organs other than adipose tissue(skeletal muscle,liver),which is called ectopic lipid deposition.The skeletal muscle is the main organ that regulates glucose tolerance in response to insulin stimulation.The development of skeletal muscle lipid deposition interferes with insulin signaling and leads to skeletal muscle insulin resistance(IR).While skeletal muscle IR is a precursor to the development of type 2 diabetes,reducing skeletal muscle ectopic lipid deposition is an important way to improve insulin sensitivity in the body.Therefore,reducing skeletal muscle lipid deposition is helpful in preventing the progression of early IR to type 2 diabetes and has become a new target for the treatment of diabetes.Chemerin,as a novel adipokine,plays an important role in the development of obesity and obesity-related diseases(such as type 2 diabetes,atherosclerosis,and metabolic syndrome)through its receptor CMKLR1.Many studies,including our group,have shown that chemerin is closely related to disorders of glucolipid metabolism and that external chemerin supplementation exacerbates glucose intolerance and reduces glucose uptake and utilization in peripheral tissues of obese mice,while adipose chemerin knockout modifies body weight,body fat percentage,and glucose tolerance in mice fed a high-fat diet.Exercise improved glycolipid metabolism in diabetic mice while also reducing their serum chemerin levels.High expression of the chemerin receptor CMKLR1 was observed in skeletal muscle,and chemerin inhibited glucose uptake by skeletal muscle cells,leading to the development of skeletal muscle IR.Few studies have been reported on whether chemerin regulates skeletal muscle lipid deposition in mice on a high-fat diet and effects the ameliorative effect of exercise on skeletal muscle lipid deposition.In this study,two different knockout mice,adipose-specific and global chemerin knockout,were used to investigate the effects of adipose-derived chemerin(adipose tissue and liver are the most important sources of chemerin)and different knockout levels of chemerin(global chemerin knockout resulting in chemerin deficiency and adipose-specific chemerin knockout resulting in reduced chemerin levels)on the regulation of skeletal muscle lipid deposition by different dietary conditions(normal and high-fat diets)and exercise.The role of key enzymes and proteins in glycolipid metabolism in skeletal muscle was then further explored,including fatty acid translocase(cluster of differentiation 36,CD36),carnitine palmitoyltransferase 1(CPT1),stearoyl-Co A desaturase 1(SCD1)1,peroxisome proliferator-activated receptor α(PPARα)and glucose transporter 4(GLUT4).This will provide insight into the mechanisms by which adipokines such as chemerin regulate glucolipid metabolism and skeletal muscle lipid deposition,and provide new perspectives on the prevention of diabetes.Method 1.Construction of chemerin knockout miceThe Cre-lox P system was used to construct global chemerin knockout mice and adipose-specific chemerin knockout mice(commissioned from Shanghai Southern Model Biotechnology).At 2 weeks of age,0.5 cm of the tails of newborn mice were taken for genetic identification to screen for the two chemerin knockouts mentioned above in pure-genic mice2.Grouping of animals and aerobic exercise programTo investigate the effect of chemerin knockout on skeletal muscle lipid deposition in mice on different diets(normal and high fat diet),8-week-old wild-type mice(WT),adipose-specific chemerin knockout mice [adipo-chemerin(-/-)] and chemerin global knockout mice [chemerin(-/-)],were divided into normal diet group(normal diet(ND)and high-fat diet(HFD)groups,namely ND+WT,ND+ adipo-chemerin(-/-),ND+chemerin(-/-),HFD+WT,HFD+ adipo-chemerin(-/-),HFD+ chemerin(-/-),6 per group,6 groups in total,feed for 12 weeks on either a normal diet(10% fat,20% protein,3.85 kcal/g)or a high-fat diet(60% fat,20% protein,5.24 kcal/g)respectively.To investigate the role of chemerin knockout in the exercise regulation of skeletal muscle lipid deposition in mice on a high-fat diet,mice fed on a high-fat diet were additionally divided into exercise control group(WT+E),adipo-chemerin(-/-)+E,chemerin(-/-)+E,6 mice in each group,for a total of 3 groups;the exercise group was started with moderate intensity aerobic exercise for 6 weeks after 6 weeks of high-fat diet feeding(during which high-fat diet feeding was maintained),with an exercise frequency of 6 times/week for 6 weeks.3.Detection of skeletal muscle lipid deposition,glucose tolerance,related enzymes,and proteins of glucolipid metabolismMice were executed after anaesthesia and their skeletal muscles were weighed and skeletal muscle lipid deposition was observed by oil red O staining;skeletal muscle TG and FFA were quantified by GPO-PAP enzyme assay and ACS-PAP assay respectively;protein levels of skeletal muscle CD36,CPT1,SCD1,PPARα and GLUT4 were measured by Western blot.Result 1.Effects of chemerin knockout on skeletal muscle lipid deposition in mice under different dietary conditions and its mechanism1)Effect on skeletal muscle mass: under a normal diet,both chemerin knockouts did not affect skeletal muscle mass in mice compared to normal mice;under a high-fat diet,chemerin(-/-)mice had increased tibialis anterior muscle weight(P<0.01),and adipose-specific chemerin knockout did not affect tibialis anterior muscle mass and ratio in mice.2)Effect on skeletal muscle lipid deposition: compared to normal mice,skeletal muscle lipid deposition was significantly increased in chemerin(-/-)mice fed a normal diet(P<0.01),but not in adipo-chemerin(-/-)mice;however,skeletal muscle lipid deposition was reduced in adipo-chemerin(-/-)mice(P<0.01)and significantly increased in chemerin(-/-)mice(P<0.01)on a high-fat diet.3)Effects on glucose tolerance and insulin sensitivity: adipo-chemerin(-/-)mice had increased insulin sensitivity on a normal diet(P<0.05);chemerin(-/-)mice had decreased insulin sensitivity(P<0.05)and increased glucose tolerance(P<0.01)on a high-fat diet.4)Effects on expression of key enzymes and proteins of glucolipid metabolism: under a normal diet,skeletal muscle GLUT4 protein expression was increased in adipo-chemerin(-/-)mice(P<0.05)and skeletal muscle PPARα and CPT1 protein expression was decreased in chemerin(-/-)mice(P<0.05);under a high-fat diet,skeletal muscle GLUT4,and CPT1 protein expression was increased and CD36 protein expression was decreased in adipo-chemerin(-/-)mice(P<0.01),and skeletal muscle CPT1 protein levels were down-regulated and SCD1 protein levels were up-regulated in chemerin(-/-)mice(P<0.01).2.Effects of adipose-specific chemerin knockout on skeletal muscle lipid deposition in mice fed a high-fat diet with exercise and its mechanism1)Effect on skeletal muscle mass: 6 weeks of aerobic exercise did not affect tibialis anterior muscle mass and ratio in high-fat fed WT and adipo-chemerin(-/-)mice.2)Effect on skeletal muscle lipid deposition: 6 weeks of aerobic exercise significantly improved skeletal muscle lipid deposition in WT mice on a high-fat diet(P<0.01),and the ameliorating effect of exercise on skeletal muscle lipid deposition in adipo-chemerin(-/-)mice was weakened.3)Effects on expression of key enzymes and proteins of glucolipid metabolism: Exercise down-regulated skeletal muscle CD36 protein expression(P<0.01)and up-regulated PPARα,CPT1,and GLUT4 protein expression(P<0.05)in WT mice;however,exercise only significantly up-regulated skeletal muscle CPT1 and PPARα protein expression in adipo-chemerin(-/-)mice(P<0.01).The regulation of skeletal muscle CD36 and GLUT4 protein expression in adipo-chemerin(-/-)mice by exercise was weakened compared to WT mice.3.Effects of global chemerin knockout on skeletal muscle lipid deposition in mice fed a high-fat diet with exercise and its mechanism1)Effect on skeletal muscle mass: 6 weeks of aerobic exercise had no effect on tibialis anterior muscle mass in WT mice,but significantly decreased tibialis anterior muscle weight in chemerin(-/-)mice.2)Effect on skeletal muscle lipid deposition: compared to WT,6 weeks of aerobic exercise showed a weakened improvement in skeletal muscle lipid deposition in chemerin(-/-)mice,as evidenced by exercise reducing skeletal muscle TG and FFA levels in WT mice(P<0.05);however,exercise only reduced skeletal muscle TG levels in chemerin(-/-)mice(P<0.05).3)Effects on glucose tolerance and insulin sensitivity: 6 weeks aerobic exercise did not affect glucose tolerance and insulin sensitivity in WT mice,but enhanced insulin sensitivity in chemerin(-/-)mice(P<0.01).4)Effects on expression of key enzymes and proteins of glucolipid metabolism: 6 weeks aerobic exercise down-regulated skeletal muscle CD36 and SCD1 protein expression(P<0.05)and up-regulated PPARα,CPT1,and GLUT4 protein expression(P<0.05)in WT mice,but exercise only up-regulated skeletal muscle CPT1 and GLUT4 protein expression and down-regulated SCD1 protein expression(P<0.05)in chemerin(-/-)mice.Compared with WT,exercise had a weakened effect on the regulation of skeletal muscle CD36 and PPARα protein expression in chemerin(-/-)mice on a high-fat diet.Conclusion 1.Adipose-specific chemerin knockout reduced skeletal muscle lipid deposition in high-fat fed mice,whereas global chemerin knockout increased skeletal muscle lipid deposition and decreased insulin sensitivity in mice;the above effects of chemerin were associated with altered levels of key enzymes and proteins of skeletal muscle glucolipid metabolism(e.g.CPT1,CD36,SCD1 or GLUT4).2.Both adipose and global chemerin knockout weakened the effect of exercise on skeletal muscle lipid deposition in mice fed a high-fat diet,with global chemerin knockout being more pronounced;chemerin knockout weakened the effect of exercise on skeletal muscle lipid deposition,which was associated with the weakened or disappeared effect of exercise on the regulation of certain skeletal muscle glycolipid metabolism-related enzymes(CD36,GLUT4 or PPARα).