Novel Mechanism Behind The Effect Of Chemerin On Aerobic Exercise-induced Impact On Glycolipid Metabolism In Male Mice

PurposeOur previous study found that the reduction of adipokine chemerin level played an important role in the aerobic exercise-induced improvement of glycolipid metabolism disorders in male diabetic mice,but the mechanism remains elusive.Recent studies have reported that chemerin could regulate androgen level,and androgen regulates glycolipid metabolism through affecting mitochondrial function,structure and biogenesis.Therefore,the present study used male mice with either exogenous chemerin intervention or chemerin knockout(adipose-specific or global knockout)to confirm whether the impact of reduced chemerin level in the exercise-induced regulation of glycolipid metabolism in male diabetic or high-fat-fed mice is achieved by enhancing androgen and androgen receptor(AR)levels,which subsequently improves mitochondrial function,structure,and biogenesis.MethodsMice with exogenous chemerin intervention:diabetic mice were replicated using high-fat diet and STZ injection.The successfully established diabetic mice were divided into diabetic control(DM),diabetic exercise(EDM)and diabetic exercise plus chemerin intervention(EDM+CHE)groups.Normal male mice were randomly divided into control(CON),exercise(E)and chemerin intervention(CHE)groups.Mice in the exercise groups were subjected to moderate intensity aerobic exercise for six weeks.Chemerin intervention was performed by intraperitoneal injection of exogenous chemerin 30 minutes before exercise.Fasting blood glucose(FBG),insulin resistance index(HOMA-IR),and blood lipids were measured.Serum testosterone and chemerin levels were detected by ELISA,and Western blot method was used to detect protein levels of AR,chemerin and CMKLR1 in gastrocnemius.In addition,the mRNA and protein levels of molecules involved in mitochondrial biogenesis(PGC-1α,NRF-1,TFAM),fusion(OPA1,MFN2)and fission(DRP1,FIS1)in gastrocnemius were also measured by RT-PCR and Western blot,respectively.Due to sample limitation,the mitochondrial structure and function in gastrocnemius were not examined.Chemerin knockout(adipose knockout or global knockout)mice:male wild-type(WT)and adipose chemerin knockout mice(heterozygous and homozygous)were fed a normal diet(ND)or a high-fat diet(HFD)for six weeks and were divided into sedentary and aerobic exercise groups,a total of 12 groups including WT,WT+E,adipo-chemerin(+/-),adipo-chemerin(+/-)+E,adipo-chemerin(-/-),and adipochemerin(-/-)+E groups under either ND or HFD.Similarly,male WT and global chemerin knockout homozygous mice were divided into sedentary and aerobic exercise groups after six weeks of food intervention with either ND or HFD to obtain WT,WT+E,chemerin(-/-)and chemerin(-/-)+E groups,eight groups in total.Parameters including body weight,body composition,blood lipids,FBG,glucose tolerance and insulin tolerance levels,serum testosterone and chemerin levels,chemerin,CMKLR1 and AR protein levels in gastrocnemius were measured.Besides,the mitochondrial number and structure(transmission electron microscopy),mitochondrial respiration rate(O2K Mitochondrial Function Assay System)and mitochondrial membrane potential level(JC-1),as well as the mRNA and protein levels of molecules involved in mitochondrial biogenesis,fusion and fission were also examined.ResultsPart Ⅰ.Chemerin regulates androgen/AR level in male mice1.Exogenous chemerin intervention reduced androgen/AR levels in exercised diabetic miceOur previous studies have demonstrated that exogenous chemerin intervention reversed the exercise-induced improvement of glycolipid metabolism in diabetic mice.Further,in the present study,we found that compared with the CON group,serum testosterone level(P<0.05)and AR protein level in gastrocnemius(P<0.01)were significantly decreased in DM group.Six weeks of aerobic exercise increased AR protein level(P<0.01)in gastrocnemius of DM mice,which was reversed by exogenous chemerin(P<0.05).Compared to the EDM group,serum testosterone concentration was significantly reduced in EDM+CHE group(P<0.05).2.Effects of adipose chemerin knockout on androgen/AR level,blood glucose and blood lipids in male mice2.1 Effect of adipose chemerin knockout on serum testosterone and AR protein level in gastrocnemius of male mice(1)Under ND:Compared to WT mice,no significant change in serum testosterone concentration was observed in adipose chemerin knockout mice(P>0.05).(2)Under HFD:Adipose chemerin knockout increased serum testosterone level in sedentary mice(P<0.05).AR protein level was significantly increased in exercised adipose chemerin knockout mice,compared to exercised WT mice(P<0.01).2.2 Effect of adipose chemerin knockout on blood glucose and blood lipids,glucose tolerance and insulin tolerance in male mice(1)Under ND:Compared with WT mice,adipose chemerin knockout mice showed improved blood lipids(reduced TC,LDL,and increased HDL level,P<0.05)and no alteration in glucose tolerance and insulin tolerance(P>0.05).Six weeks of aerobic exercise improved blood lipids in WT and adipose chemerin knockout homozygous mice(reduced TC,increased HDL,P<0.05),and the improvement in lipids was more pronounced in adipose chemerin knockout mice.(2)Unde HFD:Compared with WT mice,adipose chemerin knockout mice showed the improved blood glucose and blood lipids(decreased TC,TG,and FBG,P<0.05).Exercise significantly reduced FBG levels in WT and adipose chemerin knockout mice(P<0.01),with no significant difference in the degree of reduction between the two groups.3.Effect of global chemerin knockout on androgen level,blood glucose and lipids in male mice3.1 Effect of global chemerin knockout on serum testosterone level in male mice(1)Under ND:Global chemerin knockout didn’t change the serum testosterone level in sedentary and exercised mice(P>0.05).(2)Under HFD:Serum testosterone level was significantly increased in global chemerin knockout mice,compared with WT mice(P<0.01).Six weeks of aerobic exercise increased serum testosterone levels in WT(P<0.01)and global chemerin knockout mice(P<0.05),and the increase was more pronounced in global chemerin knockout mice.3.2 Effect of global chemerin knockout on blood glucose and lipids,glucose tolerance and insulin tolerance in male mice(1)Under ND:Global chemerin knockout increased TC level in mice(P<0.01),but had no effect on glucose tolerance and insulin tolerance levels(P>0.05).TG and FBG were significantly decreased in exercised global chemerin knockout mice,compared with exercised WT mice(P<0.05).(2)Under HFD:FBG was elevated(P<0.01),and the areas under the curve of OGTT and ITT were decreased(P<0.01)in global chemerin knockout mice.Exercise significantly reduced FBG in global chemerin knockout mice(P<0.01)and LDL level in WT mice(P<0.05),as well as the areas under the OGTT and ITT curves in WT and global chemerin knockout mice(P<0.05).Part Ⅱ Chemerin regulates the mitochondrial structure,function and biogenesis in skeletal muscle1.Effect of exogenous chemerin intervention on mitochondria related proteins in exercised diabetic miceCompared with CON mice,the mRNA and protein levels of PGC-1α,NRF-1,TFAM,MFN2,and OPA1 in gastrocnemius of DM mice were significantly decreased(P<0.05).Six weeks of aerobic exercise significantly upregulated the mRNA levels of these molecules and the protein levels of NRF-1,OPA1,and MFN2 in EDM mice(P<0.05),which were reversed by exogenous chemerin intervention(P<0.05).2.Effect of adipose chemerin knockout on mitochondrial structure,function and biogenesis-related proteins in gastrocnemius of male mice(1)Mitochondrial structure and function:High-fat diet caused a decrease in the mitochondrial number,disorganization or even loss of mitochondrial cristae,vacuolization of mitochondria,and decreased the mitochondrial membrane potential level in WT mice(P<0.05).Adipose chemerin knockout improved these mitochondrial disorders.Six weeks of exercise improved the disorders in mitochondrial number and structure with the more pronounced improvement in adipose chemerin knockout mice.Besides,exercise significantly increased the respiratory control rate in adipose chemerin knockout mice(P<0.05),but had no effect on WT mice(P>0.05).(2)Mitochondrial biogenesis-related proteins:High-fat diet reduced protein levels of PGC-1α,NRF-1 and TFAM,and decreased mRNA levels of PGC-1α,NRF-1 and OPA1 in gastrocnemius of WT mice(P<0.05).Adipose chemerin knockout not only reversed these HFD-induced detrimental effects on the above mitochondria-associated proteins,but also downregulated the mRNA and protein levels of DRP1 and FIS1(P<0.05).3.Effect of global chemerin knockout on mitochondrial structure,function,and biogenesis-related proteins in gastrocnemius of male mice(1)Mitochondrial structure and function:Under ND,compared to WT mice,mitochondrial membrane potential level was significantly increased in global chemerin knockout mice(P<0.01).Under HFD,compared with WT mice,mitochondrial density and cristae,respiratory rate of mitochondrial complex Ⅱ,and mitochondrial membrane potential were significantly increased in global chemerin knockout mice(P<0.01).Six weeks of aerobic exercise increased the number and function of mitochondria in gastrocnemius of WT and global chemerin knockout mice under both diets(P<0.05).(2)Mitochondrial biogenesis-related proteins:Under both diets,compared with WT mice,NRF-1 protein level were increased(P<0.05),DRP1(P<0.01)and FIS1(P<0.01)protein levels were decreased in gastrocnemius of global chemerin knockout mice.Compared to exercised WT mice,NRF-1(P<0.05)protein level was increased and FIS1 protein level was significantly downregulated(P<0.05)in exercised global chemerin knockout mice.Conclusions1.The impact of reduced chemerin level in aerobic exercise-induced improvement of glycolipid metabolism in male diabetic mice may be exerted by increasing serum testosterone and AR protein level,which subsequently enhances mitochondrial biogenesis in gastrocnemius.2.Chemerin negatively regulates androgen/AR level.Six weeks of aerobic exercise may improve blood glucose and lipids in high-fat-fed male chemerin knockout mice(adipose knockout and global knockout)through increasing serum testosterone and AR protein levels in gastrocnemius.3.Chemerin knockout(adipose knockout and global knockout)has improved mitochondrial structure,function and enhanced mitochondrial biogenesis in gastrocnemius of male exercised mice under high-fat diet.Given that many literatures has demonstrated the essential role of androgen/AR in promoting mitochondrial number,structure,function and mitochondrial biogenesis,as well as the important role of mitochondria in glycolipid metabolism,we propse that chemerin-induced regulation of androgen/AR level,which affects mitochondrial levels and function in skeletal muscle,is a novel mechanism for chemerin involvement in aerobic exercise-induced regulation of glycolipid metabolism in male mice.