| Purpose: Chemerin,as a novel adipokine,is believed to be associated with the onset and the impaired lipid and glucose metabolism of obesity and its related diseases.It is previously reported that the exercise-induced decrease of chemerin in serum or tissue contributed to the improvement of glycolipid metabolism in human patients or rodents with obesity or diabetes.However,little evidence about the mechanism behind has been obtained.The aerobic exercise-caused decrease of chemerin may exert positive functions on lipid and glucose metabolism in diabetic rats through upregulating peroxisome proliferators-activated receptor ?(PPAR?)-regulated key enzymes or proteins involved in glycolipid metabolism(e.g.adipose triglyceride lipase(ATGL)and lipoprotein lipase(LPL)),according to our previous study.In the current study,exogenous chemerin was applied to further verify the mechanism behind the improvement of lipid and glucose metabolism induced by decreased chemerin level in diabetic mice – relating to the PPAR? regulated expressions of key enzymes or proteins involved in lipid and glucose metabolism,such as ATGL,LPL,Glucose transporter 4(GLUT4)and Phosphoenolpyruvate carboxykinase(PEPCK)? Adipose tissue,as a lately considered endocrine organ,plays vital roles in regulating metabolic homeostasis and adipokine secretion.In addition,adipose tissue together with liver tissue are the main sources of chemerin.Chemerin and its receptor CMKLR1 are highly expressed in fat tissue.Therefore,the current study compared the conditions in obesity and its related impaired metabolism in wild type mice and adipose tissue-specific chemerin gene knockout mice fed with normal and high-fat diet.The main purpose of this study is to determine chemerin's role in lipid and glucose metabolism and further investigate the influence of chemerin gene knockout on serum chemerin,lipid and glucose metabolism in peripheral metabolic organs,expression of key enzymes or proteins(e.g.liver and skeletal muscle)involved in metabolism and their upstream molecule PPAR?.Effects and sexual differences of six-week aerobic exercise on the body fat and lipid glucose metabolism of chemerin gene knockout mice fed with high-fat diet were investigated with the basis on what mentioned above.And finally analysed whether the functions of exercise were exerted via regulating PPAR? and key lipid and glucose metabolism enzymes including ATGL,LPL and PEPCK.Methods: Part 1: Negative effects of exogenous chemerin on the exercise-induced amelioration of impaired lipid and glucose metabolism in diabetic mice and its mechanism.70 ICR mice at the age of six weeks were randomly divided into control group(n=24)and diabetes model group(n=46).Mice in the diabetes model group were intraperitoneally injected with small dose of streptozotocin(STZ,100 mg/kg)after six weeks of high-fat diet(HFD).All the successfully established diabetic mice were divided into three subgroups: diabetes group(DM,n=8),diabetes plus exercise group(EDM,n=9)and diabetes plus exercise and exogenous chemerin group(EDC,n=10),while mice in the control group were divided into three subgroups: normal control group(Con,n=8),exercise group(E,n=8)and exogenous chemerin group(C,n=8).All the mice in EDM,EDC and E groups participated in six-week modest intensity treadmill running exercise with gradually increased load,six days per week and one time per day.And all the mice in EDC and C groups were intraperitoneally injected with exogenous chemerin(with the dose of 8 ng/g)at the 4th week of exercise period.Samples were collected after the exercise intervention.Body composition,lipid metabolism indexes including TG,TC,LDL and HDL,FBG were measured and serum chemerin,fasting insulin levels(FINS)were detected by ELISA.HOMA-IR was calculated by the values of FBG and FINS.Protein levels of Chemerin,CMKLR1,PPAR?,ATGL,LPL and PEPCK were detected by Western Blot.Part 2: Incidence of obesity and impaired lipid and glucose metabolism in fat-specific chemerin gene knockout mice fed with normal diet or HFD and its underlying mechanisms.1.Incidence of obesity and lipid and glucose metabolism in fat-specific chemerin gene knockout(fabp4-cre)mice fed with normal diet or HFD Animal grouping: Wild type C57 mice(WT),flox group(flox/flox),chemerin knockout heterozygote mice group[chemerin(+/-)] and chemerin knockout homozygotes mice group[chemerin(-/-)](fabp4-cre mice was used to establish chemerin knockout mice).Both male and female were included in all the phenotype,4 groups in total with six mice each group.All mice were fed with normal diet for 11 weeks.In addition,other four groups similar to groups above were also set with the HFD.Body composition,incidence of obesity,lipid metabolism indexes,FBG,FINS,liver morphology,serum chemerin were measured as well as the protein levels of chemerin?CMKLR1?PPAR??ATGL?LPL?GLUT4 and PEPCK in liver and skeletal muscle were detected by Western Blot.2.Changes in body fat and glycolipid metabolism in other chemerin knockout(adiponectin-cre)mice fed with HFD Fabp4-cre and adiponectin-cre are tool mice for the target genes knockout in fat tissue.These fabp4-cre trangsgenic mice are a cre-lox tool useful for deletion of floxed sequences in brown and white adipose tissue,while adiponectin-cre in white adipose tissue.Animal grouping: mice were divided into several subgroups: wild type mice group(WT),flox group(flox/flox),(-/-)·fabp4 group and(-/-)·adiponectin group.Both male and female were included in all the phenotype,4 groups in total with six mice each group.All mice were fed with HFD for 11 weeks.All the parameters detected and methods used were the same to that in part 1.Part 3: Effects of aerobic exercise on the glycolipid metabolism of fat-specific chemerin knockout mice under HFD Animal grouping: mice were divided into several subgroups: wild type mice group(WT),flox group(flox/flox),(-/-)·fabp4 group and(-/-)·adiponectin group.Both male and female were included in all the phenotype,six mice each group.After five-week HFD intervention,4 groups mice were divided into HFD control group and HFD plus exercise group,WT group,respectively.Both male and female were included in all the phenotype,8 groups in total,six mice each group.All the mice in exercise groups participated in six-week modest intensity treadmill running with gradually increased load,six days per week,one time per day.All the parameters detected and methods used were the same to that in part 2.Results: 1.Exogenous chemerin reversed the exercise-induced improvement of glucose and lipid metabolism disorders in diabetic mice and its mechanism(1)Exogenous chemerin supplementation can reverse the effect of 6 weeks of aerobic exercise on the improvement of the glucose and lipid metabolism,fatty liver and the weight of liver in diabetic mice.(2)Exogenous chemerin supplementation can attenuate the reduction effect of 6-week aerobic exercise on chemerin(serum,liver,gastrocnemius,and fat)and CMKLR1(liver,gastrocnemius,and fat)protein levels in diabetic mice.(3)Exogenous chemerin can reduce the increase of PPAR?,ATGL,LPL and GLUT4 protein levels in liver,gastrocnemius and fat of 6-week aerobic diabetic mice,but has no significant effect on liver PEPCK protein levels.2.The incidence of obesity and impaired glucose and lipid metabolism in fat-specific chemerin knockout mice and mechanisms under normal diet and HFD 2.1The incidence of obesity and glucose and lipid metabolism disorders in fat-specific chemerin knockout(fabp4)mice under normal diet and HFD(1)Incidence of obesity: compared with wild-type or flox control mice,the incidence of obesity in fat-chemerin knockout mice was not different in normal diet(no obese mice were seen).However,in high-fat diets,the incidence of obesity in fat chemerin knockout mice is increased in females while decreased in males.(2)Glycolipid metabolism and fatty liver: under normal diet,compared with WT or flox mice,the fat mass and glycolipid metabolism were unchanged and still in the normal range.Under the HFD,compared with WT or flox mice,reduced insuin sensitivity,impaired lipid metabolism(increases in TC,LDL and TG),gain in liver weight and fatty liver were observed in fat-specific chemerin knockout mice.In addition,gender-dependent differences were also found in all the changes mentioned under HFD,reflected by female mice were prone to getting fat but no fatty liver,while male mice showed fatty liver without adiposity.2.2 Protein levels of chemerin,CMKLR1,PPAR? and key enzymes involved in fat-specific chemerin knockout(fabp4-cre)mice under normal and high-fat diets With normal diet,compared with WT mice,changes in fat-specific chemerin knockout mice were found:(1)serum chemerin levels are significantly reduced;(2)no changes in proteins levels of chemerin in liver,CMKLR1,PPAR?,ATGL,LPL and PEPCK protein levels were found;(3)gastrocnemius chemerin and CMKLR1 protein levels were increased significantly,and the protein levels of PPAR? were significantly decreased.With HFD,compared with WT mice,here are some changes in fat-specific chemerin knockout mice:(1)The protein levels of chemerin and CMKLR1 in gastrocnemius and liver were increased,which may be the reason for no change in serum chemerin(because in addition to fat,liver is also the source of serum chemerin).(2)Compared with WT mice,the levels of PPAR?,ATGL and LPL protein in liver and gastrocnemius muscle of male chemerin knockout mice were decreased,PEPCK in liver was increased,and only GLUT4 in gastrocnemius muscle was increased.This may be used to explain the abnormal lipid metabolism(severer impaired lipid metabolism and fatty liver)in male chemerin knockout mice.For female chemerin knockout mice,protein level of PEPCK in liver was increased and LPL was decreased in gastrocnemius muscle,but LPL in liver,and PPAR?,GLUT4 in gastrocnemius were increased.2.3 The obesity incidence,body fat,glucose and lipid metabolism,and chemerin levels in fat-specific chemerin knockout(adiponectin-cre)mice during high-fat diets and comparsion of that between two kinds of chemerin knockout mice(1)Incidence of obesity: the incidence of obesity in chemerin(-/-).adiponectin mice was decreased,male mice(67% vs 100%),that in female mice was increased(67% vs 50%),such phenomenon also appeared in chemerin(-/-)·fabp4(male: 80% vs 100%;female: 100% vs 50%).(2)Glycolipid metabolism and fatty liver: Under HFD,male(-/-)·fabp4 mice showed reduced insulin sensitivity,disturbance of lipid metabolism(increased serum TC and LDL),liver Weight gain and fatty liver;the changes in male(-/-)·adiponectin mice were different,not only increased insulin sensitivity,but also no lipid metabolism disorders and fatty liver,serum TC and TG levels were lower than wild control mice.Female(-/-)· fabp4 mice have increased glucose tolerance,decreased insulin sensitivity,and lipid metabolism disorders(serum TC,LDL increased);while female(-/-)· adiponectin has increased glucose tolerance and insulin sensitivity remains unchanged,and showed the disorders of lipid metabolism(elevated serum LDL).(3)Protein levels of chemerin and target molecules: Compared with WT mice,here are some changes in chemerin(-/-).adiponectin knockout mice fed with HFD:(1)Serum: The chemerin levels in both male and female(-/-)· fabp4 mice were significantly reduced.(2)Liver: The protein levels of chemerin,CMKLR1 and PPAR? in male(-/-)· fabp4 mice remained unchanged.And protein levels of ATGL and LPL protein were increased;(3)Gastrocnemius: The gastrocnemius chemerin and CMKLR1 of male(-/-)·fabp4 mice increased,PPAR? remained unchanged,LPL decreased,and GLUT4 increased(the gastrocnemius chemerin and CMKLR1 of male(-/-)·adiponectin mice were lower than that in(-/-)·fabp4 mice,PPAR?,LPL and GLUT4 were higher than(-/-)· fabp4 mice).In female mice,increases in chemerin,CMKLR and GLUT4 and decrease in LPL and no chnage in PPAR? were observed.Compared with WT mice,here are some changes in chemerin(-/-).fabp4 mice under HFD:(1)Serum: both male and female(-/-)· fabp4 mice had no change in serum chemerin.(2)liver: both male and female(-/-)·fabp4 mice showed the increased protein levels of chemerin and CMKLR.Decreased protein levels of PPAR?,ATGL and LPL were found,while in the female mice,PPAR? didn't change and the LPL and PEPCK were increased;(3)Gastrocnemius: both male and female(-/-)·fabp4 mice showed the increased protein levels of chemerin and CMKLR1.In male mice,PPAR? and LPL were decreased with the increased GLUT4,while female(-/-)·fabp4 mice showed the increased PPAR?,GLUT4 increased,and decreased LPL.3.Effects of exercise on the glycolipid metabolism of fat-specific chemerin knockout mice fed with HFD and its mechanism(1)Effects of exercise on fat mass of chemerin mice: six weeks of aerobic exercise reduced the body fat of male fat chemerin knockout mice,but no influences on that in female mice.(2)Effects of exercise on glycolipid metabolism of chemerin knockout mice: 6-week aerobic exercise improves glucose and lipid metabolism in male adipose chemerin knockout mice,and reduces fatty liver [fatty liver was found only in(-/-)· fabp4 mice].Exercise can also improve glucose and lipid metabolism in female fat chemerin knockout mice(reducing serum insulin and HOMA-IR,and improving lipid metabolism.)(3)Exercise reduced serum chemerin concentration of chemerin knockout mice: although under HFD,serum chemerin was reduced in(-/-).adiponectin mice but no change in(-/-).fabp4 mice was found.6 weeks of aerobic exercise reduced the serum chemerin levels of these two kinds of mice.(4)Effects of exercise on the protein levels of glycolipid metabolism related proteins: 6-week aerobic exercise improved the glycolipid metabolism related proteins both in male and female chemerin knockout mice,which was reflected as the increased protein levels of PPAR?-key enzymes involved in glycolipid metabolism(ATGL and LPL)in gastrocnemius(only in male),the up-regulated ATGL,LPL(ATGL was increased only in female)were also obtained in(-/-)·fabp4 mice.Conclusions: 1.This study verified that the decreased chemerin plays an important role in the exercise-induced improvement of glucose and lipid metabolism in diabetic mice,and this effect may be mediated by chemerin receptor(CMKLR1)and the up-regulation of PPAR?-glycolipid metabolism enzymes including ATGL,LPL and GLUT4 pathways.2.Fat-specific chemerin knockout [(-/-).adiponectin)]under high-fat diet mice showed gender-dependent differences in body fat and glycolipid metabolism(compared with control mice,male knockout mice are less prone to fat,but fatty liver may appear,and female knockout mice are more likely to be fat with increased LDL).The reason for the gender difference may be explained by the difference in protein levels of PPAR? and glucose and lipid metabolism enzymes(such as ATGL and LPL)in liver and skeletal muscle of male and female chemerin knockout mice.3.Better glycolipid metabolism was found in(-/-).adiponectin mice,compared with that in(-/-)·fabp4 mice(although low incidence of obesity,impaired lipid metabolism and fatty liver appeared)regardless of the different gender.It is also related to lower serum chemerin levels and higher expression levels of lipid and glucose metabolism enzymes in gastrocnemius or liver.4.Six weeks of aerobic exercise can still improve glucose and lipid metabolism in male and female chemerin knockout mice,and there is a gender difference in the degree of improvement.It only reduced the body fat of male mice but not in females.However,exercise improved the glycolipid metabolism both in female and male mice,and the significantly greater changes in male mice were observed.The reason for that difference may relate to the different extent of exercise-caused decrease in serum chemerin and increase in PPAR?-key enzymes involved in glycolipid metabolism.In short,this study verified the vital roles of chemerin in the HFD-induced glycolipid metabolism disorder of mice through applying exogenous chemerin and chemerin knockout mice models.In addition,we determined the effects of chemerin on exercise-related improvement in glycolipid metabolism,which might be mediated via regulating the levels of PPAR?-key enzymes involved in glycolipid metabolism(ATGL,LPL and GLUT4). |
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