| PurposeNonalcoholic fatty liver disease(NAFLD)is a common chronic liver disease characterized by the accumulation of triglyceride-dominated lipids in liver cells and pathological liver changes caused by the non-long-term large amount of alcohol consumption and non-other definite liver damage factors.NAFLD is widespread worldwide,affecting about 10% to 30% of adults worldwide.Chemerin is essential in regulating adipocyte differentiation,promoting inflammatory responses and metabolism,mainly by binding to the receptor chemokine-like-receptor1(CMKLR1),which governs glucolipid metabolism and is closely associated with the development of NAFLD.It is closely related to the development of NAFLD.Studies have shown that serum chemerin levels are significantly increased in patients with NAFLD or rats and mice and that the increased levels correspond to the degree of hepatic steatosis.Treatment of NAFLD with exercise,dietary interventions,or weight loss surgery is accompanied by a decrease in chemerin/CMKLR1 levels.Previous studies by our group have shown that adipose chemerin knockout improves glucolipid metabolism in mice on a high-fat diet by modulating the levels of key enzymes and proteins involved in glucolipid metabolism,but whether chemerin can regulate the development of NAFLD in mice on a high-fat diet(HFD)and whether it is also associated with enzymes involved in lipid metabolism has not yet been elucidated.Therefore,the first aim of this study was to verify the regulation of NAFLD by chemerin in the basal state using adipose-specific chemerin knockout mice and systemic chemerin knockout mice.Numerous papers have reported that exercise regulates the activity of key enzymes of lipid metabolism in the liver,promotes the oxidative breakdown of hepatic lipids and reduces intrahepatic fat synthesis and transport as an important way to improve NAFLD.In our group,we found that aerobic exercise-induced chemerin reduction could regulate the disruption of glucolipid metabolism by affecting key enzymes of glucolipid metabolism.Thus,we hypothesize that the role of chemerin reduction in the improvement of NAFLD in mice by exercise is likely to be mediated through the regulation of key enzymes of hepatic lipid metabolism.Therefore,the second aim of this study was to clarify the role of chemerin in regulating hepatic lipid metabolism by exercise and to investigate the mechanism of action.Method1.Mice modelling and groupingIn this study,we commissioned Shanghai Southern Model Biotechnology to construct adipose-specific chemerin knockout and systemic chemerin knockout mice using the cre-loxp system.When the mice were two weeks old,we cut their tails for DNA testing to screen out the knockout mice,and at 8 weeks old,we measured the chemerin protein levels in fat and liver for knockout validation.Eight-week-old male wild-type(WT)mice,adipo-chemerin knockout purehybrid [adipo-chemerin(-/-)] mice and systemic chemerin knockout pure-hybrid[chemerin(-/-)] mice were randomly divided into normal diet and high-fat diet groups and subjected to 12 weeks of dietary intervention to obtain ND and WT,adipo-chemerin(-/-)and chemerin(-/-)mice under HFD.In addition,HFD mice were divided into exercise control(WT+E),adipo-chemerin(-/-)+E and chemerin(-/-)+E groups,which were fed high-fat for 6 weeks followed by 6 weeks of moderate intensity running table exercise,6 times per week.2.Detection of fatty liver and liver metabolizing enzymes in miceThe weight and liver weight of each group of mice were measured.The serum levels of Alanine transaminase(ALT)and Aspartate aminotransferase(AST)were measured by Wright’s microplate method.The hepatic steatosis of mice was measured by HE staining and Oil Red O staining.Western blot method was used to detect several key enzymes of lipid metabolism in mouse liver,including Cluster of differentiation 36(CD36),Peroxisome proliferator activated receptor α(PPARα),Carnitine palmitoyltransferase 1(PPARα),Carnitine palmitoyltransferase 1(PPARα),Peroxisome proliferator activated receptor α(PPARα),and Peroxisome proliferator activated receptor α(PPARα).Carnitine palmitoyltransferase 1(CPT1),sterol regulatory element binding proteins-1(SREBP-1)and stearoyl-coenzyme A desaturase 1(SREBP-1).-Co A desaturase1(SCD1)protein levels.Results1.Effects of chemerin knockout on nonalcoholic fatty liver in untrained male mice fed with normal or high-fat diet(1)Effects of chemerin knockout on normal diet mice: WT mice and two chemerin knockout mice showed normal liver morphology and structure without obvious pathological changes.The two chemerin knockouts did not significantly affect the expression of liver lipid metabolism-related enzymes.Still,the protein expression of adipose synthesis protein SCD1 was upregulated in chemerin(-/-)mice compared with adipo-chemerin(-/-)mice(P < 0.05).(2)Effects of chemerin knockout on high-fat diet mice: compared with WT mice,adipo-chemerin(-/-)mice showed decreased obesity rate,decreased liver weight to body weight ratio(P < 0.05),decreased number of liver fat vacuoles and lipid droplets,and decreased serum ALT and AST levels(P < 0.05),indicating that HFD-fed adipo-chemerin(-/-)mice improved NAFLD;however,chemerin(-/-)mice showed increased obesity,increased number of fat vacuoles and balloon-like degeneration,suggesting worsening hepatic steatosis.At the protein level of glycolipid metabolizing enzymes,the levels of hepatic fat transport protein CD36,lipid synthesis protein SREBP-1 and SCD1 were significantly downregulated in adipochemerin(-/-)mice relative to the WT group(P < 0.05),and the expression level of oxidative catabolism rate-limiting enzyme CPT-1 was upregulated(P < 0.05);whereas chemerin(-/-)mice had up-regulated expression of CD36 and SREBP-1(P < 0.05).chemerin(-/-)mice had significantly up-regulated(P < 0.01)and decreased(P < 0.05)expression of CD36,SREBP-1,and SCD1 proteins compared with adipo-chemerin(-/-)mice.2.Effects of chemerin reduction on nonalcoholic fatty liver in exercised male mice fed with high-fat diet and the underlying mechanisms(1)Effects of global chemerin knockout on exercised HFD mice: 6weeks of aerobic exercise significantly reduced liver weight and liver specific gravity(P < 0.05),decreased the number of fat vacuoles and lipid droplets in WT and chemerin(-/-)mice,and reduced serum ALT levels(P <0.05)and AST levels(P < 0.01 and P < 0.05,respectively),but chemerin(-/-)mice showed a smaller decrease in AST levels than WT.at the protein level of glycolipid metabolizing enzymes,6 weeks of aerobic exercise decreased hepatic SREBP-1,SCD1(P < 0.05),and CD36 levels in WT mice(P < 0.01)and elevated CPT-1 levels(P < 0.05),but exercise only decreased CD36 levels in chemerin(-/-)mice(P < 0.01),with no significant effect on protein levels of the remaining metabolic enzymes.The above results suggest that the ameliorative effect of exercise on NAFLD in mice on high-fat diet is related to its regulation of key enzymes of hepatic lipid metabolism(SREBP-1,SCD1,CD36,CPT-1),and the systemic chemerin knockout mice attenuates or loses its regulatory effect on key enzymes of hepatic lipid metabolism,thus attenuating the ameliorative effect of exercise on NAFLD in mice on high-fat diet.(2)Effects of adipose-specific chemerin knockout on exercised HFD mice: 6 weeks of aerobic exercise reduced lipid accumulation in the liver of high-fat diet WT and adipose-chemerin(-/-)mice and improved the morphological structure of the liver;6 weeks of aerobic exercise significantly decreased serum ALT and AST levels in WT mice(P < 0.05,P< 0.01)and serum AST levels in adipose-chemerin(-/-)mice(P < 0.01).In terms of protein levels of glycolipid metabolizing enzymes,6 weeks of aerobic exercise reduced hepatic CD36 and SREBP-1 levels(P < 0.05,P <0.01)and increased CPT-1 levels(P < 0.05)in WT mice,and adipose-chemerin(-/-)mice further reduced protein levels of CD36 and SCD1 and increased CPT-1 levels after 6 weeks of aerobic exercise(P < 0.05,P <0.01).CPT-1 levels(P < 0.01).These results suggest that adipose se chemerin knockout enhanced the regulation of key enzymes of hepatic lipid metabolism,thereby enhancing the ameliorative effect of exercise on NAFLD in mice on a high-fat diet.Conclusions:1.In the basic state,NAFLD in HFD male mice was improved by chemerin reduction,while was exacerbated by chemerin deficiency,through regulating key hepatic lipid metabolism enzymes(such as SREBP-1,SCD-1,CD36,CPT-1).2.Exercise-induced amelioration of NAFLD in HFD mice was further enhanced in adipose-chemerin(-/-)mice,while attenuated in chemerin(-/-)mice,which were achieved by increasing and decreasing the levels of key hepatic lipid metabolism enzymes,respectively. |
