| Background:Non-alcoholic fatty liver disease(NAFLD)has become the most common liver disease cluster in the world,including several pathological stages,such as simple steatosis,inflammation,and fibrosis.It is well known that the vast majority of NAFLD cases arise from abnormal accumulation of lipid droplets(LDs).As highly dynamic intracellular depots,LDs alternate between a turnover progress of biogenesis and degradation.This dynamic process can be regulated by several stimuli,such as intracellular nutrient environment and energy state.Although the precise mechanism of LD turnover is not entirely clear yet,mounting evidence suggests that dysregulated LD turnover leads to the pathogenesis and development of NAFLD.Regular exercise training is beneficial for preventing and improving the treatment of NAFLD by regulating the hepatic lipid content.However,whether exercise-induced prevention and improvement of NAFLD is associated with the activity of LD turnover has not been elucidated,which needs to be explored.Objective: In this study,mice were fed with 15 weeks of high-fat diets(HFD)to induce NAFLD.Meanwhile,the mice performed 15 weeks of moderate treadmill exercise training.This study was aimed to explore:(1)the effect of exercise in preventing NAFLD;(2)whether the benefit of exercise on NAFLD is associated with LD turnover.Therefore,we aimed to provide new perspective for NAFLD treatment.Methods: Twenty-four C57BL/6 male mice(6 weeks of age)were used.All the mice were randomly assigned to the following groups: Normal diet sedentary(NS,n=6),Normal diet combined exercise(NE,n=6),60% High-fat diets sedentary(HS,n=6),60%High-fat diet combined exercise(HE,n=6)with a period of 15 weeks.All the mice had access to water and food ad libitum.Group HS and Group HE received a one-week adaption period of HFD.In addition,Group NE and Group HE received a 5-day period of adaptive exercise training.During the formal training period,the mice were subjected to a training protocol of 60 min/day,and 5 days/week for 15 weeks.Each day,mice were trained in a sequence in the following order: 5 min for warming up(7 m/min),60 min for formal training(13m/min),and 5 min for relaxing(7 m/min).Body weight was measured weekly until the mice were sacrificed.One week before the end of the exercise intervention,ITT and body composition analysis was carried out.Serum from overnight fasted mice were collected for biochemical tests.The same area of liver tissues from mice were taken for histomorphology examination,biochemical examination,and LD turnover-related PCR and WB examination.Results:(1)From the perspective of metabolism,compared with Group NS,the body weight(P<0.01),fat mass(P<0.01),i WAT mass(P<0.01),e WAT mass(P<0.01),BAT mass(P<0.01),ITT AUC(P<0.01),fasting blood glucose(P<0.01),serum LDL-c(P<0.01),serum TC(P<0.01)of Group HS were significantly increased,the lean body mass of Group HS was significant decreased(P=0.0363).There was no significant difference in serum TG between the two group.Compared with Group NS,the body weight(P<0.05)and lean body mass(P<0.01)of Group NE were increased,the fat body mass(P=0.0135),i WAT mass(P<0.01),e WAT mass(P<0.05)and ITT AUC(P<0.01)of Group NE were significantly decreased.There was no significant difference in other indicators between the two group.Compared with Group HS,the body weight(P<0.01),fat mass(P<0.01),i WAT mass(P<0.01),e WAT mass(P<0.01),BAT mass(P<0.01),ITT AUC(P<0.01),fasting blood glucose(P<0.01),serum LDLc(P<0.01)and serum TC(P<0.01)of Group HE were significantly decreased,the lean body mass of Group HE was significantly increased(P<0.01).There was no significant difference in serum TG between the two group.Compare with Group NE,lean mass of Group HE was significantly decreased(P<0.05),the fat mass,i WAT mass(P<0.05),e WAT(P<0.05),fasting blood glucose(P<0.05)were significantly increased.(2)From the perspective of liver pathological phenotype.Compared with Group NS,the liver weight(P<0.01),liver TG(P<0.01),serum AST(P<0.01),serum ALT(P<0.01),score of steatosis(P<0.01),score of inflammation(P<0.01),score of hepatocyte ballooning(P<0.01),score of NAFLD(P<0.01),fibrosis positive area(P<0.01)of Group HS were significantly increased.There was no significant difference in liver TC between the two group.Compared with Group NS,the liver TG was significantly decreased.There was no significant difference in other indicators between the two group.Compared with Group HS,the liver weight(P<0.01),liver TG(P<0.01),serum AST(P<0.01),serum ALT(P<0.01),score of steatosis(P<0.05),score of inflammation(P<0.05),score of hepatocyte ballooning(P<0.05)and score of NAFLD(P<0.05)of Group HE were significantly decreased.There was no significant difference in fibrosis positive area and liver TC between the two group.The results of Western blots shown that,compared with Group NS,the protein level of hepatic Collagen I(P<0.01),ASC(P<0.01),IL-1 pro(P<0.01),IL-1 mature(P<0.01)of Group HS were significantly increased.There was no significant difference in protein levels of NLRP3 between the two group.There was no significant difference in these indicators above between the Group NS and Group NE.Compared with Group HS,the protein levels of ASC(P<0.05)and IL-1 mature(P<0.01)of Group HE was significantly decreased,and there was no significant difference in other indicators between the two group.(3)From the perspective of liver lipid droplet morphology and structure.Compared with Group NS,the positive of Oil red O staining(P<0.01),amount of LD(P<0.01),and amount of large-size LD(P<0.01)of Group HS were significantly increased,the amount of small-size LD was significant decreased(P<0.01).Compared with Group NS,the positive area of Oil red O staining(P<0.01)of Group NE was significantly decreased(P<0.01),while there was no significant difference in other indicators between the two group.Compared with Group HS,the positive of Oil red O staining(P<0.01),amount of LD(P<0.01),and amount of large-size LD(P<0.01)of Group HE were significantly decreased,the amount of small-size LD was significantly increased(P<0.01).The results of Western blots shown that,compared with Group NS,the hepatic PLIN2 protein level of Group HS was significantly increased(P<0.05),while there was no significant difference in the protein level of PLIN3 between the two group.Compared with Group NS,the hepatic PLIN3 protein level of Group NE was significantly increased(P<0.05),while there was no significant difference in the protein level of PLIN2 between the two group.Compared with Group HS,the hepatic PLIN2 protein level of Group HE was significantly decreased(P<0.05),while the hepatic PLIN3 protein level of Group HE was significantly increased(P<0.05).The results of PCR shown that,there was no significant difference in the m RNA level of PLIN3 and PLIN2 between the Group NS and Group NE.Compared with Group NS,the hepatic PLIN3 m RNA level of Group HS was significantly increased(P<0.05),while there was no significant difference in the m RNA level of PLIN2 between the two group.Compared with Group HS,the hepatic PLIN2 m RNA level of Group HE was significantly decreased(P<0.05),while there was no significant difference in the m RNA level of PLIN3 between the two group.(4)From the perspective of hepatic lipid droplet biogenesis.Compared with Group NS,the hepatic gene expression of DGAT2(P<0.05),CIDEA(P<0.01),FSP27(P<0.05)was significant increased in Group HS,while there was no significant difference in the gene expression of DGAT1,SEIPIN,FITM2 between the two group.Compared with Group NS,the m RNA level of SEIPIN was significantly decreased in Group NE.there was no significant difference in DGAT1,DGAT2,FITM2,FSP27,and CIDEA between the Group NS and Group NE.Compared with Group HS,the hepatic gene expression of CIDEA(P<0.01)and FSP27(P<0.01)was significantly decreased in Group HE,while there was no significant difference in the gene expression of other indicators between the two group.(5)From the perspective of hepatic lipid droplet biogenesis.Compared with Group NS,the protein levels of ATGL(P<0.05),LC3II/I(P<0.05),p62(P<0.05)and CTSD-pro/mat(P<0.01)in Group HS was significantly increased,the protein levels of LAMP2(P<0.05)and LAL(P<0.01)in Group HS was significantly decreased,while there was no significant difference in the protein levels of Beclin1,Atg5 and LAMP1 between the Group NS and Group HS.Compared with Group NS,the protein level of Atg5 was significantly increased in Group NE,while there was no significant difference in other indicators between the two group.Compared with Group HS,the protein levels of ATGL(P<0.01),p62(P<0.05)and CTSD-pro/mat(P<0.01)in Group HE was significantly decreased,the protein levels of Atg5(P<0.05),LAMP1(P<0.05),LAMP2(P<0.05)and LAL(P<0.01)in Group HE was significantly increased,while there was no significant difference in the protein levels of Beclin1 and LC3II/I between the Group HS and Group HE.The result of immunofluorescence shown that,compared with Group NS,the colocalization of hepatic LAMP1 and PLIN2 was significantly decreased in Group HS(P<0.01),and was significantly increased in Group NE(P<0.01).Compared with Group HS,the colocalization of hepatic LAMP1 and PLIN2 was significantly increased in Group HE(P<0.01).Conclusion:(1)15 weeks of HFD can lead to a NAFLD characterization in mice,mainly manifested as significant hepatic lipid accumulation,inflammation,bubble-like change,and fibrosis.HFD can increase the area and number of hepatic fat droplets,which is manifested as the increase of large size fat droplets and the decrease of small size fat droplets.The remodeling of lipid droplet population may be related to that HFD can increase the fusion and expansion of liver lipid droplet and inhibit the degradation of lipid droplet through autophagy.(2)15 weeks of treadmill training can significantly reduce liver steatosis,inflammation and bubble-like change in HFD mice,but it can not significantly reduce the degree of liver fibrosis.Exercise significantly reduced the area and number of hepatic lipid droplets,increased the number of small lipid droplets,and decreased the number of large lipid droplets in HFD mice.The mechanism may involve inhibiting the abnormal fusion and expansion of lipid droplets and promoting lipid droplets degradation through lysosome pathway.The remodeling of lipid droplet population by exercise enhances the turnover of lipid droplet formation and removal. |
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