The Mechanisms Of Tumor Necrosis Factor ?-induced Fat Accumulation In Hepatocytes

ObjectiveAlcoholic liver disease(ALD)is a progressively aggaravated liver disease induced by excessive ethanol intake,which compose a spectrum of liver disease ranging from alcoholic fatty liver(AFL),alcoholic hepatitis(steatohepatitis),liver fibrosis and cirrhosis,and the hepatocellular carcinoma(HCC).Alcoholic fatty liver disease is usually reversible when the individual concerned abstains from alcohol consumption,is the optimal stage for the intervention of ALD.The onset of ALD is complex and both the disturbance of hepatic fat metabolism and extrahepatic adipose fat mobilization may be involved.Recent studies have suggested that M1-polarized Kupffer cells(KCs),the resident macrophages in the liver,play an important role in the development of ALD.Targeting the polarization of KCs is considered as a promising research direction for the prevention and treatment of ALD.TNF-?,an improtant pro-inflammatory cytokine released by M1 Kupffer cells,is well known to be involved in the inflammatory response,while its roles in the pathogenesis of AFL remains to be studied.Our previous study revealled that KCs inhibitor(GdCl3)and TNF-? antagonist(etanercept)could reduce hepatocyte fat accumulation caused by acute alcohol exposure,which might be attributed to the suppression of mobilization of white adipose tissues by TNF-? mediated activation of lipolysis.In addition to mobilization of white adipose tissues,TNF-? may also regulate fatty acid synthesis and catabolism in hepatocytes.Thus,in this study,in vivo experiments and in vitro experiments were used to investigate the effect of TNF-? on hepatic triglyceride(TG)accumulation and to explore its possible mechanism.In the in vivo experiment,male ICR mice were exposed to TNF-a with or without an lipolytic inhibitor,phenylisopropyl adenosine(PIA),and then sacrificed at 1.5,3 h,6 h,and 24 h time points.The levels of hepatic TG and protein levels of sterol regulatory element-binding protein 1c(SREBP-1c)and peroxisome proliferators-activated receptor a(PPAR-?)in liver,and the porotien levels of perilipin,adipose triglyceride lipase(ATGL),hormone sensitive lipase(HSL)were detected.In the in vitro experiment,human hepaotcarcinoma celline HepG2 were exposed to different doses of TNF-a,and then the protein levels of sterol regulatory element-binding protein 1c(SREBP-1c)and peroxisome proliferators-activated receptor a(PPAR-?)were detected.The results of our study will be helpful to illustrate the molecular mechansims of AFL,and thus provide potential molecular targets for the prevention and treatment of ALD.Methods1.In vivo experimentSPF male ICR mice(20 ± 2 g),were maintained on a 12-h light/dark cycle at constant temperature(22 ± 2?)and humidity(40%-70%).After 3 days adaptive feeding,mice were randomly divided into 4 groups:control group,TNF-? group,lipolytic inhibitor phenylisopropyl adenosine(PIA)group,and TNF-? + PIA group(n=24).TNF-a was injected by i.p.at 0.166 mg/kg bw to mice in TNF-? and TNF-? +PIA groups,while the mice in control and PIA groups received the same volume of saline at the same time of day.Where indicated,animals were given intraperitoneal injection of 0.15 ?mol/kg bw PIA in saline or an equal volume of saline alone(control)30 min before TNF-a administration.Six animals were sacrificed at 1.5 h,3 h,6 h and 24 h after administration.Blood was collected from the orbital vein,and the liver and adipose tissue samples were dissected for subsequent experiments.Oil red O staining was performed to observe the accumulation of fat in frozen section of liver;TG was detected by triglyceride kit;free fatty acid was detected by free fatty acid detection kit;glycogen accumulation in liver cells was observed by glycogen periodic acid Schiff(PAS)staining;The levels of triglyceridea,lanine transaminase(ALT),aspartate transaminase(AST)were detected by automated biochemical analyzers.The proteins levels involved in fatty acid metabolism were detected by Western blotting methods.2.In vitro experimentsThe human hepatocellular carcinoma cell line HepG2 was routinely cultured in DMEM high glucose medium with 10%fetal bovine serum(FBS)and 1%penicillin and streptomycin in an incubator under an atmosphere of 5%CO2 at 37?.The cells starvated for 12 hours in 0.2%bovine serum albumin(BSA)-DMEM,and then exposed to vaious concentration of TNF-?(0.5 h,1 h,3 h,6 h,12 h,20ng/ml)for different times..The cytotoxicity of TNF-a on HepG2 cells was detected by CCK-8 assay kits.Cell slides were prepared and oil red O staining was used to observe the accumulation of fat in cells.TG kit was used to detect intracellular TG levels.The proteins levels involved in fatty acid metabolism were detected by Western blotting methods.Results1.Effect of TNF-ca treatment on Liver injury in miceAfter a single dose TNF-ca injection in ICR mice,compared with the control group,the activity of ALT in the serum of TNF-a treated group were significantly increased at both the 3 h and 6 h time points(P<0.05),the activity of AST in the serum of TNF-?treated group were significantly increased at 3 h time point(P<0.05).The ratio of AST/ALT decreased significantly at both 3 h and 6 h time points(P<0.05).The lipolysis inhibitor PIA had no significant effect on serum transaminase activity.2.Effect of TNF-a treatment on hepatic triglyceride accumulationAt 3 h and 6 h after a single dose TNF-a treatment,the liver coefficient of mice treated with TNF-a significantly was increased compared with the control mice(P<0.05).Compared with the control group,the concentration of serum FFA in TNF-a treated group was significantly increased at 3 h(P<0.05).Compared with the control group,the contents of triglyceride in the liver and serum triglyceride of TNF-a treated group were significantly increased at 6 h(P<0.05).Compared with the mice in the TNF-a treated group,the contents of liver triglyceride in TNF-? + PIA group were significantly decreased(P<0.05).At four time points,lipid droplets of the liver section in each group were not significantly altered.3.Effect of TNF-a treatment on SREBP-lc regulated fat synthesis pathway in liverCompared with the control group,the protein levels of precursor and mature of liver SREBP-lc in the TNF-a treated group showed a decreased trend at 1.5 h,3 h,and 6 h after a single dose TNF-? treatment.Compared with the TNF-? exposure group,the protein levels of precursor and mature of liver SREBP-lc in the in the liver of TNF-a+PIA treated group mice was not found significant changes(P>0.05)at 1.5 h,3 h,6 h,and 24 h after TNF-? exposure.No significant changes of FAS protein levels were found in TNF-? treated group at four time points after TNF-? treatment.4.Effect of TNF-? treatment on PPAR-a regulated fatty acid oxidative metabolism pathway in liverCompared with the control group,the protein levels of the liver PPAR-a in the TNF-a treated group were slightly decreased at 1.5 h,3 h,6 h,24 h after a single dose TNF-a treatment(P>0.05).Compared with the TNF-?exposure group,the protein levels of the liver PPAR-a in the TNF-? +PIA treated group were increased at 1.5 h after a single dose TNF-? treatment(P<0.05).Compared with the control group,the protein levels of the liver ACOX1 in the TNF-? treated group were slightly decreased at 1.5 h,but there was not significantly changes.5.Effect of TNF-? treatment on fat mobilization in epididymal adipose tissueCompared with the control group,the protein levels of perilipin of the epididymal adipose tissue in the TNF-? treated group were significantly decreased at 1.5 h after a single dose TNF-? treatment(P<0.05);the protein levels of ATGL of the epididymal adipose tissue in the TNF-? treated group were significantly increased at 3 h after a single dose TNF-? treatment(P<0.05).Compared with the TNF-? treated group,the proteins levels of perilipin of the epididymal adipose tissue in the TNF-? + PIA treated group showed a trend of increase at 1.5 h after TNF-? treatment,but there was no significant difference(P>0.05);the proteins levels of ATGL in the TNF-? + PIA treated group showed a trend of decrease at 3 h after TNF-a treatment,but there was no significant difference(P>0.05).Compared with the control group,the protein levels of p-HSL(Ser563)of the epididymal adipose tissue in the TNF-? treated group were significantly increased at 3 h and decreased at 6 h and 24 h after a single dose TNF-a treatment(P<0.05);the protein levels of p-HSL(Ser565)in the TNF-a treated group were significantly increased at both the 3 h and 6 h after a single dose TNF-a treatment(P<0.05);the protein levels of p-HSL(Ser660)in the TNF-a treated group were slightly increased at both the 1.5 h and 3 h after a single dose TNF-a treatment,but there was not significantly changes.Compared with the TNF-a treated group,the proteins levels of p-HSL(Ser563),p-HSL(Ser565)and p-HSL(Ser660)in the TNF-? + PIA group had no significantly change at these time points after a single dose TNF-? treatment(P>0.05).6.Cytotoxicity of TNF-a treatment on HepG2 cellsThe cytotoxicity of TNF-a on HepG2 cells was determined by CCK-8 assay.The viability of HepG2 cells in each group exposed different dose TNF-a was above 90%(P>0.05),and cell activity was not significantly affected.7.Effect of TNF-a treatment on triglyceride accumulation in HepG2 cellsHepG2 cells treated with different doses of TNF-a for 12 hours showed no significant changes in triglyceride content.After treated with different doses of TNF-?for 24 hours and 48 hours,no significant changes were observed in HepG2 cells.8.Effect of TNF-a treatment on SREBP-lc regulated fat synthesis pathway in HepG2 CellsTime experiment:Compared with the control group,the protein levels of mature SREBP-lc in HepG2 cells were significantly increased at 1 h after exposed TNF-a(P<0.05),the protein levels of FAS and ACC regulated by SREBP-1c showed an significant increasing trend at 6 hours after exposed TNF-a(P<0.05).Dose experiment:Compared with the control group,the protein levels of mature SREBP-lc in HepG2 cells in the TNF-a treated group were significantly increased after a dose of 20 ng/ml and 40 ng/ml TNF-? treatment(P<0.05).Compared with the control group,the protein levels of downstream proteins FAS regulated by SREBP_lc showed a trend of increase after a dose of 40 ng/ml TNF-a treatment(P<0.05),the protein levels of downstream proteins ACC were significantly increased after TNF-a treatment at dose 20 ng/ml and 40 ng/ml(P<0.05).9.Effect of TNF-a on PPAR-a regulated fatty acid oxidative metabolism pathway in HepG2 cellsCompared with the control group,the protein levels of PPAR-a expression showed a trend of decrease after TNF-a treatment,but there was no significant difference(P>0.05).There was no significant difference in the protein levels of ACOX1 expression in HepG2 cells treated with TNF-a(P>0.05).Conclusion1.TNF-?-induced fat accumulation in mouse hepatocytes may be mainly attributed to the extrahepatic lipolysis:TNF-? may enhance the lipid hydrolysis of peripheral adipose tissue by promoting the phosphorylation of HSL,and then promote the accumulation of triglyceride in mouse liver.2.TNF-a can up-regulate SREBP-lc regulated proteins and promote de novo synthesis of fatty acids in HepG2 cells.