Effects Of Hypoxia On Lipid Metabolism In L02Cell Line And Its Possible Mechanism

Background and ObjectiveWith the living standards improving rapidly, NAFLD has increasedrapidly year by year, which affects the people’s life seviously. It is knownthat people with Obesity, hyperlipemia,insulin resistance or type2diabetesare susceptive to NAFLD.However, recently,chronic liver injury duringObstructive Sleep Apnea has been reported,and the liver injury in OSAcould also be the result of direct hypoxia which could influence the lipidmetabolism of liver[1-2]. However, whether a causal link exists betweenhypoxia and NAFLD and its exact mechanism remain unclear. HIF-2is oneof the important components of the cellular oxygen-signalingpathway[4].We therefore decided to examine the effects of hypoxic exposureon the fatty degeneration,and to detect the expression ofSREBP-1,FAS,ADFP and HIF-2αin L02cell line to explore the possiblemechanism.Methods1. Cell culture: L02cells were cultured with RPMI1640mediumcontaining10%fetal bovine serum. 2.Experiment groups:Control group（under21%O₂）,Hypoxiagroup[3]（under1%O₂）.3. The hypoxia culture time was selected by MTT assay.4. The content of triglyceride and the accumulation of lipid droplets inthe hepatocytes were observed by biochemical assays and oil red O stainingrespectively.The expression of HIF-2α,SREBP-1mRNA andSREBP-1,FAS,HIF-2α,ADFP protein were detected by RT-PCR andWestern blot respectively.Results1. Steatosis models in L02cell were established successfully throughhypoxia exposure. Accompanied with time prolonging, steatosis hepatocytewas more aggravated.1) Hypoxic model: L02cells were cultured in a Forma3131incubatorwith1%O₂,5%CO₂,balance N2（94%N2） at37℃for12,24,48hoursrespectively.2) TG content: Compared with the control groups,the TG contentincreased significantly in hypoxia groups at24,48hours and also itincreased gradually accompanied with extension of hypoxia time （P<0.01）.3) Only small lipid droplets were observed in control groups. Therewere much more visible orange lipids accumulations in hypoxia groups.Integration with lipid drops could be observed along with the prolonging of the hypoxia eoposure in hypoxia groups.2. The expression of SREBP-1,FAS in groupsCompared with control group，the expression of SREBP-1mRNA、SREBP-1protein and FAS protein in hypoxia groups at12,24,48hour wasremarkably downregulated （P<0.01）.3. The expression of HIF-2α,ADFP in groupsWe can detect expression of HIF-2αmRNA in each group，but there isno significant difference among groups（P>0.05）.There was little or noexpression of HIF-2α on immunoblots under baseline conditions,whileafter continuous exposure of L02cells to1%O₂for6h it reached apeak,then decreased gradually.The expression of ADFP protein in hypoxiagroups at12,24and48h was higher than that of nomoxia group（allP<0.05）,it increased gradually accompanied with the prolonging of thehypoxia time（F=167.49,P<0.05）.Conclusion1. The development of steatosis in L02cells was not a consequence ofincreased lipid synthesis by way of SREBP-1-FAS.2. Exposoure to hypoxia might cause lipidosis via HIF-2-ADFP inL02cells.3. HIF-2α maybe a novel therapeutic target in fatty liver disease.