Study On The Relationship Between Very Long Chain Fatty Acid Elongase And Lipotoxicity

When the accumulation of long-chain fatty acids excess the storage capacity of adiposetissue, lipotoxicity occurs as the pathological consequence of lipid overfow in non-adipose tissue,which may even lead to lipid-induced cell death (lipoapoptosis). Lipotoxicity is related to manymetabolic diseases. Very long-chain fatty acids elongases involve in FA fluxes, and are closelyinteracted with membrane lipid metabolism. However, the relationship between fatty acidselongases and lipotoxicity is unclear. The yeast Saccharomyces cerevisiae has three FA elongases(Elo1, Elo2and Elo3). Of the three yeast FA elongases, Elo2and Elo3are involved in theelongation of LCFA to VLCFA, and play essential roles in the regulation of various life activitiesand functions. The FA elongation reactions and responsible enzymes are conserved amongeukaryotes. Therefore, the yeast Saccharomyces cerevisiae is a good model to study the role ofVLCFA elongases in lipotoxicity.In this study, a colorimetric method for rapidly detecting neutral lipid content in yeast wasdeveloped firstly. Using the colorimetric method and thin layer chromatography (TLC) toanalyse lipids of wide-type and FA elongases mutants, we found the mutants contained a higheramount of neutral lipid than wild-type. After the treatment with0.5mM oleic acid, the neutrallipid content was increased significantly compared to untreated cells. Analysis of the componentsof total fatty acids using GC-MS showed that the C26species were absent in the elo3△strain,with the higher level of C20and C22. Sharply reduced levels of C26were found in the elo2△strain. In the wide-type strain, the proportion of VLCFAs increased in response to OLA, that wascontrary to elo2△and elo3△.The ratio of unsaturated to saturated FA of mutants in ELO2orELO3gene increased after the treatment with0.5mM OLA.The yeast strains lack of ELO2or ELO3gene were strikingly sensitive to oleic acid whilethere was no detectable change on the growth of strains deleted ELO1gene. The growthinhibition of elo2△and elo3△could be reduced by two popular antioxidant agents NAC and VC. The ROS level of mutants in ELO2or ELO3gene increased significantly compared tocontrols, respectively, after the treatment with0.5mM OLA for18h. The activities ofantioxidant enzyme increased in wide-type strain and elo1△in response to OLA. However, theactivities of CAT and SOD decreased in elo2△and elo3△when treated with0.5mM OLA,compared to the untreated controls, respectively. The intracellular reduced glutathione (GSH)concentration decreased after0.5mM OLA treatment. Analysis of lipid peroxidation and proteinoxidation showed increased level of MDA and PCO, especially in elo2△and elo3△, indicatingthat the OLA treatment caused oxidative damage. The elo2△and elo3△mutants weresusceptible to antifungal agent such as amphotericin B, with lower ergosterol content than WTstrain. By PI staining, the membrane permeability of wide type and elo mutants increasedsignifcantly after0.5mM OLA treated compared to untreated controls, respectively.Interestingly, mutants disrupted in ELO2or ELO3gene showed increased percentages of cellswith permeable membranes. The results showed that the lack of VLCFA elongases not onlycaused the modification of cellular fatty acids composition and reduced the resistance tooxidative damage induced by OLA, but also decreased the membrane ergosterol amount and themembrane stability.In addition, the high expression of the essential gene pool of Saccharomyces cerevisiae inelo3△was constructed. By screening the pool on plates with high concentration of oleic acid,some strains were found to be resistant to oleic acid stress. After sequencing of plasmid DNA, aseries of resistant genes was obtained, including replication factor, subunit involved in DNAreplication repair and transcriptional silencing, ATPase involved in the degradation ofubiquitinated substrates, ferredoxin involved in heme A biosynthesis and so on. The screeningconfirmed the key effect of oxditive stress on lipotoxicity. Meanwhile, it indicated the essentialroles of VLCFA elongases in maintaining the stability of membrane system and resisting to oleicacid stress. Further studies on the function of these resistant genes will need to be done to shedlight on the lipidtoxicity mechanism.