Potential Mechanism Of Lipid Droplets In Protecting Cells Against Oxidative Stress By Regulating Microfliament Remodeling And Mitochondria Activity

Lipids obtained by cells from the outside or produced by themselves can be stored in lipid droplets(LDs),which is reported to be the important site for lipid metabolism.LDs are approximately spherical organelles composed of a neutral lipid core and a single layer of phospholipid membrane on the surface,and many related proteins are localized to the surface.LDs can interact with a variety of organelles and participate in a variety of cellular processes.In addition,LDs move rapidly in the cytoplasm and are a highly dynamic organelle.Studying LD function is great significant and valuable in investigating the mechanism of intramuscular fat metabolism.However,the function of LDs in cells and their molecular mechanisms involved in various cellular processes have not been fully clarified.This study found that cells with higher LDs can resist oxidative stress and apoptosis induced by hydrogen peroxide.LD-related protein mass spectrometry detection revealed that LDs participate in cytoskeleton and mitochondrial-related biological processes.Furthermore,several methods were utilized includinglabelling and isolation of subcellular organelles,Laser confocal microscopy observation,immunoprecipitation combined mass spectrometry analysis,co-immunoprecipitation combined Western Blot detection,signaling pathway analysis,real-time fluorescence quantitative detection and live cell workstation observation.The result revealed that surface protein Actinin(ACTN)and Perilipin5(PLIN5)of LDs regulate cellular microfilament remodeling and mitochondrial activity,thereby regulating cellular oxidative stress.In this study,the molecular mechanism of LDs regulating cell oxidative stress was investigated from the perspective of organelle interactions.In particular,the regulatory role of LDs on cytoskeleton-related biological process was discovered and the molecular mechanism was investigated.The novelevidences and ideas would promote subsequent studies on lipid metabolism in pigs.The main findings are as follows: 1.Lipid droplets regulate the remodeling of cellular microfilament skeleton and maintain its stability,thereby resisting oxidative stress.(1)Lipid droplets maintain the microfilament structure of porcine skeletal muscle satellite cells under oxidative stress.Porcine skeletal muscle satellite cells(PMSCs)were incubated with oleic acid to increase lipid droplet content.Hydrogen peroxide stimulation and phase contrast microscopy observations revealed that cells with high lipid droplet content maintained good morphology and had fewer dead cells.The microfilaments of cells were found to be more intact after being labeled with microdroplets.(2)Lipid droplets regulate cell microfilament remodeling.In mouse myoblasts C2C12,after cytochalasin B was used to destroy the microfilament skeleton structure,the number of lipid droplets increased during the process of microfilament remodeling,and the cell microfilament skeleton with higher lipid droplets remodeled more quickly.fast.ACTN3 was transferred to the microfilament during the remodeling of the microfilament skeleton.Microfilaments were distributed and co-localized with lipid droplets,and the level of ACTN3 on the surface of lipid droplets decreased during the remodeling of the microfilament skeleton.Next,we constructed a lipid droplet targeting ACTN3 fluorescent labeling carrier.Using laser confocal microscopy,we found that ACTN3 on the surface of the lipid droplets was transferred to the microfilaments during the remodeling of the microfilament skeleton,and the immunoblotting detection after subcellular organelle separation also supported the fluorescence observation results.(3)ACTN3 is recruited on the surface of lipid droplets by binding lipid droplet proteins ACSL3 and LPCAT1.We used immunoprecipitation combined with mass spectrometry to screen the lipid droplet proteins Acyl-Co A Synthetase Long Chain Family Member 3(ACSL3)and Lysophosphatidylcholine Acyltransferase 1(LPCAT1)in combination with ACTN3,and further detected the Spectrin repeat sequence of Actinin(SR domain)binds to these two lipid droplet proteins,and interfering with ACSL3 and LPCAT1 significantly reduces ACTN3 levels on the lipid droplet surface.In addition,we also found that lipid droplets delayed ACTN3 degradation through protein half-life tests.(4)ARF1 membrane vesicle transport system mediates the transport of ACTN3 protein to microfilaments on the surface of lipid droplets.We further analyzed the co-localization of lipid droplets and ARF1-COPI membrane vesicles and found that the contact between lipid droplets and membrane vesicles increased after disrupting the microfilament skeleton structure.Interfering with ARF1 and simultaneous treatment with Brefeldin A significantly inhibited the number of ARF1-COPI membrane vesicles,resulting in During the remodeling of the microfilament skeleton,the transfer of ACTN3 to the microfilament on the lipid droplet surface was inhibited.(5)Lipid droplets promote myoblast differentiation by enhancing cell migration and fusion.Microfilament remodeling occurred during the differentiation of C2C12 cells,ACTN3 on the surface of lipid droplets was transferred to the microfilaments during differentiation,and overexpression of ACTN3 targeted by lipid droplets could also promote muscle cell differentiation.Lipid droplets promote the expression of My HC and the formation of multinucleated myotubes.The q PCR results showed that lipid droplets promoted the expression of the cell fusion genes Myomaker and Caveolin,and live cell workstation observations revealed that cells with more lipid droplets formed more filamentous,spine-like,and sheet-like pseudopods,and had stronger cell migration capabilities.(6)The stability of the microfilament skeleton affects the level of intracellular oxidative stress.After treatment with cytochalasin,ROS levels increased,and oxidative stress increased;while JASP,a cytoskeleton stabilizer,could resist oxidative stress induced by hydrogen peroxide.2.The lipid droplet protein PLIN5 resists oxidative stress by enhancing mitochondrial activity.(1)Lipid droplets reduce mitochondrial damage of PMSCs under oxidative stress.In the aforementioned PMSC test,flow cytometry showed that cells with high lipid droplet content had higher mitochondrial membrane potentials,lower ROS levels,and lower apoptotic rates.(2)PLIN5 expression regulates ROS levels in cells.PLIN5 expression was up-regulated after treatment with lipopolysaccharide or hydrogen peroxide,and intracellular ROS levels decreased after PLIN5 was overexpressed in cells.In addition,overexpression of PLIN5 could resist the increase of ROS levels induced by hydrogen peroxide.Interference with PLIN5 has the opposite effect.(3)PLIN5 regulates mitochondrial activity.Overexpression of PLIN5 can inhibit the release of mitochondrial cytochrome c and resist the reduction of mitochondrial membrane potential induced by hydrogen peroxide.Interference with PLIN5 has the opposite effect.(4)PLIN5 regulates the expression of mitochondrial functional genes.By detecting the expression level of mitochondrial activity-related genes,it was found that overexpression of PLIN5 can up-regulate the expression of mitochondrial respiratory enzyme genes COX,CS,etc.,and regulate the expression of antioxidant genes in some cells.Interference with PLIN5 has the opposite effect.(5)The expression of PLIN5 under oxidative stress is regulated by the JNK-p38-ATF signaling pathway.Further analysis of the upstream signaling pathway that regulates the expression level of PLIN5 revealed that hydrogen peroxide treatment can activate JNK-p38 signal,and downstream ATF expression was activated accordingly,while ATF1,ATF3 and ATF4 can be combined with PLIN5 promoter region to activate PLIN5 transcription.The pathway inhibitor GS-4997 can inhibit the up-regulation of hydrogen peroxide-induced PLIN5 expression.(6)Regulation of PLIN5 on lipid droplets and mitochondria under oxidative stress.It was found by fluorescent labeling that PLIN5 can promote the formation of lipid droplets,and the increased expression of PLIN5 mediated by hydrogen oxide treatment can promote the contact between lipid droplets and mitochondria.(7)Effect of PLIN5 on oxidative stress-related diseases.PLIN5 is up-regulated in non-alcoholic fatty liver tissue of mice.In addition,low expression of PLIN5 is associated with poor prognosis in some cancers.The above results prove that LDs play an important role in the regulation of cellular oxidative stress,can promote the remodeling of microfilament skeleton and maintain the stability of cell morphology,and promote mitochondrial activity,reduce mitochondrial damage and reduce ROS levels.LDs reduce cell oxidative stress damage and promote cell survival.