Mechanism Of Nuclear Lipid Droplets Recruiting A-KGDH Complex To Promote Phospholipid Synthesis

Intramuscular fat is one of the important economic traits in pigs,and lipids in adipocytes are stored in organelles called lipid droplets.Lipid droplets are composed of monolayer phospholipids,neutral lipids and surface proteins,which are highly conserved in different species,tissues and cells.Lipid droplets are not only responsible for the storage of cellular lipids,but also regulate the synthesis,decomposition and transport of cellular lipids.They are the center of cellular lipid metabolism and have important research value in the field of cellular lipid metabolism.Recent studies have found that there are lipid droplets in the nucleus,which have the same structure as cytoplasmic lipid droplets,but their function is not clear.This study found that blocking the pathway of cellular phospholipid synthesis led to the accumulation of lipid droplets in the nucleus.By extracting the surface protein of lipid droplets in the nucleus for mass spectrometry analysis,and using immunoprecipitation,laser confocal microscopy,realtime fluorescence quantification,Western blotting and other technologies,it revealed the molecular mechanism of lipid droplets in the nucleus regulating the expression of genes related to cellular phospholipid synthesis by regulating the level of histone succinylation.The main results are as follows:(1)Excess fatty acids promote the formation of lipid droplets in the nucleus.Treatment of cells with concentration and time gradients of oleic acid(unsaturated fatty acid)significantly increased the number of lipid droplets in the nucleus.(2)Blocking the phospholipid synthesis pathway leads to the accumulation of lipid droplets in the nucleus.Using choline-deficient medium or exogenous addition of miltefosine(a phosphatidylcholine transferase inhibitor)to block the cellular phospholipid synthesis pathway,respectively,significantly increased the number of lipid droplets in the nucleus.(3)Α-KGDH complex is recruited on the surface of lipid droplets in the nucleus.Cytoplasmic lipid droplets and nuclear lipid droplets were separated,and surface proteins were extracted for mass spectrometry.It was found that compared with cytosolic lipid droplets,α-KGDH complexes were recruited on the surface of nuclear lipid droplets,which consisted of OGDH,DLD and DLST.It is involved in the synthesis of succinyl-Co A.(4)DLD mediates the recruitment of α-KGDH complex on the surface of nuclear lipid droplets.Through interference,overexpression and protein truncation experiments,it was found that DLD in OGDH,DLD and DLST has lipid droplet targeting ability and mediates the localization of α-KGDH complex on the surface of lipid droplets in the nucleus.(5)Intranuclear lipid droplets regulate the level of histone succinylation.By inducing and inhibiting the formation of nuclear lipid droplets,extracting cellular histones,and using kucc-pan antibody to detect the level of histone succinylation modification,the results show that the increase of nuclear lipid droplets leads to the decrease of histone succinylation level.As detected by co-immunoprecipitation assay,it was found that increased lipid droplets in the nucleus lead to a decrease in the level of α-KGDH complex bound to histones,thereby reducing the level of succinylation.(6)Histone succinylation inhibits the expression of phospholipid synthesis-related genes.Through interference and overexpression of α-KGDH,KAT2 A and other genes,the level of histone succinylation was activated or inhibited,and the expression of phospholipid synthesis-related genes PCYT1 A,LPCAT,FASN,etc.was detected by real-time fluorescence quantification and western blotting,and it was found that histone amber elevated levels of acylation lead to decreased expression levels of these genes.(7)Intranuclear lipid droplets regulate cellular oxidative stress.The oxidative stress of cells was induced by exogenous addition of hydrogen peroxide,and the levels of ROS and DNA damage were detected.The results showed that cells with more intranuclear lipid droplets had decreased ROS levels and reduced DNA damage.Based on the above test results,the increase of lipid droplets in the nucleus recruits the α-KGDH complex in the nucleus,reduces its binding to histones,reduces the level of histone succinylation modification,and then promotes the expression of phospholipid synthesis-related genes and maintains lipid stability.state and reduce the level of cellular oxidative stress.This study analyzed the function and mechanism of lipid droplets in the nucleus,and provided new clues and theoretical basis for the mechanism of cellular lipid metabolism.