The Role And Mechanism Of LSDP5in Lipid Droplet And Lipid Metabolism Of Liver

Background：Liver steatosis or fatty liver which is believed to be caused by abnormalmetabolism of fats is one of the most common metabolic disorders in China.Nearly Chinese20-30%people have fatty livers. The typical pathological featureof liver steatosis is the lipid deposition in liver cells in the form of lipid droplets(LDs). With regard to etiology analysis, the researches on metabolism andformation of lipid droplets are involved little. Lipid droplets are neutral lipidstorage surrounded by a phospholipid monolayer, which are now recognized tobe functional subcellular organelles rather than metabolically inactive lipiddepots. They are involved in multiple intracellular processes including lipidmetabolism, vesicle traffic, and cell signaling through interactions with otherorganelles so that important roles in lipid homeostasis are likely. In mammaliancells, LDs contain a class of proteins in their surface layers that contribute to thelipolysis, synthesis and fusion of LDs and share a homologous sequence calledthe PAT domain, including perilipin, adipophilin/adipose differentiation-related protein (ADRP), a tail-interacting protein of47kDa (TIP47), and S3-12. Lipidstorage droplet protein5(LSDP5) is a newly identified member of PAT family. Initialidentifications and characterizations of LSDP5as a lipid droplet binding protein werereported by three independent groups as myocardial lipid droplet protein (MLDP),oxidative tissue-enriched PAT protein (OXPAT), and LSDP5respectively. All thesereports are in agreement that LSDP5is expressed in tissues that exhibit high levels offatty acid oxidation, including heart, skeletal muscle, and liver. However, as a newmember of PAT family, the exact function of LSDP5has not been elucidated so far.Objective:We initiate the current study to determine the subcellular localization ofLSDP5protein and to further characterize the LD-localization signal sequenceswithin LSDP5. We investigate the effects of LSDP5on the numbers and sizes ofLD, synthesis and hydrolysis of triglycerides (TG). We indentify theLSDP5-interacting proteins and facilitate the illustration of the molecularmechanism of LSDP5.Methods:1) Immunofluorescence was used to detect the the distribution and subcellularlocalization of LSDP5protein.2) The biological functions of LSDP5were observed by upregulating anddownregulating the expression of LSDP5in hepatic cell line AML12andprimary mouse hepatocytes.3) The interacting-proteins of LSDP5were screened through the mouse livercDNA library with the prey protein LSDP5using yeast two-hybrid.Co-immunoprecipitation and subcellular localization of LSDP5and the interacting-protein were employed to confirm the specificity of theinteractions.Results:1) LSDP5protein was located in the surface of lipid droplets.By using immunofluorescence assay, we observed that LSDP5was localizedto the surface of lipid droplets in hepatocytes. Serial deletion analyses showedthat the lipid droplet targeting domain and the domain directing lipid dropletclustering were overlapped and localized to the188amino acid residues at theN-terminal region of LSDP5.2) LSDP5enhances triglyceride storage in hepatocytes.Overexpression of LSDP5could enhance lipid accumulation in primaryhepatocytes and hepatic cell line AML12. Moreover, knock-down of LSDP5significantly decreased the size of lipid droplets and reduced the contents oftriglyceride.3) LSDP5could inhibit lipolysis and interacted with esterase1(ES1).Knock-down of LSDP5significantly stimulated lipolysis in cells, and furtherincreased fatty acid β-oxidation and mitochondrial number. Real-time PCRanalysis revealed that the depletion of LSDP5was associated with increasedmRNA level of enzymes in lipolysis and fatty acid oxidation. In addition, weperformed yeast two-hybrid using LSDP5as a bait protein. Among all thesepositive clones we got, ES1was choosed for the further investigation. ES1isa65-kDa glycoprotein present in liver able to hydrolyze a variety of estersincluding triglycerides, cholesteryl esters and fatty acid esters.Co-immunoprecipitation and lmmunofluorescent co-localization furtherlyproved that LSDP5and ES1possessed physical interactions. ConclusionsOur data clearly demonstrate that LSDP5, a novel lipid droplet protein,contributes to triglyceride accumulation by negatively regulating lipolysis andfatty acid oxidation in hepatocytes may be through mechanisms of the interactionof LSDP5and ES1. This study makes an experimental foundation for the role andmechanism of LSDP5in lipid metabolism and may provide new thinking for thetreatment of fatty liver.