Intracellular lipid droplets (LDs) are found in a wide variety of cell types and have been recognized as organelles with unique spherical structures. Although LDs are not stable lipid-depots, they are active sites of neutral lipid metabolism, and comprise neutral lipid or cholesterol cores surrounded by phospholipid monolayers containing specialized proteins. However, sizes and protein compositions vary between cell and tissue types. Proteins of the perilipin family have been associated with surfaces of LDs and all carry a conserved 11-mer repeat motif. Accumulating evidence indicates that all perilipins are involved in LD formation and that all play roles in LD function under differing conditions. In this brief review, we summarize current knowledge of the roles of perilipins and lipid metabolizing enzymes in a variety of mammalian cell types.

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3-to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C.These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells. The appearance of cytosolic lipid droplets is observed in many cell types in physiological and pathological conditions. Adipocytes, the major lipid-storing cells in mammals, are full of lipid droplets. Foam cells in atherosclerotic lesions and parenchymal cells in fatty liver are lipid dropletcontaining cells associated with disease states. These cells accumulate massive amounts of cholesteryl ester and/or triacylglycerol (TG) as lipid droplets in their cytoplasmic space. Recently, we and others have shown that there is a distinct set of proteins specifically localized in lipid droplets (1-5), suggesting that the lipid droplet is an organized intracellular structure.Adipose differentiation-related protein (ADRP), also called adipophilin, is known to be a lipid droplet-associating protein. ADRP was originally found as a major protein induced in the early stages of adipocyte differentiation (6), although it is now known that ADRP is expressed in a variety of tissues and cells (7-13) in addition to macrophage-derived foam cells in atherosclerotic lesions (14). ADRP is the most abundant protein in lipid droplets in hepatic cells (3) and macrophages (Y. Fujimoto, et al., unpublished data).Expression of ADRP in macrophages during foam cell formation has been demonstrated by several studies. The gene-chip technique was used to show that ADRP is one of the most strongly induced genes in macrophages after incubation with oxidized LDL (OxLDL) (15). Addition of VLDL stimulated foam cell formation and ADRP mRNA expression in cultured macrophages (16,17). Expression of ADRP mRNA in human atherosclerotic lesions has been demonstrated by in situ hybridization experiments (14). Overexpression of the ADRP gene in cultured cells resulted in increased fatty a...

BackgroundOxidized phosphatidylcholines (oxPC) and lysophosphatidylcholine (lysoPC) generated during the formation of oxidized low-density lipoprotein (oxLDL) are involved in atherosclerotic lesion development. We investigated the time course-changes in phosphatidylcholine (PC) molecular species during oxidation of LDL to determine how those atherogenic PCs are produced.MethodsHuman and rabbit LDLs were pretreated with or without a selective platelet-activating factor acetylhydrolase (PAF-AH) inhibitor. LDL was oxidized by incubation with copper sulfate, and PC profiles were analyzed by liquid chromatography-tandem mass spectrometry.ResultsWhen human LDL was oxidized, the peak areas for polyunsaturated fatty acid (PUFA)-containing PC species dramatically decreased after a short lag period, concomitantly lysoPC species increased sharply. Although a variety of oxPC species containing oxidized fatty acyl groups or cleaved acyl chains are formed during LDL oxidation, only a few oxPC products accumulated in oxLDL: 1-palmitoyl-2-(9-oxo-nonanoyl) PC and long-chain oxPC with two double bonds. Pretreatment of LDL with the PAF-AH inhibitor greatly reduced lysoPC production while it had no effect on lipid peroxidation reactions and oxPC profiles. Rabbit LDL, which has a different composition of PC molecular species and needs a longer time to reach achieve full oxidation than human LDL, also accumulated lysoPC during oxidation. The increase in lysoPC in rabbit oxLDL was suppressed by pretreatment with the PAF-AH inhibitor. The major oxPC species formed in rabbit oxLDL were almost the same as human oxLDL.ConclusionsThese results suggest that lysoPC species are the major products and PAF-AH activity is crucial for lysoPC generation during oxidation of LDL. The oxPC species accumulated are limited when LDL is oxidized with copper ion in vitro.

Supplementary key words fatty liver • hepatocytes • HuH-7 • lipid droplets • NASH • phospholipase C • protein kinase A • sucrose density gradient centrifugationIntracellular lipid droplets (LD) are cellular organelles observed ubiquitously in physiological and pathological conditions. LDs are composed of a core of neutral lipids containing cholesteryl esters and/or triacylglycerol (TG), a surrounding surface monolayer of phospholipids, and several associated proteins. LDs are considered to be a storage site of neutral lipids and to play a role in wholebody energy homeostasis ( 1-3 ).A number of proteomic studies on LD in various cells have revealed the unique protein profi les of LD ( 4-8 ). A Abstract Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-defi cient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage. -Takahashi, K., N. Sasabe, K. Ohshima, K. Kitazato, R. Kato, Y. Masuda, M. Tsurumaki, T. Obama, S-i. Okudaira, J. Aoki, H. Arai, T. Yamaguchi, and H. Itabe. MEXT, 2005MEXT, -2009 Abbreviations: Ab, antibody; ADRP, adipose differentiation-related protein; ER, endoplasmic reticulum; GRP78/BiP, glucose-regulated protein of 78-kDa/binding protein; iLD, intermediate-density lipid droplet; LD, lipid droplet; MCD, methione/choline defi cient; MTP, microsomal triacylglycerol transfer protein; NASH; nonalcoholic steatohepatitis; OA, oleic acid; PAT, perilipin-ADRP-TIP47; PLP, 4% paraformaldehydelysine-sodium periodate; PC, phosphatidylcholine; PKA, protein kinase A; PKI, PKI14-22amide; PLC, phospholipase C; PNS, post-nuclear supernatant; PPAR, peroxisome proliferator-activated receptor; Rp, Rp-8-Br-cAMPS; SCD...

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