Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRPexpressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells. Eukaryotes store lipid in cytosolic lipid droplets, which consist of neutral lipid cores surrounded by phospholipid monolayers (1-3). In mammals, lipid droplets are most abundant in adipose tissue, where stored triacylglycerol (TAG) provides the primary energy reserve for the organism. Lipid droplets in steroidogenic cells contain cholesteryl esters used in the synthesis of steroid hormones. Most other mammalian cells contain smaller lipid droplets, whose function remains unclear. They may serve as local energy reserves or sources of lipid for membrane synthesis. Furthermore, they may protect cells from the harmful effects of excess lipid accumulation by sequestering toxic lipid species away from pathways that lead to cell death (4, 5).Mechanisms controlling the synthesis and turnover of lipid droplets are only partially understood. According to one model of lipid droplet biogenesis, newly synthesized neutral lipids accumulate inside the endoplasmic reticulum membrane, forming a disk that eventually buds into the cytoplasm surrounded by an endoplasmic reticulumderived phospholipid monolayer (2, 6). Conversely, lipid droplet turnover occurs via the hydrolysis of stored neutral lipids by cytosolic lipases. Much of what we know about the regulation of lipolysis stems from studies in adipocytes. In response to hormone stimulation, protein kinase A phosphorylates two key substrates: hormone-sensitive lipase (HSL) (7) and perilipins (8, 9). Phosphorylation of HSL stimulates both its activity and its association with lipid droplets, in a manner that depends on perilipins.Perilipins regulate TAG hydrolysis in two...

Lipid droplets (LDs), also called adiposomes, are found in many eukaryotic cells, and are highly upregulated in lipid-storage cells, such as adipocytes. The mechanism by which adiposomes and their component neutral lipids are degraded is an important health issue with the rapidly spreading epidemic of obesity. Recently, a novel triglyceride lipase (adipose triglyceride lipase (ATGL)) that catalyses the initial step in triglyceride hydrolysis in adipocyte LDs was identified. Here, we show that ATGL also functions in non-adipocyte cells, and has an important role in LD degradation in these cells. Overexpression of wild-type ATGL causes a marked decrease in LD size, whereas a catalytically inactive mutant retains the ability to localize to LDs, but is unable to decrease their size. Depletion of ATGL by RNA interference leads to a significant increase in the size of LDs. These results show that ATGL has an important role in LD/adiposome turnover in mammalian cells.

Members of the Patatin-like Phospholipase Domain containing Protein A (PNPLA) family play key roles in triglyceride hydrolysis, energy metabolism, and lipid droplet (LD) homoeostasis. Here we report the identification of two distinct LD targeting motifs (LTM) for PNPLA family members. Transient transfection of truncated versions of human adipose triglyceride lipase (ATGL, also known as PNPLA2), PNPLA3/adiponutrin, or PNPLA5 (GS2-like) fused to GFP revealed that the C-terminal third of these proteins contains sequences that are sufficient for targeting to LDs. Furthermore, fusing the C-termini of PNPLA3 or PNPLA5 confers LD localization to PNPLA4, which is otherwise cytoplasmic. Analyses of additional mutants in ATGL, PNPLA5, and Brummer Lipase, the Drosophila homolog of mammalian ATGL, identified two different types of LTMs. The first type, in PNPLA5 and Brummer lipase, is a set of loosely conserved basic residues, while the second type, in ATGL, is contained within a stretch of hydrophobic residues. These results show that even closely related members of the PNPLA family employ different molecular motifs to associate with LDs.

Despite the ubiquity of neutral lipid storage droplets and their importance in obesity, very little is known about how their synthesis and turnover are controlled. A major candidate for regulating these processes is adipocyte differentiation‐related protein (ADRP; also known as adipophilin). To determine the mechanism through which ADRP affects lipid storage, we stably expressed the protein in HEK 293 cells. ADRP was targeted to the surface of lipid droplets in 293 cells and increased triacylglycerol (TAG) mass under both unsupplemented and oleate‐supplemented conditions. At least part of the increased mass resulted from a 2‐fold decrease in the rate of TAG hydrolysis in ADRP‐expressing cells. Furthermore, ADRP expression induced a significant decrease in association of adipose triglyceride lipase (ATGL) with lipid droplets. Reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP‐expressing cells. This work was supported by a National Institutes of Health (NIH) Ruth L. Kirschstein National Research Service Award (1 F32 GM074453‐01, to L. L. L.), and NIH grant GM47897 (to D. A. B.).

Recently, a new family of cytoplasmic lipases named Patatin‐like Phospholipase Domain Containing Proteins (PNPLA), whose members include ATGL/PNPLA2, Adiponutrin/PNPLA3, and PNPLA5/GS2‐like, has been discovered to play a key role in the hydrolysis of stored triglycerides and regulation of lipid droplet (LD) metabolism. Our studies have focused on the role of PNPLA5 (a.k.a. GS2‐like) and its targeting to LDs. Using affinity‐purified antibodies, we found that PNPLA5 is present in many murine tissues, with highest expression in kidney and heart, and much less in adipose. However, PNPLA5 expression was induced >11‐fold during differentiation of NIH 3T3‐L1 cells to adipocytes. Expression of GFP‐labeled PNPLA5 in HeLa cells revealed that it was localized to LDs, along with ATGL/PNPLA2, ADRP, and TIP47. Importantly, over‐expression of PNPLA5 reduced the volume of LDs, with an associated decrease in stored triglycerides, comparable to that achieved by over‐expression of ATGL/PNPLA2. Interestingly, over‐expression of a catalytically inactive mutant version of PNPLA5 resulted in the generation of significantly enlarged, but fewer, LDs. Analysis of truncated and mutant versions of PNPLA5 revealed a four amino acid LD targeting motif (LTM) in the C‐terminal third of the protein, whose sequence is conserved among members of the PNPLA family. This work supported by Hatch Project NYC‐165415 and NIH grant DK51596 to WJB