Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies ( E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44–57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function.

(LD) in lipid metabolism, cell signaling, and membrane trafficking is increasingly recognized, yet the role of the LD phospholipid monolayer in LD protein targeting and function remains unknown. To begin to address this issue, two populations of LD were isolated by ConA sepharose affinity chromatography: 1) functionally active LD enriched in perilipin, caveolin-1, and several lipolytic proteins, including ATGL and HSL; and 2) LD enriched in ADRP and TIP47 that contained little to no lipase activity. Coimmunoprecipitation experiments confirmed the close association of caveolin and perilipin and lack of interaction between caveolin and ADRP, in keeping with the separation observed with the ConA procedure. The phospholipid monolayer structure was evaluated to reveal that the perilipin-enriched LD exhibited increased rigidity (less fluidity), as shown by increased cholesterol/phospholipid, Sat/Unsat, and Sat/ MUFA ratios. These results were confirmed by DPH-TMA, NBDcholesterol, and NBD-sphingomyelin fluorescence polarization studies. By structure and organization, the perilipin-enriched LD most closely resembled the adipocyte PM. In contrast, the ADRP/TIP47-enriched LD contained a more fluid monolayer membrane, reflecting decreased polarizations and lipid order based on phospholipid fatty acid analysis. Taken together, results indicate that perilipin and associated lipolytic enzymes target areas in the phospholipid monolayer that are highly organized and rigid, similar in structure to localized areas of the PM where cholesterol and fatty acid uptake and efflux occur. lipolysis; caveolae; caveolin; concanavalin-A; adipose differentiationrelated protein IT IS INCREASINGLY CLEAR THAT LIPID DROPLETS (LD) are dynamic organelles with regulatory roles in many cellular processes besides lipid metabolism, including cell signaling, immune function, membrane trafficking, and regulation of longevity (42,70). Whereas the mechanism of these processes is only partially understood, much less is known about how the structure of the LD phospholipid monolayer may affect LD protein targeting and function. Embedded in the monolayer that surrounds the LD neutral lipid (NL) core are proteins, such as perilipins (65) and adipose differentiation-related protein (ADRP) (16,41), that coat the LD surface and lipids such as cholesterol (4, 80) that help to define the LD structure. Perilipin and ADRP are two members of the PAT [perilipin, ADRP, tail-interacting protein 47 (TIP47)] family from the Plin group of genes (56) that were initially thought to act simply as barriers to protect the NL core against lipolytic action (63, 65). Current evidence suggests a more complex mechanism, one that requires coordination of perilipin with several lipases, including adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and the activator molecule comparative gene identification-58 (CGI-58), a protein with no lipolytic activity that increases ATGL activity to promote triacylglyerol hydrolysis (11,14,44). Perilipin and HSL upon hormonal stimu...

Although a link between excess lipid storage and aberrant glucose metabolism has been recognized for many years, little is known what role lipid storage droplets and associated proteins such as Plin2 play in managing cellular glucose levels. To address this issue, the influence of Plin2 on glucose uptake was examined using 2-NBD-Glucose and [3H]-2-deoxyglucose to show that insulin-mediated glucose uptake was decreased 1.7- and 1.8-fold, respectively in L cell fibroblasts overexpressing Plin2. Conversely, suppression of Plin2 levels by RNAi-mediated knockdown increased 2-NBD-Glucose uptake several fold in transfected L cells and differentiated 3T3-L1 cells. The effect of Plin2 expression on proteins involved in glucose uptake and transport was also examined. Expression of the SNARE protein SNAP23 was increased 1.6-fold while levels of syntaxin-5 were decreased 1.7-fold in Plin2 overexpression cells with no significant changes observed in lipid droplet associated proteins Plin1 or FSP27 or with the insulin receptor, GLUT1, or VAMP4. FRET experiments revealed a close proximity of Plin2 to SNAP23 on lipid droplets to within an intramolecular distance of 51 Å. The extent of targeting of SNAP23 to lipid droplets was determined by co-localization and co-immunoprecipitation experiments to show increased partitioning of SNAP23 to lipid droplets when Plin2 was overexpressed. Taken together, these results suggest that Plin2 inhibits glucose uptake by interacting with, and regulating cellular targeting of SNAP23 to lipid droplets. In summary, the current study for the first time provides direct evidence for the role of Plin2 in mediating cellular glucose uptake.

Although ADRP (adipose differentiation‐related protein, also known as perilipin 2) has been shown to bind fatty acids (FA) and other lipids with high affinity, the structure and location of the FA binding site has not been examined in detail. This is of some importance since ADRP's affinity for lipids and especially FA may be related to lipid droplet formation, triacylglycerol accumulation, and FA uptake and metabolism in the cell. The purpose of the present investigation was to characterize ADRP and several truncated mutants using fluorescence binding assays and circular dichroism (CD) to assess the structural requirements for binding. Results presented here show that removal of 120 amino acids from the N‐terminus, a high homology region common to other lipid droplet proteins increased the FA binding affinity (Kd) 43%. In contrast, deleting 254 residues decreased the Kd 24%. Removal of 173 amino acids from the C‐terminus, however did not significantly change the protein's ability to bind FA. Conformational changes upon binding were confirmed by CD. Taken together, these results indicate that residues 121–254 appear essential for highest affinity binding and show that the ability of ADRP to bind FA may depend on the proper folding of several domains, the partial deletion of which leads to altered, but not ablated ligand binding. This work was supported by NIH Grant DK70965 (BPA).