Objectives-Adipocyte differentiation-related protein (ADRP)-containing lipid droplets have an essential role in the development of insulin resistance and atherosclerosis. Such droplets form in a cell-free system with a diameter of 0.1 to 0.4 m, while the droplets present in cells vary in size, from small to very large, suggesting that the droplets can increase in size after being assembled. We have addressed this possibility. Methods and Results-Experiments in NIH 3T3 cells demonstrated that the lipid droplets could increase in size independentlyof triglyceride biosynthesis. NIH 3T3 cells were either microinjected with ADRP-GFP (green fluorescent protein) or stained with Nile Red and followed by confocal microscopy and time-lapse recordings. The results showed that lipid droplets formed complexes with each other, with a volume equal to the sum of the merging particles. The formation of complexes could be inhibited by the nocodazole-induced depolymerization of the microtubules; thus, the process is dependent on microtubules.The presence of dynein on ADRP-containing droplets supports a role for this motor protein. Key Words: lipid droplets Ⅲ lipid droplet fusion Ⅲ adipocyte differentiation protein Ⅲ microtubules C ytosolic lipid droplets are organelles involved in the storage and turnover of triglycerides and cholesterol esters. Excessive accumulation of triglycerides, particularly in the liver and skeletal muscle, is associated with metabolic disorders such as insulin resistance and type 2 diabetes, 1,2 with a strongly increased risk of cardiovascular diseases. Moreover, the accumulation of lipid droplets in macrophages is a key feature of both early and late stages of the atherosclerotic lesion. 3 The PAT domain protein 4,5 ADRP (adipocyte differentiation-related protein) both promotes the formation of lipid droplets 6 (L.A., P.B., M.R., J.E., Marchesan D, Magnusson B, Ruiz M, P.H., Asp L, M.A.F., J.B., S.-O. O., unpublished data, 2005) and is an important constituent of the lipid droplets formed in liver, muscles, 8 and macrophages. 9 Thus, ADRP-containing lipid droplets are important in the pathogenesis of these diseases. In addition, lipid droplets have been proposed to have a central role in the inflammatory response, 10 which may further link the structures to the development of both insulin resistance and atherosclerosis. 11 Phospholipase D (PLD), which catalyzes the conversion of phosphatidylcholine to phosphatidic acid, promotes the assembly of ADRP-containing lipid droplets from intracellular membranes 12 (L.A., P.B., M.R., J.E., Marchesan D, Magnusson B, , unpublished data, 2005). The newly formed ("primordial") droplets have a diameter of 0.1 to 0.4 m, 12 whereas the mature droplets present in a cell can be more than 10 to 50ϫ larger (even larger in adipocytes) and vary considerable in size, indicating that the primordial lipid droplets increase in size after being formed. This increase appears to be an important step in the process leading to the accumulation of cytosolic lipid droplets in the ce...

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Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor.Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.-Li, L., P. Stillemark-Billton, C. Accumulation of triglycerides, particularly in the liver and skeletal muscle, is associated with metabolic disorders such as insulin resistance and type 2 diabetes (1, 2), diseases that are strong risk factors for cardiovascular diseases. Accumulation of triglycerides in the liver is also the landmark of nonalcoholic fatty liver disease, which results in inflammation and liver damage (3). Moreover, the stores of triglyceride in the liver fuel the assembly and secretion of VLDLs (3-6). Thus, it is obvious that elucidation of the mechanism of the storage of lipid in the cytosol is important for our understanding of the pathogenesis of major metabolic diseases.Triglycerides are stored in the cytosol in the form of lipid droplets (also referred to as lipid bodies) (for reviews, see 7, 8). These droplets are formed from microsomes (9; see also 7, 8 for reviews) as a primordial droplet with a diameter of z0.1 Am It has been demonstrated that the fatty acids used for the biosynthesis of VLDL triglycerides are derived from triglycerides stored in the liver (3). The triglycerides are hydrolyzed and reesterified into new triglycerides before being assembled into VLDL (4-6). VLDLs are assembled in a complex series of events (for reviews, see 11-16), starting with the formation of a primordial particle (pre-VLDL) during the translation and translocation of apolipoprotein B-100 (apoB-100) to the lumen of the endoplasmic reticulum. This step is catalyzed by the microsomal triglyceride transfer protein (MTP). The pre-VLDL par-

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