Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3-and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER Fat storage has become an area of great interest because as a population we are experiencing significant increases in adiposity and its associated metabolic complications. There is a direct correlation between levels of adiposity and fat accumulation outside adipocytes, which is associated with a vast array of pathologies. However, the mechanisms underlying cellular fat deposition are not well understood. One example of a metabolically important but poorly understood process is how fat gets into intracellular droplets and how the single membrane leaflet of amphipathic proteins and lipids assembles around these droplets. In previous work, we have demonstrated that when cells are given fatty acids they rapidly synthesize triacylglycerol (TG), 2 and a set of proteins moves from the cytosol to coat the nascent TG droplets (1-5). These fat coat proteins are members of the PAT family, and the name is an acronym derived from the first letter of the nonsystematic names of the original three family members, Perilipin/ADRP/TIP47 (6). Later, two other proteins, S3-12 and OXPAT, were added based on similar sequence and lipid binding behaviors (2, 4). We will use the newly described systematic nomenclature for the PAT proteins (7) as follows: perilipin 1 for perilipin; perilipin 2 for adipophilin/ADRP; perilipin 3 for PP17/TIP47; perilipin 4 for S3-12, and perilipin 5 for MLDP/LSDP5/OXPAT. Unlike some lipid droplet proteins, PAT proteins do not have an ER targeting signal and are not trafficked to lipid droplets through the secretory pathway (8 -10). PAT proteins have been described as either constitutively lipid-associated proteins (CPATs) or exchangeable lipid-binding proteins (EPATs) (5). Perilipin 1 and perilipin 2 are CPATs, because they are stabilized by lipid binding, and thus are almost always bound to lipid. Perilipin 3, perilipin 4, and perilipin 5 are EPATs, because they are stable when not bound to lipid, and thus can exchange between the cytosol and lipid droplet based on the metabolic state of the cells. For example, when the lipid surface is expanded by fatty acid-driven TG synthesis, perilipin 3, perilipin 4, and perilipin 5 move from cytosol to the lipid droplet membrane leaflet (5).The PAT proteins appear to play an important role in regulating intracellular lipid storage, and CPATs in particular appear to protect lipid stores from unregulated hydrolysis. Studies in cultured cells and whole animals show that CPAT

We have developed a reliable, rapid, and economical assay for the quantification of triacylglycerol (TG) in cells and animal tissues. In a few hours, this assay quantifies microgram amounts of TG from tens or even hundreds of samples. The protocol includes an organic extraction to partition TG away from proteins and other hydrophilic molecules found in cells and tissues that may interfere with the colorimetric enzyme-linked TG detection method. In addition, this assay is economical, as no expensive reagents, supplies, or equipment are needed. Another benefit of this assay is that it does not require environmentally unfriendly halogenated solvents. Excessive intracellular triacylglycerol (TG) storage is the hallmark of obesity. Complications of obesity include type 2 diabetes, vascular disease, nonalcoholic steatohepatitis, and cardiomyopathy (1, 2). These significant health problems have resulted in an increased interest in understanding TG metabolism, including the enzymes that catalyze TG synthesis, the proteins that package TG for storage and secretion, and the regulators of TG levels in cells or tissues. These studies often require TG quantification in cells and in animal tissues. In fact, a high-throughput assay that measures the cellular uptake and oxidation of fatty acids, which are the main components of TG, was developed recently (3).Several strategies have been used to quantify TG in biological samples over many decades. The most rigorous methods resolve organically extracted lipids and compare the signal from TG in the samples with known amounts of similarly resolved TG (4-6). Each of these measurements requires extraction and resolution of the samples, making these measurements involved and tedious; thus, these methods are economically constraining when large data sets are needed.More recently, basic researchers have used clinical kits designed to quantify TG emulsified in lipoproteins in the sera of patients. These kits use lipoprotein lipase (LPL) to hydrolyze the fatty acids from TG, releasing glycerol. Then, through a series of reactions, electrons from glycerol reduce a dye, increasing the dye's absorbance. This absorbance change is proportional to the moles of TG in the sample (Fig. 1). These kits are simple to use and sensitive. However, LPL's biological role is to target specific nutrients to the appropriate tissue by hydrolyzing a defined set of lipids from lipoproteins. Adapting these kits to measure intracellular TG requires care and thought. Intracellular TG is enclosed in protein-coated droplets that control lipase's access to the TG (4, 7-10). Thus, it is unclear whether the LPL used in these kits can hydrolyze TG in intact intracellular lipid droplets. To further complicate the situation, experimental manipulation can alter lipid droplet coat proteins (11-16), and changing the lipid droplet coat proteins likely changes TG vulnerability to lipases. These lipid droplet coat proteins may block the quantitative measurement of TG, making the results reflective of changes in TG acce...

PE are best depicted as filling defects when displayed with a modified window referenced to the right or left main PA attenuation. Standard mediastinal windows are useful for identifying vessels that are occluded completely with PE.

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