Lipid droplet proteins of the PAT (perilipin, adipophilin, and TIP47) family regulate cellular neutral lipid stores. We have studied a new member of this family, PAT-1, and found that it is expressed in highly oxidative tissues. We refer to this protein as "OXPAT.

Animals have evolved mechanisms to maintain circulating nutrient levels when energy demands exceed feeding opportunities. Mammals store most of their energy as triacylglycerol in the perilipin-coated lipid droplets of adipocytes. How newly synthesized triacylglycerol is delivered to perilipin-coated lipid droplets is poorly understood. Perilipin is a member of the evolutionarily related family of PAT proteins (Perilipin, Adipophilin, TIP47), which is defined by sequence similarity and association with lipid droplets. We previously showed that S3-12, which is also a member of this family, associates with a separate pool of lipid droplets that emerge when triacylglycerol storage is driven by adding oleate to the culture medium of adipocytes. Our current data extend these findings to demonstrate that nascent lipid droplets emerge with a coat composed of S3-12, TIP47, and adipophilin. After 100 min of oleate treatment, the nascent lipid droplets are more heterogeneous: S3-12 and TIP47 coat smaller, peripheral droplets and adipophilin coats a more medial population of droplets. Fractionation of untreated and oleate-treated adipocytes shows oleate-dependent redistribution of TIP47 and adipophilin from cytosolic fractions to the lipid droplet fraction. Inhibition of protein synthesis with cycloheximide does not block the oleate-induced formation of the nascent lipid droplets, nor does it prevent TAG accumulation. We suggest that the non-lipid droplet pools of S3-12, adipophilin, and TIP47 constitute a ready reservoir of coat proteins to permit rapid packaging of newly synthesized triacylglycerol and to maximize energy storage during nutrient excess.The ability to store energy as triacylglycerol (TAG) 1 and later mobilize energy from this reservoir as free fatty acids and glycerol allows animals to survive prolonged periods of fasting. Higher animals store TAG in specialized cells, adipocytes, which are able to efficiently synthesize TAG from fatty acid and glucose and package TAG into large storage droplets (10 -100-m diameter). Other cell types have a more limited capacity to store TAG and rarely accumulate large lipid droplets. The robust ability of the adipocyte to store TAG is not without limit. It has been hypothesized that, when the visceral adipose tissue depot fills up, excess lipid "overflows" into other tissues that are less able than adipocytes to package and store TAG. Fatty acids not esterified to glycerol are toxic even at low concentrations (1). In fact, failure to efficiently sequester lipid into the lipid storage droplets of adipocytes has been implicated in the pathogenesis of insulin resistance, nonalchoholic fatty liver disease, ␤-cell failure, and cardiomyopathy (2).Little is known of mechanisms by which cells, and adipocytes in particular, package newly synthesized TAG into storage droplets (3, 4). The structure of intracellular lipid droplets is similar to that of lipoproteins, a neutral lipid core is surrounded by a phospholipid monolayer and a protein coat. This structural organization is con...

Much knowledge of adipocyte biology has been learned from cell culture models, most notably 3T3-L1 cells. The 3T3-L1 model has several limitations, including the requirement of 2 weeks to generate adipocytes and the waning of adipogenic potential in culture. We have investigated the capacity of OP9 cells, a line of bone marrowderived mouse stromal cells, to recapitulate adipogenesis. When OP9 cells are given any one of three adipogenic stimuli, they rapidly accumulate triacylglycerol, assume adipocyte morphology, and express adipocyte late marker proteins, including glucose transporter 4 and adiponectin. OP9 cells can differentiate into adipocytes within 2 days. This rapid rate of differentiation allows for the detection of transiently expressed proteins in mature OP9 adipocytes. Adipogenesis in OP9 cells involves the master transcriptional regulator of adipocyte differentiation, peroxisome proliferator-activated receptor g (PPARg). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins a and b, PPARg, sterol-regulatory element binding protein-1, S3-12, and perilipin. OP9 differentiation is not diminished by maintenance in culture at high cell density or by long periods in continuous culture, thereby facilitating the generation of stable cell lines that retain adipogenic potential. Thus, the unique features of OP9 cells will expedite the study of adipocyte biology. The increase in obesity and the identification of adipocyte-secreted proteins that regulate energy metabolism (1) have generated interest in adipocyte biology. Adipocytes are the primary storage site for energy in vertebrate animals. During fasting, adipocytes release energy-rich molecules that provide metabolic fuels to other tissues. Adipocytes also secrete hormones that orchestrate the storage, release, and oxidation of energy-rich molecules throughout the body and that control behavior, including feeding (2). Primary adipocytes maintain a large dynamic triacylglycerol (TAG) pool and express a specific set of proteins to maintain circulating metabolic fuel levels. Primary adipocytes and adipose tissue have been used to study basic adipocyte biology. However, these systems have several limitations: they do not propagate in culture, they are difficult to transfect with DNA, they have a huge TAG store that interferes with biochemistry and microscopy, they vary as a result of the genetics and conditions of the animals from which they are isolated, and the isolation procedure is tedious and introduces variation. In addition, harvesting primary adipocytes or adipose from animals generally requires the euthanasia of a vertebrate animal and the expense of specialized facilities and protocols. For these reasons and likely others, cell lines have been developed that can be induced to store TAG, to express proteins that are hallmarks of adipocytes, and presumably to recapitulate key events in adipocyte ontogeny.Three decades ago, Green and colleagues (3-...