A single centrifugation method for isolating fat droplets from cells and tissues.

Fat droplets (FDs) have important roles in cellular energy regulation. Isolating FDs from either cells or tissue continues to be important for studying these organelles. Here, we describe a procedure wherein whole homogenates of cultured cells or tissue are fractionated with a single centrifugation step in a standard microcentrifuge. This procedure reproducibly yields three fractions highly enriched in either FDs, soluble cellular components, or sedimentable organelles/membranes.

CD36 protein is involved in store-operated calcium flux, phospholipase A2 activation, and production of prostaglandin E2.

The scavenger receptor FAT/CD36 contributes to the inflammation associated with diabetes, atherosclerosis, thrombosis, and Alzheimer disease. Underlying mechanisms include CD36 promotion of oxidative stress and its signaling to stress kinases. Here we document an additional mechanism for the role of CD36 in inflammation. CD36 regulates membrane calcium influx in response to endoplasmic reticulum (ER) stress, release of arachidonic acid (AA) from cellular membranes by cytoplasmic phospholipase A(2)α (cPLA(2)α) and contributes to the generation of proinflammatory eicosanoids. CHO cells stably expressing human CD36 released severalfold more AA and prostaglandin E(2) (PGE(2)), a major product of AA metabolism by cyclooxygenases, in response to thapsigargin-induced ER stress as compared with control cells. Calcium influx after ER calcium release resulted in phosphorylation of cPLA(2) and its translocation to membranes in a CD36-dependent manner. Peritoneal macrophages from CD36(-/-) mice exhibited diminished calcium transients and reduced AA release after thapsigargin or UTP treatment with decreased ERK1/2 and cPLA(2) phosphorylation. However, PGE(2) production was unexpectedly enhanced in CD36(-/-) macrophages, which probably resulted from a large induction of cyclooxygenase 2 mRNA and protein. The data demonstrate participation of CD36 in membrane calcium influx in response to ER stress or purinergic receptor stimulation resulting in AA liberation for PGE(2) formation. Collectively, these results identify a mechanism contributing to the pleiotropic proinflammatory effects of CD36 and suggest that its targeted inhibition may reduce the acute inflammatory response.

Diacylglycerol enrichment of endoplasmic reticulum or lipid droplets recruits perilipin 3/TIP47 during lipid storage and mobilization.

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). Blocking membrane trafficking with AlF(4)(-) during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF(4)(-) activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF(4)(-) perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF(4)(-) redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.

OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.

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." Physiologic lipid loading of mouse liver by fasting enriches OXPAT in the lipid droplet tissue fraction. OXPAT resides on lipid droplets with the PAT protein adipophilin in primary cardiomyocytes. Ectopic expression of OXPAT promotes fatty acid-induced triacylglycerol accumulation, long-chain fatty acid oxidation, and mRNAs associated with oxidative metabolism. Consistent with these observations, OXPAT is induced in mouse adipose tissue, striated muscle, and liver by physiological (fasting), pathophysiological (insulin deficiency), pharmacological (peroxisome proliferator-activated receptor [PPAR] agonists), and genetic (muscle-specific PPARalpha overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARgamma agonist treatment induces adipose OXPAT mRNA. Further, adipose OXPAT mRNA negatively correlates with BMI in nondiabetic humans. Our collective data in cells, mice, and humans suggest that OXPAT is a marker for PPAR activation and fatty acid oxidation. OXPAT likely contributes to adaptive responses to the fatty acid burden that accompanies fasting, insulin deficiency, and overnutrition, responses that are defective in obesity and type 2 diabetes.

Perilipin 5-Driven Lipid Droplet Accumulation in Skeletal Muscle Stimulates the Expression of Fibroblast Growth Factor 21.

Perilipin 5 (PLIN5) is a lipid droplet protein and is highly expressed in oxidative tissue. Expression of the PLIN5 gene is regulated by peroxisome proliferator-activated receptor-α, fasting, and exercise. However, the effect of increased muscle PLIN5 expression on whole-body energy homeostasis remains unclear. To examine this, we developed a mouse line with skeletal muscle PLIN5 overexpression (MCK-Plin5). We show that MCK-Plin5 mice have increased energy metabolism and accumulate more intramyocellular triacylglycerol but have normal glucose and insulin tolerance. MCK-Plin5 mice fed high-fat chow manifest lower expression of inflammatory markers in their liver and increased expression of "browning" factors in adipose tissue. This muscle-driven phenotype is, at least in part, mediated by myokines; the MCK-Plin5 mice have 80-fold higher FGF21 gene expression in muscle and increased serum FGF21 concentration. The increase in FGF21 occurs mainly in muscles with a predominance of fast-twitch fibers, suggesting that fiber type-specific lipid storage may be part of the mechanism conferring metabolic protection in MCK-Plin5 mice. In conclusion, upregulating the PLIN5 level in skeletal muscle drives expression of the FGF21 gene in fast-twitch fibers and is metabolically protective. These findings provide insight into the physiology of PLIN5 and the potential contribution of its upregulation during exercise.

Imaging of neutral lipids and neutral lipid associated proteins.

Intracellular fat droplets are large and have a distinct morphology, which makes their imaging at the light level simple and informative. We detail how to image the fat droplet core by metabolic labeling with fluorescent fatty acids or lipophilic fluorochromes. Further, we describe the use of indirect immunostaining to image fat droplet proteins and fat cores in the same field. We also address the use of appropriate controls for determining signal specificity and other practical considerations for optimizing image quality.

Perilipin 1 moves between the fat droplet and the endoplasmic reticulum.

Perilipin 1, unlike the other perilipins, is thought to be restricted to the fat droplet. We reassessed its cellular distribution using the fat droplet marker CGI-58 in OP9 and 3T3-L1 adipocyte lines and in brown adipose tissue (BAT). As expected, we found perilipin 1 in the fat droplet-enriched floating fraction from centrifuged adipocyte or BAT homogenates. However, about half of perilipin 1 was suspended in the cytosol/infranate or pelleted with cellular membranes. In these fractionations, most of the fat droplet-associated protein CGI-58 was in the floating fraction. In BAT and OP9 adipocytes about a third of perilipin 1 pellets, compared with a much smaller fraction of CGI-58. Co-imaging perilipin 1 and smooth endoplasmic reticulum (ER) markers reveals both ER and fat droplet associated perilipin 1 in OP9 adipocytes. Consistent with these observations, perilipin 1 overexpressed in COS7 cells mostly fractionates with cellular membranes and imaging shows it on the ER. In 3T3-L1 adipocytes almost half of perilipin 1 floats, half is suspended as infranate and small amounts pellet. Finally, driving rapid fat droplet synthesis in OP9 adipocytes increases the intensity of perilipin 1 on fat droplets, while decreasing non-fat droplet immunolabeling. Confirming the morphological findings, fractionation shows perilipin 1 moving from the pelleted to the floated fractions. In conclusion, this study documents an expanded intracellular distribution for perilipin 1 and its movement from ER to fat droplet during lipid synthesis.

OP9 mouse stromal cells rapidly differentiate into adipocytes: characterization of a useful new model of adipogenesis.

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 marrow-derived 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 gamma (PPARgamma). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins alpha and beta, PPARgamma, 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.

S3-12, Adipophilin, and TIP47 package lipid in adipocytes.

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.

Adipocyte protein S3-12 coats nascent lipid droplets.

Most animals store lipid intracellularly in protein-coated droplets. The protein coat usually contains at least one member of the PAT (perilipin, adipose differentiation-related protein, and TIP47) family. Evidence suggests that PAT proteins control access to the lipid they enclose. The protein S3-12, which has sequence similarity to the PAT proteins, was found in a screen for adipocyte-specific proteins. The adipocyte expression of S3-12 and its similarity to the PAT proteins suggest that S3-12 is involved in adipocyte lipid storage. To test this hypothesis, we supplemented 3T3-L1 adipocytes with fatty acids and assessed the distribution of S3-12 by immunofluorescence microscopy. Prior to fatty acid incubation, S3-12 was distributed diffusely throughout the cytoplasm on punctate structures of heterogeneous size. After 10 min of lipid loading, S3-12 localized to 500-nm structures concentrated at the adipocyte periphery. After longer incubations, S3-12 coated the surface of lipid droplets up to several micrometers in diameter. Initially, these droplets were distinct from those droplets surrounded by perilipin; but by 240 min, most perilipin-coated droplets had some S3-12 on the surface as well. We additionally report that the formation of S3-12-coated droplets 1) required glucose and fatty acids that can be incorporated into triacylglycerol, 2) was blocked by an inhibitor of triacylglycerol synthesis, and 3) was insulin-dependent. This study reports for the first time the early morphological events in the genesis and maturation of adipocyte lipid droplets.