Nathan E. Wolins

A proposed model of fat packaging by exchangeable lipid droplet proteins

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3–12, and OXPAT/MLDP/PAT‐1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue‐specific complement of these proteins determines how tissues manage lipid stores.

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.

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 PPARα overexpression) perturbations that increase fatty acid utilization. In humans with impaired glucose tolerance, PPARγ 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.

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.

Packaging of Fat: An Evolving Model of Lipid Droplet Assembly and Expansion

Lipid droplets (LDs) are organelles found in most types of cells in the tissues of vertebrates, invertebrates, and plants, as well as in bacteria and yeast. They differ from other organelles in binding a unique complement of proteins and lacking an aqueous core but share aspects of protein trafficking with secretory membrane compartments. In this minireview, we focus on recent evidence supporting an endoplasmic reticulum origin for LD formation and discuss recent findings regarding LD maturation and fusion.

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 γ (PPARγ). OP9 cells are late preadipocytes in that, before the addition of adipogenic stimuli, they express the adipocyte proteins CCAAT/enhancer binding proteins α and β, PPARγ, 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.

Molecular Characterization of the Protein Encoded by the Hermansky-Pudlak Syndrome Type 1 Gene

Hermansky-Pudlak syndrome (HPS) comprises a group of genetic disorders characterized by defective lysosome-related organelles. The most common form of HPS (HPS type 1) is caused by mutations in a gene encoding a protein with no homology to any other known protein. Here we report the identification and biochemical characterization of this gene product, termed HPS1p. Endogenous HPS1p was detected in a wide variety of human cell lines and exhibited an electrophoretic mobility corresponding to a protein of ∼80 kDa. In contrast to previous theoretical analysis predicting that HPS1p is an integral membrane protein, we found that this protein was predominantly cytosolic, with a small amount being peripherally associated with membranes. The sedimentation coefficient of the soluble form of HPS1p was ∼6 S as inferred from ultracentrifugation on sucrose gradients. HPS1p-deficient cells derived from patients with HPS type 1 displayed normal distribution and trafficking of the lysosomal membrane proteins, CD63 and Lamp-1. This was in contrast to cells from HPS type 2 patients, having mutations in the β3A subunit of the AP-3 adaptor complex, which exhibited increased routing of these lysosomal proteins through the plasma membrane. Similar analyses performed on fibroblasts from 10 different mouse models of HPS revealed that only the AP-3 mutants pearl and mocha display increased trafficking of Lamp-1 through the plasma membrane. Taken together, these observations suggest that the product of the HPS1gene is a cytosolic protein capable of associating with membranes and involved in the biogenesis and/or function of lysosome-related organelles by a mechanism distinct from that dependent on the AP-3 complex.

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 AlF4− during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF4− activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF4− perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF4− 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.

A simple and rapid method to assay triacylglycerol in cells and tissues

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.

Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation.

Lipid droplets are organelles found in most mammalian cells, as well as various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol into which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation.