Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylumbelliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets.Adipose tissue contains the largest energy reserve in the body, stored as triacylglycerol in the intracellular lipid droplets of adipocytes. Triacylglycerol stores are hydrolyzed to mobilize energy during fasting and extended exercise. Catecholamines bind to ␤-adrenergic receptors on the plasma membranes of adipocytes to initiate a G-protein-mediated signaling cascade that activates adenylyl cyclase, thus increasing intracellular cAMP levels. Protein kinase A (or cAMP-dependent protein kinase) is then activated and phosphorylates several proteins required for the hydrolysis of triacylglycerol and consequent mobilization of fatty acids. Following phosphorylation by protein kinase A, hormone-sensitive lipase translocates from the cytosol onto lipid droplets (1-5), where it hydrolyzes triacylglycerol and diacylglycerol (6 -8). Perilipin A is another major substrate for protein kinase A (9, 10).Perilipin A localizes to lipid droplets in adipocytes and plays roles in facilitating both the storage and hydrolysis of triacylglycerol. In adipocytes, two isoforms of perilipin are derived from the alternate splicing of mRNA transcribed from a single gene (10, 11); perilipin A is the predominant protein isoform, whereas perilipin B is a minor isoform. Perilipins A and B share a common sequ...

Lipid droplets are cellular organelles, structurally similar to lipoprotein particles. Lipid droplets include a neutral lipid core composed largely of triglycerides, surrounded by a phospholipid monolayer and coated with surface proteins that provide an interface for various aspects of lipid metabolism, including lipid transport, lipogenesis, and lipolysis (1-5). Lipolysis is an important mechanism by which cells release energy stored in lipid droplets; its impairment has been linked to cellular lipotoxicity and insulin resistance (6). Studies are needed to gain an understanding of the underlying molecular mechanisms regulating lipolysis. Although all cells are equipped to perform lipolysis, the extent of lipid accumulation and specific components of the lipolytic pathway are variable, depending on the type of cell.Numerous recent studies have led to consensus that members of the PAT family of proteins, originally named for Perilipin, Adipose differentiation-related protein (ADFP) 4 and Tail Interacting Protein 47 (TIP47), play conserved structural and functional roles on lipid droplets (6 -9). Proteomic studies have identified a "signature" composition for lipid droplets from a variety of types of cells that includes at least one PAT family member. In mammalian cells, the PAT family includes perilipin

Perilipin A is the most abundant protein associated with the lipid droplets of adipocytes and functions to control both basal and stimulated lipolysis. Under basal or fed conditions, perilipin A shields stored triacylglycerols from cytosolic lipases, thus promoting triacylglycerol storage. When catecholamines bind to cell surface receptors to initiate signals that activate cAMP-dependent protein kinase (PKA), phosphorylated perilipin A facilitates maximal lipolysis. Mutagenesis studies have revealed that central sequences of moderately hydrophobic amino acids are required to target nascent perilipin A to lipid droplets and provide an anchor into the hydrophobic environment of lipid droplets. Sequences of amino acids in the unique carboxyl terminus of perilipin A and those in amino terminal sequences flanking the first hydrophobic stretch are required for the barrier function of perilipin A in promoting triacylglycerol storage. Site-directed mutagenesis studies of serine residues within six PKA consensus sites of perilipin A reveal functions for phosphorylation of at least three of the sites. Phosphorylation of one or more of the serines within three amino terminal PKA sites is required to facilitate hormone-sensitive lipase access to lipid substrates. Phosphorylation of serines within two carboxyl terminal sites is also required for maximal lipolysis. Phosphorylation of serine 492 (site 5) triggers a massive remodeling of lipid droplets, whereby large peri-nuclear lipid droplets fragment into myriad lipid micro-droplets that scatter throughout the cytoplasm. We hypothesize that perilipin A binds accessory proteins to provide assistance in carrying out these functions.

Perilipin A controls lipolysis in adipocytes and is phosphorylated by protein kinase A (PKA) on six serine residues, promoting hormone‐sensitive lipase activity, and triggering remodeling of lipid droplets, whereby large droplets fragment into myriad micro‐droplets. Phosphorylation of perilipin Ser492 initiates this process and is required for maximal lipolysis. We hypothesize that perilipin A forms a dynamic scaffold on lipid droplets that organizes lipid metabolic enzymes; perilipin conformation responds to the metabolic status of the adipocyte. CGI‐58 binds to the carboxyl terminus of perilipin A in adipocytes under basal conditions. Humans with neutral lipid storage disorder (NLSD) have mutations in CGI‐58 and store excessive triacylglycerol (TAG) in many cells and tissues; hence, CGI‐58 plays a role in cellular TAG homeostasis. Recombinant CGI‐58 shows Coenzyme A‐dependent lysophosphatidic acid acyltransferase activity, and is important for channeling fatty acids released from triacylglycerol hydrolysis to phospholipid synthesis. CGI‐58 is a substrate for PKA and is phosphorylated on Ser239, yet mutation of Ser239 to Ala does not alter the function of CGI‐58 in reducing TAG levels of NLSD fibroblasts. This PKA‐mediated phosphorylation facilitates release of CGI‐58 from the perilipin scaffold. Supported by DK54797 from NIH and an American Heart Association Established Investigator Award.