A highly sensitive adenylate cyclase assay method has been developed which employs sequential chromatography on columns of Dowex cation exchange resin and aluminum oxide. With the use of [a-"'PIATP as substrate, this method permits the nearly complete separation of cyclic ["'PIAMP formed from the substrate and other "P-containing compounds, i.e., "P in the assay blanks was barely detectable. In comparative studies, this method was found to be considerably more sensitive than previously reported methods. The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies.

Perilipin coats the lipid droplets of adipocytes and is thought to have a role in regulating triacylglycerol hydrolysis. To study the role of perilipin in vivo, we have created a perilipin knockout mouse. Perilipin null (peri ؊/؊ ) and wild-type (peri ؉/؉ ) mice consume equal amounts of food, but the adipose tissue mass in the null animals is reduced to Ϸ30% of that in wild-type animals. Isolated adipocytes of perilipin null mice exhibit elevated basal lipolysis because of the loss of the protective function of perilipin. They also exhibit dramatically attenuated stimulated lipolytic activity, indicating that perilipin is required for maximal lipolytic activity. Plasma leptin concentrations in null animals were greater than expected for the reduced adipose mass. The peri ؊/؊ animals have a greater lean body mass and increased metabolic rate but they also show an increased tendency to develop glucose intolerance and peripheral insulin resistance. When fed a high-fat diet, the perilipin null animals are resistant to diet-induced obesity but not to glucose intolerance. The data reveal a major role for perilipin in adipose lipid metabolism and suggest perilipin as a potential target for attacking problems associated with obesity.

ABSTACT Cell surface adenosine receptors mediate either stimulation or inhibition of adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1], and the receptors that mediate these different responses can be discriminated with selected adenosine analogs. 5'-N-Ethylcarboxamideadenosine is a more potent agonist at stimulatory receptors (Ra) than is N8-phenylisopropyladenosine, whereas the reverse potency order is seen with inhibitory receptors (Ri). The potency of adenosine is intermediate between the potencies of these two analogs. The relative potencies of adenosine receptor agonists are maintained in physiological responses in intact cells, such as steroidogenesis and inhibition of lipolysis. As with adrenergic receptors, subelasses of adenosine receptors differ functionally and pharmacologically. Adenosine modifies the physiological function and cyclic AMP concentration in a large variety of cell types by interacting with external receptors (1-3), the basic properties of which were described by Sattin and Rall (4). In plasma membrane preparations from many different cell types, adenosine and several purine-modified analogs stimulate adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] (1-5), whereas in adipocyte membranes the adenosine receptor mediates decreased activity (1,3,6). Previously published studies suggested that, despite their superficial similarities, stimulatory and inhibitory adenosine receptors might differ. Thus, whereas N6-phenylisopropyladenosine (PIA) and N6-methyladenosine were equipotent in stimulating the Leydig tumor cell adenylate cyclase (5), the former analog was far more potent that N6-methyladenosine in inhibiting the adipocyte adenylate cyclase (7). However, the fat cell studies were performed in the presence of adenosine deaminase, added to metabolize adenosine resulting from breakdown of the substrate, ATP. In this case, the analogs might have acted either at the receptor or by inhibiting the adenosine deaminase, which has a rather broad substrate specificity (8). Another method to circumvent interference from "intrinsic" adenosine is the use of dATP as the cyclase substrate (9). Metabolism of this substrate yields 2'-deoxyadenosine, which has little activity at adenosine receptors. In this report we present a pharmacological investigation of stimulatory and inhibitory receptors associated with adenylate cyclases, using dATP as substrate in the absence of adenosine deaminase. From a screening of numerous adenosine analogs we have selected two that demonstrate the existence of subclasses of adenosine receptors: PIA and 5'-N-ethylcarboxamideadenosine (NECA). The relative potencies of the analogs in the adenylate cyclase studies are maintained in physiological studies in intact cells.* Fig. 1 presents a comparison of the concentration dependencies of adenosine, PIA, and NECA in their actions on three adenylate cyclase systems: liver and 1-10 Leydig cell enzymes, which are activated by adenosine, and rat adipocyte enzyme, which is inhibit...

Akey step in lipolytic activation of adipocytes is the translocation of hormone-sensitive lipase (HSL) from the cytosol to the surface of the lipid storage droplet. Adipocytes from perilipin-null animals have an elevated basal rate of lipolysis compared with adipocytes from wild-type mice, but fail to respond maximally to lipolytic stimuli. This defect is downstream of the β-adrenergic receptor–adenylyl cyclase complex. Now, we show that HSL is basally associated with lipid droplet surfaces at a low level in perilipin nulls, but that stimulated translocation from the cytosol to lipid droplets is absent in adipocytes derived from embryonic fibroblasts of perilipin-null mice. We have also reconstructed the HSL translocation reaction in the nonadipocyte Chinese hamster ovary cell line by introduction of GFP-tagged HSL with and without perilipin A. On activation of protein kinase A, HSL-GFP translocates to lipid droplets only in cells that express fully phosphorylatable perilipin A, confirming that perilipin is required to elicit the HSL translocation reaction. Moreover, in Chinese hamster ovary cells that express both HSL and perilipin A, these two proteins cooperate to produce a more rapidly accelerated lipolysis than do cells that express either of these proteins alone, indicating that lipolysis is a concerted reaction mediated by both protein kinase A–phosphorylated HSL and perilipin A.

No abstract

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/ 503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6 -30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 M triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.Lipid droplets in adipocytes store the body's major energy reserves as triacylglycerols. These structures contain a large core of neutral lipid, primarily triacylglycerol, covered by a phospholipid monolayer. The intracellular mechanisms that control the storage and release of triacylglycerols are largely uncharacterized, yet are likely to be fundamental to understanding the regulation of energy metabolism in the body. Recent studies have shown that lipid droplets are covered with a proteinaceous coat; the functions and identities of the component proteins have not been fully elucidated. The first identified lipid droplet-specific proteins are the perilipins (1-7), a family of proteins coating the surfaces of lipid droplets of adipocytes and steroidogenic cells of adrenal cortex, testes, and ovaries, but lacking in other types of cells and in other cellular compartments. The perilipins are encoded by a single copy gene that gives rise to multiple mRNAs by alternative splicing mechanisms 1 ; these mRNAs are translated to yield the three described protein isoforms (2, 4). Perilipin A is the predominant isoform in both adipocytes and steroidogenic cells, perilipin B is found primarily in adipocytes, and perilipin C is unique to steroidogenic cells. Perilipin A is the most abundant protein on highly purified lipid droplets isolated from fully differentiated cultured 3T3-L1 adip...

Intracellular neutral lipid storage droplets are essential organelles of eukaryotic cells, yet little is known about the proteins at their surfaces or about the amino acid sequences that target proteins to these storage droplets. The mammalian proteins Perilipin, ADRP, and TIP47 share extensive amino acid sequence similarity, suggesting a common function. However, while Perilipin and ADRP localize exclusively to neutral lipid storage droplets, an association of TIP47 with intracellular lipid droplets has been controversial. We now show that GFP-tagged TIP47 co-localizes with isolated intracellular lipid droplets. We have also detected a close juxtaposition of TIP47 with the surfaces of lipid storage droplets using antibodies that specifically recognize TIP47, further indicating that TIP47 associates with intracellular lipid storage droplets. Finally, we show that related proteins from species as diverse as Drosophila and Dictyostelium can also target mammalian or Drosophila lipid droplet surfaces in vivo. Thus, sequence and/or structural elements within this evolutionarily ancient protein family are necessary and sufficient to direct association to heterologous intracellular lipid droplet surfaces, strongly indicating that they have a common function for lipid deposition and/or mobilization.

The PAT family of proteins has been identified in eukaryotic species as diverse as vertebrates, insects, and amebazoa. These proteins share a highly conserved sequence organization and avidity for the surfaces of intracellular, neutral lipid storage droplets. The current nomenclature of the various members lacks consistency and precision, deriving more from historic context than from recognition of evolutionary relationship and shared function. In consultation with the Mouse Genomic Nomenclature Committee, the Human Genome Organization Genomic Nomenclature Committee, and conferees at the 2007 FASEB Conference on Lipid Droplets: Metabolic Consequences of the Storage of Neutral Lipids, we have established a unifying nomenclature for the gene and protein family members. Each gene member will incorporate the root term PERILIPIN (PLIN), the founding gene of the PAT family, with the different genes/proteins numbered sequentially.