Functional Studies on Native and Mutated Forms of Perilipins

Abstract

Perilipin A coats the lipid storage droplets in adipocytes and is polyphosphorylated by protein kinase A (PKA); the fact that PKA activates lipolysis in adipocytes suggests a role for perilipins in this process. To assess whether perilipins participate directly in PKAmediated lipolysis, we have expressed constructs coding for native and mutated forms of the two major splice variants of the perilipin gene, perilipins A and B, in Chinese hamster ovary fibroblasts. Perilipins localize to lipid droplet surfaces and displace the adipose differentiation-related protein that normally coats the droplets in these cells. Perilipin A inhibits triacylglycerol hydrolysis by 87% when PKA is quiescent, but activation of PKA and phosphorylation of perilipin A engenders a 7-fold lipolytic activation. Mutation of PKA sites within the N-terminal region of perilipin abrogates the PKAmediated lipolytic response. In contrast, perilipin B exerts only minimal protection against lipolysis and is unresponsive to PKA activation. Since Chinese hamster ovary cells contain no PKA-activated lipase, we conclude that the expression of perilipin A alone is sufficient to confer PKA-mediated lipolysis in these cells. Moreover, the data indicate that the unique C-terminal portion of perilipin A is responsible for its protection against lipolysis and that phosphorylation at the N-terminal PKA sites attenuates this protective effect.Acute mobilization of adipose triacylglyerol stores for energy is regulated primarily by the activation state of cAMP-dependent protein kinase (PKA) 1 (1). Historically, this stimulation has been attributed to phosphorylation and activation of hormonesensitive lipase (HSL), but as noted previously (2, 3), the meager doubling of HSL activity upon phosphorylation in vitro falls far short of explaining the 30 -100-fold activation of cellular lipolysis upon elevation of PKA activity in isolated primary adipocytes. Although some the of differences between the magnitude of the in vitro and in vivo responses may be attributed to the PKA-induced translocation of HSL from the cytosol to the lipid storage droplets within adipocytes (4), it is likely that additional factors, notably the perilipins, contribute to the cellular response.The perilipins are a class of proteins found exclusively at the limiting surface of lipid storage droplets, i.e. at the lipid/aqueous interface, in adipocytes and in steroidogenic cells (5-7). These proteins are the most abundant PKA substrates in adipocytes (5), and both their subcellular location and polyphosphorylation by PKA suggest a role for the perilipins in PKAmediated lipolysis. In adipocytes, alternative mRNA splicing gives rise to perilipins A and B, the former at much higher levels than the latter (8). Thus far, functional studies point to a role for the perilipins in protecting stored TAG from hydrolysis by cellular lipases. Ectopic expression of perilipin A in 3T3-L1 adipoblasts results in perilipin A-coated lipid droplets and increases the half-life of stored TAG deposits by a factor of 4...

Perilipin A Mediates the Reversible Binding of CGI-58 to Lipid Droplets in 3T3-L1 Adipocytes

“…The perilipin B cDNA subcloned into the pSR␣MSVtkneo retroviral expression vector (15) was kindly provided to us by Dr. John Tansey (Otterbein College, Westerville, OH). Ecotropic retroviral stocks were produced in transfected 293T cells as described previously (26), and used to infect cultured 3T3-L1 preadipocytes.…”

“…Studies in perilipin knockout mice have established an important role for perilipins in regulating lipolysis in adipocytes, and hence, controlling the mass of triacylglycerol deposited in adipose tissue (10, 11). The mechanisms by which perilipins maintain control of adipocyte triacylglycerol metabolism are poorly understood, although recent mutagenesis studies have identified structural motifs important to perilipin function in triacylglycerol storage and lipolysis (12)(13)(14)(15)(16)). An attractive model consistent with these data suggests that perilipins restrict the access of cytosolic lipases to triacylglycerol stored within lipid droplets, and thus suppress the rate of basal lipolysis under conditions when triacylglycerol storage is favored.…”

Lipase-selective Functional Domains of Perilipin A Differentially Regulate Constitutive and Protein Kinase A-stimulated Lipolysis

“…This view, however, has recently been challenged by the following observations: 1) HSL-deficient mice are not obese, suggesting significant activity of lipase(s) other than HSL (10,12,19); 2) adipocytes derived from epididymal fat pads or embryonic fibroblasts of HSL null mice retain 50% of basal TAG lipase activity and are responsive to PKA activation (10,11,12,19); and 3) cell lines lacking endogenous HSL, i.e. 3T3-L1 preadipocytes (21), NIH 3T3 fibroblasts (22), and Chinese hamster ovary cells (23), exhibit significant TAG hydrolysis under basal and PKA-stimulated conditions. Thus, both HSL and non-HSL(s) play important roles in basal and PKA-stimulated lipolysis.…”

“…1). Under nonstimulated (basal) conditions, Peri A acts as a barrier to HSL (22) and non-HSL(s) (21)(22)(23), thereby decreasing basal lipolysis (22,23) and increasing intracellular (TAG) storage (21,22). Upon lipolytic stimulation, Peri A is phosphorylated at up to six sites by PKA (25,28) (Fig.…”

“…Recent data have suggested a model of Peri A function in which the phosphorylation of N-terminal PKA sites (PKA1, PKA2, and PKA3; Fig. 1) promotes PKA-stimulated lipolysis (22,23), and the C terminus of Peri A (aa 406 -517) acts as a barrier to all lipases (inhibits basal lipolysis) (23). In contrast, novel data in the present study identify multiple, lipase-selective domains within Peri A that function coordinately to regulate basal and stimulated lipolysis.…”