The lipolytic stimulation of 3T3-L1 adipocytes promotes the translocation of hormone-sensitive lipase to the surfaces of lipid storage droplets

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Perilipins, the major structural proteins coating the surfaces of mature lipid droplets of adipocytes, play an important role in the regulation of triacylglycerol storage and hydrolysis. We have used proteomic analysis to identify CGI-58, a member of the ␣/␤-hydrolase fold family of enzymes, as a component of lipid droplets of 3T3-L1 adipocytes. CGI-58 mRNA is highly expressed in adipose tissue and testes, tissues that also express perilipins, and at lower levels in liver, skin, kidney, and heart. Both endogenous CGI-58 and an ectopic CGI-58-GFP chimera show diffuse cytoplasmic localization in 3T3-L1 preadipocytes, but localize almost exclusively to the surfaces of lipid droplets in differentiated 3T3-L1 adipocytes. The localization of endogenous CGI-58 was investigated in 3T3-L1 cells stably expressing mutated forms of perilipin using microscopy. CGI-58 binds to lipid droplets coated with perilipin A or mutated forms of perilipin with an intact C-terminal sequence from amino acid 382 to 429, but not to lipid droplets coated with perilipin B or mutated perilipin A lacking this sequence. Immunoprecipitation studies confirmed these findings, but also showed co-precipitation of perilipin B and CGI-58. Remarkably, activation of cAMP-dependent protein kinase by the incubation of 3T3-L1 adipocytes with isoproterenol and isobutylmethylxanthine disperses CGI-58 from the surfaces of lipid droplets to a cytoplasmic distribution. This shift in subcellular localization can be reversed by the addition of propanolol to the culture medium. Thus, CGI-58 binds to perilipin Acoated lipid droplets in a manner that is dependent upon the metabolic status of the adipocyte and the activity of cAMP-dependent protein kinase.Lipid droplets, organelles that store neutral lipids, are found in nearly all types of eukaryotic cells. The most highly studied lipid droplet-associated proteins are members of the PAT 1 (for perilipin, adipophilin (also called adipose differentiation-related protein or ADRP), and TIP47) family of proteins (1) that includes perilipin, adipophilin, TIP47, and a related but structurally divergent protein, S3-12 (2, 3). Three protein isoforms of perilipins, named perilipins A, B, and C, are translated from alternatively spliced forms of mRNA. Perilipin A and S3-12 localize to lipid droplets in adipocytes (3-5), where they are the most abundant structural proteins of large mature and small nascent lipid droplets, respectively. In contrast, most other types of cells have small lipid droplets covered with adipophilin (6, 7) and TIP47 (1, 8, 9). Perilipins A and C, and adipophilin, coat the lipid droplets of steroidogenic cells (6) that store cholesterol ester precursors for steroid hormone synthesis.The members of the PAT family comprise the major structural proteins of lipid droplets. 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 per...

Section: Figmentioning

confidence: 99%

Section: Cgi-58 Localizes To Cytosol In 3t3-l1 Preadipocytes and To Tmentioning

confidence: 99%

Section: Cgi-58 Localizes To Cytosol In 3t3-l1 Preadipocytes and To Tmentioning

confidence: 99%

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

Subramanian

Rothenberg

Gómez

et al. 2004

275

290

“…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.…”

mentioning

confidence: 99%

Functional Studies on Native and Mutated Forms of Perilipins

Tansey¹,

Huml

Vogt

et al. 2003

Self Cite

194

107

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...

“…The activity of HSL is principally determined by the intracellular level of cAMP, which controls the activity of protein kinase A that phosphorylates HSL on critical serine residues (7). In the adipocyte, this triggers translocation of HSL from the cytosol to the surface of the lipid droplet (8) and, possibly, also a change in specific activity. HSL has a broad substrate specificity, being able to hydrolyze, among others, mono-, di-, and triglycerides and cholesteryl esters.…”

mentioning

confidence: 99%

A Novel Hormone-sensitive Lipase Isoform Expressed in Pancreatic β-Cells

Lindvall¹,

Nevsten²,

Ström³

et al. 2004

Hormone-sensitive lipase (HSL) is a key enzyme in fatty acid mobilization in many cell types. Two isoforms of HSL are known to date, namely HSL adi (84 kDa in rat) and HSL tes (130 kDa in rat). These are encoded by the same gene, with exons 1-9 encoding the parts that are common to both and an additional 5 -exon encoding the additional amino acids in HSL tes . HSL of various tissues, among these the islet of Langerhans, is larger than HSL adi , but not as large as HSL tes , indicating that there may be other 5 -coding exons. Here we describe the molecular basis for a novel 89-kDa HSL isoform that is expressed in ␤-cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1-9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon. The additional 5 -base pairs encode a 43-amino acid peptide, which is highly positively charged. Conglomerates of HSL molecules are in close association with the secretory granules of the ␤-cell, as determined by immunoelectron microscopy with antibodies targeting two separate regions of HSL. We have also determined that the human genomic sequence upstream of exon A has promoter activity in INS-1 cells as well as glucose sensing capability, mediating an increase in expression at high glucose concentration. The minimal promoter is present within 170 bp from the transcriptional start site and maximal glucose responsiveness is conferred by sequence within 850 bp from the transcriptional start site.Because of the established association between type 2 diabetes and obesity, lipids have received much attention in research on the pathophysiology of type 2 diabetes. Lipids are believed to cause insulin resistance in tissues and to alter ␤-cell function. The effect of lipids on ␤-cells has been studied extensively, but the picture that emerges is complex. Different effects are seen depending on parameters such as duration of exposure, concentration, and fatty acid chain length and saturation. Briefly, the presence of free fatty acids is essential for normal glucosestimulated insulin secretion (1, 2) and also has an amplifying action on glucose-stimulated insulin secretion. On the other hand, a prolonged exposure (24 -48 h) to high concentrations (ϳ1 mM) of free fatty acids is detrimental to ␤-cell function and survival (3, 4).In view of this, knowledge of lipid mobilization, lipid storage, and the regulation of these in the ␤-cell are of great interest. Recently, we showed that hormone-sensitive lipase (HSL) 1 is expressed and active in islets of Langerhans and clonal ␤-cells (5). HSL is a key enzyme in fatty acid mobilization in the adipocytes and presumably also in several non-adipocyte cell types (see Ref. 6 for review). It is unique among known lipases in that its activity is acutely controlled by hormones. Catabolic hormones, such as glucagon and catecholamines, activate the enzyme and anabolic hormones (e.g. insulin) decrease its activity. The activity of HSL is principally determined by the intracellular level of cAMP, which con...