Molecular Mechanisms Regulating Hormone-Sensitive Lipase and Lipolysis

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...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Functional Studies on Native and Mutated Forms of Perilipins

Tansey¹,

Huml

Vogt

et al. 2003

194

107

“…TG hydrolysis (lipolysis) is mediated by a TG-lipase that has been purified from the cytosol (7). Like hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step in mobilization of adipose tissue fatty acids (8,9), the TG-lipase from M. sexta fat body is an enzyme that can be phosphorylated. The end product of insect lipolysis is sn-1,2-diacylglycerol (DG) that is released into the hemolymph (10,11) and loaded into the hemolymph lipoprotein, lipophorin.…”

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confidence: 99%

Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body

Patel

Soulages

Hariharasundaram

et al. 2005

102

127

The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak ϳ10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.Insects rely on lipid reserves to survive during physiological non-feeding periods or to meet the energy requirements of developing eggs, flight, and starvation. The fat body is the principal site for storage of both glycogen and lipids. The fat body synthesizes most of the proteins found in the hemolymph while also serving as the main storage site of triglycerides (TG), 1 which constitute Ͼ90% fat body lipids. Therefore, functionally, the fat body accomplishes roles that in vertebrates are carried out by both liver and adipose tissue (1).The tobacco hornworm, Manduca sexta, feeds constantly, and the content of fat body TG increases continuously until the end of the larval development. During the larval period (ϳ20 days), the content of TG in the fat body increases from a few micrograms to ϳ80 mg (2). During subsequent development, the lipid reserves are used to sustain the life of the adult insect, which feeds occasionally (2-5). Due to these metabolic features, M. sexta represents an excellent model for studying the basic mechanisms involved in either the synthesis/deposition o...

“…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.…”

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confidence: 99%

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

Lindvall¹,

Nevsten²,

Ström³

et al. 2004

Self Cite

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...