The Turnover of Cytoplasmic Triacylglycerols in Human Fibroblasts Involves Two Separate Acyl Chain Length-dependent Degradation Pathways

Hilaire, Nathalie; Salvayre, Robert; Thiers, Jean-Claude; Bonnafé, Marie-José; Nègre‐Salvayre, Anne

doi:10.1074/jbc.270.45.27027

Cited by 43 publications

(60 citation statements)

References 45 publications

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“…In contrast, a dramatic accumulation of TAG in humans results in severe defects as observed with patients suffering from a metabolic disorder named neutral lipid storage disease (38). In cultured neutral lipid storage disease fibroblasts TAG with long acyl chains (C 12 and longer) remained undegraded, whereas short/medium chain (C 4 to C 10 ) TAG species and endogenous STE were mobilized at a normal rate (39). This result is reminiscent of the slight substrate specificity of the yeast Tgl3p in vivo (see Fig.…”

Section: Fig 2 Sequence and Hydropathy Blot Of Tgl3pmentioning

confidence: 89%

YMR313c/TGL3 Encodes a Novel Triacylglycerol Lipase Located in Lipid Particles of Saccharomyces cerevisiae

Athenstaedt

Daum

2003

Journal of Biological Chemistry

194

161

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Previous work from our laboratory (Athenstaedt, K., Zweytick, D., Jandrositz, A., Kohlwein, S. D., and Daum, G. (1999) J. Bacteriol. 181, 6441-6448) showed that the gene product of YMR313c (named Tgl3p) is a component of yeast lipid particles, and deletion of this gene led to an increase in the cellular level of triacylglycerols (TAG). These observations suggested that TGL3 may encode a TAG lipase of Saccharomyces cerevisiae. Here we demonstrate by cell fractionation and by microscopic inspection of a strain bearing a Tgl3p-GFP hybrid that this polypeptide is highly enriched in the lipid particle fraction but virtually absent from other organelles. The entire TAG lipase activity of lipid particles is attributed to Tgl3p, because the activity in this organelle is completely absent in a ⌬tgl3 deletion mutant, whereas it is significantly enhanced in a strain overexpressing Tgl3p. A His 6 -tagged Tgl3p hybrid purified close to homogeneity from a yeast strain overexpressing this fusion protein exhibited high TAG lipase activity. Most importantly, experiments in vivo using the fatty acid synthesis inhibitor cerulenin demonstrated that deletion of TGL3 resulted in a decreased mobilization of TAG from lipid particles. The amino acid sequence deduced from the open reading frame YMR313c contains the consensus sequence motif GXSXG typical for lipolytic enzymes. Otherwise, Tgl3p has no significant sequence homology to other lipases identified so far. In summary, our data identified Tgl3p as a novel yeast TAG lipase at the molecular level and by function in vivo and in vitro.

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Section: Fig 2 Sequence and Hydropathy Blot Of Tgl3pmentioning

confidence: 89%

YMR313c/TGL3 Encodes a Novel Triacylglycerol Lipase Located in Lipid Particles of Saccharomyces cerevisiae

Athenstaedt

Daum

2003

Journal of Biological Chemistry

194

161

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“…Mutations of human CGI-58 are responsible for excessive triacylglycerol storage in many types of cells (23)(24)(25). Studies of triacylglycerol metabolism in fibroblasts from NLSD patients show that CGI-58 is required for normal rates of triacylglycerol turnover (34 -36); however, triacylglycerol lipase activity in NLSD cell lysates was comparable to that of control cells (34). Additionally, the rate of triacylglycerol clearance increased in NLSD cells incubated with triacsin C, an inhibitor of acyl CoA synthetases, although not quite to the higher rates observed in control cells (35).…”

Section: Fig 6 Cgi-58 Localizes To Lipid Droplets In 3t3-l1 Preadipmentioning

confidence: 99%

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

Subramanian

Rothenberg

Gómez

et al. 2004

Journal of Biological Chemistry

275

290

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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...

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“…Instead, fibroblasts from CDS patients seem to lack the ability to hydrolyze stored TG, even though cellular lipase and carboxylesterase activities are normal (20)(21)(22). As a result of this paradoxical finding, it has been speculated that TG lipases in these cells may be missing some critical cofactor, be mislocalized, or be unable to gain access to the LD surface (23,24). This paradox may be partially explained by the fact that CDS fibroblasts exhibit abnormal recycling of TG-derived monoacylglycerols or diacylglycerols into major PL species (25,26).…”

mentioning

confidence: 90%

“…Indeed, in 2001, the genetic mutation and the speculated "missing cofactor" (23,24) was identified as Comparative Gene Identification-58 (CGI-58) (27), also known as a/b-hydrolase domain-containing 5. Several studies have since confirmed that multiple loss-of-function mutations in CGI-58 are causative of CDS (28)(29)(30)(31).…”

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confidence: 99%

CGI-58 facilitates the mobilization of cytoplasmic triglyceride for lipoprotein secretion in hepatoma cells

Brown

Chung

Das

et al. 2007

Journal of Lipid Research

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Comparative Gene Identification-58 (CGI-58) is a member of the a/b-hydrolase family of proteins. Mutations in the human CGI-58 gene are associated with ChanarinDorfman syndrome, a rare autosomal recessive genetic disease in which excessive triglyceride (TG) accumulation occurs in multiple tissues. In this study, we investigated the role of CGI-58 in cellular lipid metabolism in several cell models and discovered a role for CGI-58 in promoting the packaging of cytoplasmic TG into secreted lipoprotein particles in hepatoma cells. Using both gain-of-function and loss-of-function approaches, we demonstrate that CGI-58 facilitates the depletion of cellular TG stores without altering cellular cholesterol or phospholipid accumulation. This depletion of cellular TG is attributable solely to augmented hydrolysis, whereas TG synthesis was not affected by CGI-58. Furthermore, CGI-58-mediated TG hydrolysis can be completely inhibited by the known lipase inhibitors diethylumbelliferyl phosphate and diethyl-p-nitrophenyl phosphate, but not by p-chloro-mercuribenzoate. Intriguingly, CGI-58-driven TG hydrolysis was coupled to increases in both fatty acid oxidation and secretion of TG. Collectively, this study reveals a role for CGI-58 in coupling lipolytic degradation of cytoplasmic TG to oxidation and packaging into TG-rich lipoproteins for secretion in hepatoma cells. The ability to store excess energy in the form of triglyceride (TG) is a critical defense mechanism to help organisms survive extended periods of fuel deprivation. Therefore, elegant systems have evolved to facilitate the storage of excess energy into adipose tissue in the form of TG-rich lipid droplets (LDs) or "adiposomes" (1). Although adipocytes have the unique ability to store large amounts of cytoplasmic TG, almost all tissues can synthesize and store TG in smaller cytoplasmic LDs under normal and pathologic conditions (2-4). The metabolic fate of TG in cytoplasmic LDs differs among cell types (3,(5)(6)(7)(8). Hepatocytes in the liver and enterocytes in the intestine are lipoprotein-producing cells, having the unique ability to efficiently package lipid cargo into apolipoprotein B (apoB)-containing lipoproteins that ultimately deliver these lipids to all peripheral tissues. This assembly of apoB-containing lipoproteins is widely accepted as a twostep process (7,8). The first step involves the cotranslational transfer of a small amount of phospholipids (PLs) and TGs to apoB by the actions of microsomal triglyceride transfer protein (MTP). This step results in a small dense precursor particle (?25 nm) residing within the endoplasmic reticulum (ER) lumen. The more elusive second step involves the bulk addition of TG to these precursor particles to form large (30-80 nm) secretion-competent VLDLs. This second step expansion occurs through an obscure process involving 1) the lipolysis of cytoplasmic LD-associated TG, 2) the movement of liberated acylglycerols toward the ER, and 3) the reesterification of acylglycerols to form a large lumenal TG-rich dro...

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The Turnover of Cytoplasmic Triacylglycerols in Human Fibroblasts Involves Two Separate Acyl Chain Length-dependent Degradation Pathways

Cited by 43 publications

References 45 publications

YMR313c/TGL3 Encodes a Novel Triacylglycerol Lipase Located in Lipid Particles of Saccharomyces cerevisiae

YMR313c/TGL3 Encodes a Novel Triacylglycerol Lipase Located in Lipid Particles of Saccharomyces cerevisiae

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

CGI-58 facilitates the mobilization of cytoplasmic triglyceride for lipoprotein secretion in hepatoma cells

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