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J. Biol. Chem., Vol. 279, Issue 39, 40683-40689, September 24, 2004

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Carboxylesterase 3 (EC 3.1.1.1) Is a Major Adipocyte Lipase^*

Krishnakant G. Soni, Richard Lehner, Pavel Metalnikov¶, Paul O'Donnell¶, Meriem Semache, Wenhui Gao, Keith Ashman||, Alexey V. Pshezhetsky**, and Grant A. Mitchell

From the Division of Medical Genetics, Ste-Justine Hospital, Montréal, Québec H3T 1C5, Canada, Canadian Institutes of Health Research Group on Molecular and Cell Biology of Lipids, University of Alberta, Edmonton, Alberta T6G 2S2, Canada, ^¶Samuel Lunenfeld Institute, Toronto, Ontario M5G 1X5, Canada, and ^||MDS Sciex, Concord, Ontario L4K 4V8, Canada

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Received for publication, January 17, 2004 , and in revised form, June 23, 2004.

^* This work was supported in part by Canadian Institutes of Health Research Grant 18961 (to G. A. M.) and a Canadian Foundation for Innovation equipment grant (to A. V. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** National Researcher of the Fonds de Recherche en Santé du Quebec.

Senior Researcher of the Fonds de Recherche en Santé du Quebec. To whom correspondence should be addressed: Division of Medical Genetics, Sainte-Justine Hospital, 3175 Côte Ste-Catherine Rd., Montréal, Québec H3T 1C5, Canada. Tel.: 514-345-4931 (ext. 4727); Fax: 514-345-4766; E-mail: grant.mitchell{at}recherche-ste-justine.qc.ca.

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