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Originally published In Press as doi:10.1194/jlr.M700050-JLR200 on March 22, 2007

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Journal of Lipid Research, Vol. 48, 1280-1292, June 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7

Yasuyuki Fujimoto1,*, Hiroyuki Itabe{dagger}, Tetsuaki Kinoshita*, Koichi J. Homma*, Jun Onoduka*, Masahiro Mori*, Shinji Yamaguchi*, Minoru Makita*, Yusuke Higashi§, Atsushi Yamashita** and Tatsuya Takano2,*

* Department of Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Sagamihara, Kanagawa 199-0195, Japan
{dagger} Department of Biological Chemistry, School of Pharmaceutical Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan
§ Section of Cardiology, Department of Medicine, Tulane University Health Sciences Center, New Orleans, LA 70112
** Department of Molecular Health Sciences, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Sagamihara, Kanagawa 199-0195, Japan

Published, JLR Papers in Press, March 22, 2007.

1 Present address of Y. Fujimoto: School of Pharmacy, Iwate Medical University, Yahaba, Iwate 028-3694, Japan (yfujimot{at}iwate-med.ac.jp).

2 To whom correspondence should be addressed. e-mail: t_takano{at}pharm.teikyo-u.ac.jp

Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, ~80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with 14C-labeled OA or palmitic acid resulted in [14C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [14C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.

Supplementary key words triacsin C • triacylglycerol • acyl-coenzyme A

Abbreviations: ACSL, long-chain acyl-coenzyme A synthetase; ADRP, adipose differentiation-related protein; CE, cholesteryl ester; DG, diacylglycerol; DGAT, acyl-coenzyme A:diacylglycerol acyltransferase; ER, endoplasmic reticulum; FC, free cholesterol; LD, lipid droplet; LDH, lactate dehydrogenase; MAM, mitochondria-associated membrane; NSDHL, NAD(P)H steroid dehydrogenase-like protein; OA, oleic acid; PNS, postnuclear supernatant; TG, triacylglycerol


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