Involvement of long chain acyl-CoA synthetase in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7

Yasuyuki Fujimoto, Hiroyuki Itabe, Tetsuaki Kinoshita, Koichi J. Homma, Jun Onoduka, Masahiro Mori, Shinji Yamaguchi, Minoru Makita, Yusuke Higashi, Atsushi Yamashita, and Tatsuya Takano

Molecular Pathology, Teikyo University, Sagamihara, Kanagawa 199-0195

Corresponding Author: 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-CoA synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line, HuH7. In the current 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 increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased following addition of an ACSL inhibitor, triacsin C, and was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with 14C-labeled oleic acid 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 quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [14C]-oleoyl-CoA resulted in radioactive triglyceride and cholesteryl esters. These results suggest that lipid droplet ACSL activity is involved in local synthesis of neutral lipids and LD formation.

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