出版社：American Society for Biochemistry and Molecular Biology
摘要：Lipid droplets (LDs) function as intracellular storage depotsof neutral lipids. Recently, we identified long-chain acyl-coenzymeA synthetase 3 (ACSL3) as a major LD-associated protein in thehuman hepatocyte cell line HuH7. In this study, we investigatedwhether droplet-associated ACSL is involved in lipid metabolismin LDs. Addition of oleic acid (OA) to culture medium was shownto enhance the intracellular accumulation of LDs in the cells,which was accompanied by an increase of droplet ACSL3. WhenLD-enriched cells induced by OA were further incubated withoutOA for 3 days, 80% of LDs were retained in the cells. Conversely,cellular LD content was greatly decreased after the additionof an ACSL inhibitor, triacsin C. This was accompanied by aconcomitant decrease of the droplet ACSL3. Incubation of isolatedLD fractions with 14C-labeled OA or palmitic acid resulted in[14C]acyl-CoA generation in vitro, indicating the presence ofACSL activity in LDs. The droplet ACSL activity varied accordingto the quantity of LDs in their emergence and disappearancein cells. Incubation of the LD fraction with [14C]oleoyl-CoAresulted in radioactive triacylglycerol and cholesteryl esters.These results suggest that LD ACSL activity is involved in localsynthesis 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
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
