Abstract
Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 mum [1-(14)C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, beta-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and beta-oxidation. The increase in [1,2,3-(3)H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [(14)C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [(14)C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.
Highlights
Long-chain acyl-CoA synthetases (ACSLs)3 catalyze the first step in fatty acids (FA) metabolism by converting long-chain FA into acyl
In ACSL1 heart-specific transgenic mice, TAG and PL accumulate in heart muscle in the absence of changes in CE or -oxidation [9], and when ACSL5 is overexpressed in rat hepatoma McArdle-RH777 cells, it partitions exogenously derived FA toward TAG synthesis and storage, but not toward PL or CE synthesis [10]
Studies suggest that ACSL1 is important for TAG synthesis in adipocytes and fibroblasts [4, 11], conflicting data exist concerning the function of ACSL1 in liver
Summary
Materials—DNA restriction endonucleases and ligase for recombinant adenovirus construction were from New England Biolabs. Cell Labeling and Lipid Extraction and Analysis—Twentyone h after adenoviral infection (Ad-GFP or Ad-Acsl1) at 20 m.o.i., hepatocytes (1.5 ϫ 106 cells in 60-mm dishes) were labeled with 3 ml of MEM containing 1.0 Ci of [1-14C]oleate bound to bovine serum albumin (essentially FA free) in a 3:1 molar ratio for 3 h [10]. After a 3-h incubation, the cells were either collected (pulse) as described above, or washed twice with 1% bovine serum albumin in PBS and incubated for an additional 14 h in MEM-DA without added oleate or glycerol (chase). Cell Homogenate Preparations for ACS Activity and ACSL Protein Assays—Hepatocytes infected with Ad-Acsl or Ad-GFP were washed twice with cold PBS and collected in cold Medium A (10 mM Tris, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM dithiothreitol, and Protease Inhibitor Mixture (Sigma))
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