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Originally published In Press as doi:10.1074/jbc.M604427200 on October 6, 2006

J. Biol. Chem., Vol. 281, Issue 48, 37246-37255, December 1, 2006
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Overexpression of Rat Long Chain Acyl-CoA Synthetase 1 Alters Fatty Acid Metabolism in Rat Primary Hepatocytes*

Lei O. Li{ddagger}, Douglas G. Mashek{ddagger}1, Jie An§, Scott D. Doughman{ddagger}, Christopher B. Newgard§, and Rosalind A. Coleman{ddagger}2

From the {ddagger}Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina 27599 and the §Departments of Medicine and Pharmacology & Cancer Biology, and the Sarah W. Stedman Nutrition and Metabolism Center, Duke University, Durham, North Carolina 27710

Received for publication, May 9, 2006 , and in revised form, September 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM [1-14C]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 [1,2,3-3H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [14C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [14C]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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Long-chain acyl-CoA synthetases (ACSLs)3 catalyze the first step in FA metabolism by converting long-chain FA into acyl-CoA thioesters. Acyl-CoAs enter both anabolic and catabolic pathways (1), and the disturbance of these pathways is linked to disorders such as hepatic steatosis, hyperlipidemia, and insulin resistance. Five ACSL isoforms, each the product of a separate gene, have been cloned and characterized in mammals (2, 3). Even though individual ACSL isoforms have different substrate preferences, enzyme kinetics, and cellular and subcellular locations, and are regulated uniquely (4-6), the significance of this diversity is unknown. We hypothesized that, instead of being redundant, individual ACSL isoforms might channel FA into distinct metabolic pathways.

Evidence for the importance of ACSL in FA channeling comes from studies with triacsin C, an inhibitor of recombinant ACSL1, -3, and -4, but not ACSL5 or -6 (5, 6). For example, in hepatocytes, in which Acsl1, -3, -4, and -5 are abundant (7), triacsin C inhibits TAG synthesis 70% but inhibits oleate incorporation into phospholipids and beta-oxidation only 34% (8). More direct evidence comes from studies that overexpress individual ACSL isoforms. In ACSL1 heart-specific transgenic mice, TAG and PL accumulate in heart muscle in the absence of changes in CE or beta-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). However, the exact function of other ACSL isoforms in liver remains largely unexplored, in part because we lack inhibitors that can exclusively inhibit one isoform without affecting others.

Although 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. Supporting a role for ACSL1 in TAG synthesis is its location in the ER and MAM, which are sites of TAG synthesis, and its absence from mitochondria, the major site of FA beta-oxidation (12). Further, Acsl1 mRNA is induced when previously fasted rats are refed with a high fat or high sucrose diet that favors lipogenesis (13, 14), and hepatic ACS specific activity and Acsl1 mRNA are enhanced in obese and hypertriglyceridemic rats that have fatty livers (15, 16). On the other hand, in support of a role for ACSL1 in providing FA for beta-oxidation are data showing that PPAR{alpha} agonists, which up-regulate genes for both FA beta-oxidation and de novo FA synthesis (17), increase total ACS activity in rat liver (18) and Acsl1 mRNA expression in rat liver (18) and in the rat liver-derived cell line AML-12 (19). This up-regulated transcription is mediated by a PPAR-responsive element in the promoter of the Acsl1 gene (20).

To elucidate the specific role of ACSL1 in hepatic FA metabolism, we overexpressed rat ACSL1 in rat primary hepatocytes. We hypothesized that ACSL1 would channel FA toward some specific lipid metabolic pathways and away from others.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—DNA restriction endonucleases and ligase for recombinant adenovirus construction were from New England Biolabs. Human embryonic kidney 293 and Chinese hamster ovary cells were from the American Type Culture Collection. MEM, nonessential amino acids, FBS, and tissue culture dishes were from Invitrogen. Rat-tail collagen I was from Collaborative Biomedical Products. Silica gel G plates were from Whatman (cat. No. 4865-821). [1-14C]Acetate, [1,2,3-3H]glycerol, and [1-14C]oleate were obtained from PerkinElmer Life Sciences, and [14C]palmitate was from PerkinElmer Life Sciences. Lipid standards were from Sigma and Avanti%20Polar%20Lipids">Avanti Polar Lipids. Polyacrylamide stock was from National Diagnostics. Lab-TekTM II Chamber SlidesTM were from Nunc. RNeasy kit was from Qiagen. Chemicals were from Sigma-Aldrich unless otherwise indicated.

Construction of pACCMV-Acsl1FLAG Adenovirus—A fulllength rat Acsl1 cDNA with a C-terminal FLAG epitope (DYKDDDDK) was subcloned from a previously constructed pFLAG-CTC plasmid (5) into a shuttle vector, pACCMVpLpA at the BamHI and SalI sites. Expression of the inserted Acsl1FLAG cDNA is driven by the cytomegalovirus promoter. The inserted ACSL1FLAG in the pACCMV-Acsl1FLAG construct was verified by restriction enzyme analysis and confirmed by DNA sequencing at the University of North Carolina (UNC) DNA sequencing facility. Expression and activity of the pACCMV-Acsl1FLAG construct were confirmed by transient transfection into Chinese hamster ovary cells for 24 h, followed by ACS activity assay and anti-FLAG Western blot. The pACCMV-Acsl1FLAG construct was co-transfected with an adenoviral DNA, pJM17, into human embryonic kidney 293 cells for homologous recombination to form recombinant adenovirus carrying Acsl1FLAG cDNA (Ad-Acsl1) (21). After plaque purification, virions were further purified and amplified by the UNC Vector Core Facility. A virus containing a GFP gene under control of the cytomegalovirus promoter (Ad-GFP) was used for control infections (10).

Hepatocyte Isolation and Adenovirus Infection—Animal protocols were approved by the UNC Institutional Animal Care and Use Committee. Male Wistar rats (250-300 g) were housed in a 12:12-h light-dark cycle and were allowed free access to food (Prolab® Rat/Mouse/Hamster 3000 diet, Labdiet) before hepatocyte isolation. Primary hepatocytes were isolated by collagenase perfusion by the UNC Cellular Metabolism and Transport Core. Cell viability, determined by trypan blue exclusion, exceeded 90%. Hepatocytes were seeded at a density of 1.5 x 106 cells per 60 mm or 4.5 x 106 cells per 100-mm collagen-coated dish in MEM supplemented with 10% FBS (v/v), 50 units/ml penicillin, and 50 mg/ml streptomycin (22). After cells attached (4-5 h), recombinant adenoviruses (Ad-GFP or Ad-Acsl1) were added for 2 h at 37 °C in serum-free MEM. Infection medium was removed and replaced by MEM containing 10% FBS, 10 nM dexamethasone, and 0.1 mM nonessential amino acid (MEM-DA).

For dose-dependent expression, hepatocytes were infected with Ad-Acsl1 at m.o.i. values of 5, 10, 20, or 50 for 2 h. Uninfected cells or Ad-GFP-infected cells (20 m.o.i.) served as controls. After 18 h, cells were washed with cold PBS, and homogenates were collected as described below. For the time course, hepatocytes were infected with Ad-GFP or Ad-Acsl1atan m.o.i. of 20. Homogenates were collected after 12, 18, 24, 26, or 36 h of incubation for Ad-Acsl1-infected cells and after 18 h of incubation for Ad-GFP-infected cells.

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 x 106 cells in 60-mm dishes) were labeled with 3 ml of MEM containing 1.0 µCi of [1-14C]oleate bound to bovine serum albumin (essentially FA free) in a 3:1 molar ratio for 3 h (10). Although ACS activity with oleate was ~20% lower than that with palmitate in both GFP and Acsl1 adenovirus-infected cells (data not shown), oleate was used for labeling to avoid lipotoxicity (23, 24). The radiolabeling medium, which included 1 mM carnitine, contained a final concentration of 100 µM or 750 µM oleate (22). In some experiments cells were incubated with 250 µM [1,2,3-3H]glycerol (1.1 µCi) or 2.5 mM [1-14C]acetate (1.0 µCi). The medium was collected for ASM measurement or extracted to measure radiolabel incorporation into secreted lipids (10, 22). Hepatocytes were washed twice with 1% bovine serum albumin in PBS at 37 °C, and cellular lipids were extracted (25). For pulse-chase experiments, hepatocytes were infected with adenovirus for 21 h (as above) and incubated with 750 µM [1-14C]oleate in the presence of 250 µM unlabeled glycerol, or with 250 µM [1,2,3-3H]glycerol in the presence of 750 µM unlabeled oleate. 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 then incubated for an additional 14 h in MEM-DA without added oleate or glycerol (chase). The medium and cells were collected, and lipids were extracted as described.

Aliquots of the lipid extracts from the cells and media were separated by TLC on 0.25-mm silica gel G plates in either hexane:ethyl ether:acetic acid (80:20:1, v/v) for neutral lipids (10) or in chloroform:methanol:acetic acid:water (50:37.5:3.5:2, v/v) for PL (26), together with authentic lipid standards in parallel. The 14C- or 3H-labeled lipids were detected and quantified with a Bioscan 200 Image System.

Cell Homogenate Preparations for ACS Activity and ACSL Protein Assays—Hepatocytes infected with Ad-Acsl1 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, 1mM dithiothreitol, and Protease Inhibitor Mixture (Sigma)) and homogenized on ice with 10 up-and-down strokes with a Teflon-glass motor-driven homogenizer. Homogenate aliquots were stored at -80 °C until use. Protein concentrations were determined by the BCA method (Pierce). ACS specific activity was determined by measuring the production of [14C]acyl-CoA in the presence of 175 mM Tris-HCl, pH 7.4, 8 mM MgCl2, 5 mM dithiothreitol, 10 mM ATP, 0.25 mM CoA, and 50 µM [14C]palmitic acid in 0.5 mM Triton X-100, 0.01 mM EDTA. The assays were performed in a total volume of 200 µl at 37 °C for 5 min. The reaction was started by adding 0.5-1.5 µg of homogenate protein, terminated with 1 ml of Dole's reagent (isopropanol, heptane, 1 M H2SO4, 80:20:2, v/v), and extracted (27). Enzyme assays measured initial rates.

Western Blot Analysis—Homogenates from the dose-dependent infection and time-course incubations (10 µg) were separated by electrophoresis on a 10% polyacrylamide gel with 0.1% SDS and transferred to a polyvinylidene fluoride membrane (Bio-Rad). Immunoreactive bands were detected by incubating the membranes with anti-FLAG M2 monoclonal antibody (Sigma), horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G, and SuperSignal West Pico Chemiluminescent Reagent (Pierce) (28).

Quantitative Real-time-PCR—Hepatocytes were plated at 4.5 x 106cells per 100-mm dish and infected with Ad-GFP or Ad-Acsl1 at an m.o.i. of 20 for 2 h as described above. Thirty hours after infection, RNA was isolated (RNeasy, Qiagen) and stored at -80 °C until use. Samples were analyzed on an ABI Prism 7700 sequence detection system (Applied Biosystems). Primers and corresponding FAM probes are listed in Table 1 except for CPT1 (Applied Biosystems, Rn00580702_m1). Data were analyzed using the relative standard curve method (10).


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  		TABLE 1
Primer and probe sequences used for gene expression by quantitative reverse transcription-PCR for rat primary hepatocytes

 
Immunocytochemistry—Primary hepatocytes were plated on 8-well chamber slides (100,000 cells/well) and infected with Ad-Acsl1 for 18 h as described above. After washing three times with PBS at room temperature, cells were fixed at room temperature with 3.7% formaldehyde in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. Next, cells were washed three times with PBS and blocked in 10% FBS in PBS (blocking buffer) for 30 min at room temperature. Cells were then doubly labeled with anti-FLAG M2 monoclonal antibody (1:400) and individual organelle markers (fluorescein isothiocyanate-concanavalin A (1:500, Miles-Yeda Ltd.) and anti-calnexin polyclonal antibody (1:100, Abcam) for ER, anti-VDAC polyclonal antibody (1:100, ABR) for mitochondria, or rhodamine phalloidin (1:100, Molecular Probes) for actin fiber) in blocking buffer for 1 h at room temperature. Cells were washed three times and then incubated in blocking buffer containing Alexa Fluor® 488 or Alexa Fluor® 568 goat anti-mouse IgG-specific antibodies (1:1000, Molecular Probes) and Alexa Fluor® 568 goat anti-rabbit IgG-specific antibodies (1:1000, Molecular Probes) or cellular stains for 1 h at room temperature. After washing three times for 5 min in PBS, mounting media (Biomedia) was added to the chambers on the detached glass slide and fitted with a no.1 cover glass before being sealed and imaged. Confocal microscopy was performed using a Zeiss 510 Meta Inverted Laser Scanning Confocal Microscope, and LSM 510 META software (Zeiss) for image acquisition.

Cellular Free Cholesterol Measurement—Hepatocytes (4.5 x 106 cells per 100-mm dish) infected for 21 h with Ad-GFP or Ad-Acsl1 at m.o.i. 20 were incubated with oleate at 0, 100, or 750 µM for 3 h, followed by lipid extraction. Free cholesterol was determined using an enzymatic colorimetric assay (Free Cholesterol E, COD-DAOS, Wako).

Statistical Analysis—Data from each group were expressed as means ± S.E. Data were analyzed by Student's t test, and significance was declared at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Adenoviral Overexpression of Acsl1 Increased ACS Activity in Rat Primary Hepatocytes—We infected rat primary hepatocytes with Ad-GFP or an adenovirus containing rat Acsl1 with a FLAG epitope at the C terminus (Ad-Acsl1) and measured the ACS specific activity with palmitate at different virus doses (Fig. 1A) and incubation times (Fig. 1B). After an 18-h infection with Ad-GFP, ACS specific activity did not change (167.8 ± 4.9 versus 155.0 ± 2.6 nmol/min/mg protein in uninfected cells). In contrast, Ad-Acsl1 (18 h) increased ACS specific activity 86-236% at 5-50 m.o.i., with a linear increase through 20 m.o.i. Ad-Acsl1 at 20 m.o.i. increased ACS specific activity linearly for 36 h. Western blotting with anti-FLAG primary antibody showed a band of ~75 kDa, confirming that the increase in ACS specific activity was due to the overexpression of ACSL1. Consistent with the increase in activity, the density of the immunoreactive band increased with the time of incubation and the adenovirus dose. For labeling experiments, we chose 20 m.o.i. and 24 h when ACS specific activity was increased 3.7-fold. Acsl4or Acsl5 are two other major Acsl genes expressed in rat liver. Overexpressed Acsl1 increased Acsl5 mRNA expression 2-fold, but Acsl4 mRNA expression was not altered (data not shown).

Overexpressed ACSL1 Co-localized with ER but Not Mitochondria or Plasma Membrane—Previous studies used subcellular fractionation to show that endogenous ACSL1 in rat liver is present in ER fractions and MAM fraction but not in purified mitochondrial fractions (8, 12). We used confocal microscopy to characterize the intracellular localization of overexpressed ACSL1. FLAG antibody detection of ACSL1-FLAG strongly co-localized with the ER markers concanavalin A (Fig. 2A) and calnexin (Fig. 2B) but not with the mitochondrial membrane marker VDAC (Fig. 2C). Further, staining of actin fibers, which revealed cellular morphology, implied the absence of ACSL1 on the plasma membrane (Fig. 2D). We were unable to use an antiphosphatidylethanolamine N-methyltransferase antibody to identify MAM in hepatocytes.


Figure 1
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  		FIGURE 1.
ACS activity increased in rat hepatocytes overexpressing ACSL1. Rat primary hepatocytes (1.5 x 106 cells/60-mm dish) were uninfected (control) or infected with adenoviruses carrying either GFP (Ad-GFP) or rat Acsl1 (Ad-Acsl1). A, cells were infected with Ad-Acsl1 at different m.o.i. as indicated. After an 18-h incubation, cells were scraped and homogenized; B, cells were infected with Ad-GFP or Ad-Acsl1 (m.o.i. = 20) and homogenates were collected at the indicated times. Total ACS activity was measured, and Western blotting with anti-FLAG monoclonal antibody was performed as described under "Experimental Procedures." Data are reported as means ± S.E. from triplicate dishes. All Ad-Acsl1 versus Ad-GFP, p < 0.001.

 
The ER marker concanavalin A labels lumen-oriented mannose sugars on proteins in ER and Golgi compartments. The polyclonal calnexin antibody labels epitopes on the luminal side of this ER resident protein. Calnexin co-distributed best with ACSL1-FLAG, possibly because both are integral ER proteins. Because ACSL1-FLAG did not co-distribute with equal intensity on ER membranes labeled with concanavalin A, we stained cells for other ER proteins. The chaperone-like luminal ER proteins protein disulfide isomerase and BiP also co-localized with ACSL1-FLAG but reflected staining patterns similar to concanavalin A (data not shown). The anti-rabbit Alexa-568 antibody (Fig. 2, B-D, red) was preferred for the detection of endogenous proteins due to lower background levels in secondary antibody controls (data not shown). These data provided evidence for exogenous ACSL1 localization to ER membranes. Thus, the subcellular location of overexpressed ACSL1 in primary hepatocytes was consistent with the endogenous ACSL1 detected in liver fractionation studies (12).

Ad-Acsl1 Increased [1-14C]Oleate Incorporation into DAG but Not TAG and Decreased [1-14C]Oleate Incorporation into CE—To determine the effects of overexpressed ACSL1 on lipid metabolism, we incubated the adenovirus-infected hepatocytes with 100 or 750 µM [1-14C]oleate, representing physiological concentrations of exogenous FA under fed or fasting conditions, respectively. At 100 µM oleate, incorporation of [1-14C]oleate into [14C]DAG doubled with Ad-Acsl1 (Fig. 3A). Increasing exogenous oleate to 750 µM resulted in a 1.7-fold increase of [14C]DAG in cells infected with Ad-GFP, and this was doubled again by the presence of Ad-Acsl1. Surprisingly, however, despite the Ad-Acsl1-mediated increase in [14C]oleate incorporation into DAG, the incorporation of oleate into TAG remained unchanged at both oleate concentrations (Fig. 3B). The addition of 750 µM oleate increased [14C]TAG 5-fold in both Ad-GFP- and Ad-Acsl1-infected cells. Cellular TAG mass also remained unchanged by the presence of excess ACSL1 (data not shown). Decreasing the adenovirus dose to 10 m.o.i. did not change the incorporation pattern (data not shown), thereby excluding the possibility that cell toxicity related to excess Ad-Acsl1 had inhibited TAG synthesis. Further, the mRNA level of DGAT2, the major enzyme that converts DAG to TAG in liver (29), was unchanged in Ad-Acsl1-infected cells (data not shown).

Acyl-CoAs are also substrates for cholesterol esterification. Compared with Ad-GFP control hepatocytes, the incorporation of [1-14C]oleate into CE was 70% lower in Ad-Acsl1 cells at both 100 and 750 µM (Fig. 3C). To determine whether the amount of cholesterol might be limiting in cells that overexpress ACSL1, we measured cellular free cholesterol after cells were incubated with 0, 100, and 750 µM oleate. Overexpression of ACSL1 did not change free cholesterol content, nor did higher concentration of oleate deplete cholesterol, suggesting that ACSL1 channels acyl-CoAs away from cholesterol esterification (Fig. 3D).

ACSL1 Overexpression Increased [1-14C]Oleate Incorporation into Specific PL—In addition to its effects on neutral lipids, Ad-Acsl1 increased [1-14C]oleate incorporation into total cellular PL 70% and 21% with 100 and 750 µM oleate, respectively (Fig. 4A). At 100 µM oleate, Ad-Acsl1 increased oleate incorporation into PE 164%, PI 104%, and PC 54% (Fig. 4, B-D). At 750 µM oleate, Ad-Acsl1 overexpression increased labeled PE 99% and labeled PI 56%, with no change in labeled PC. Thus, it appears that overexpression of ACSL1 enhanced oleate incorporation predominantly into PE and PI, phospholipids that originate from DAG and phosphatidic acid, respectively.

ACSL1 Overexpression Did Not Affect the Use of [1-14C]Oleate for beta-Oxidation—To determine whether overexpressed ACSL1 provided acyl-CoAs for FA oxidation, we measured labeled ASM in the medium as an indicator of FA beta-oxidation (22). ASM is considered a more accurate measure of beta-oxidation than is CO2 production (30). The incorporation of [1-14C]oleate into ASM was equal in Ad-Acsl1-infected cells and Ad-GFP control cells at both 100 and 750 µM oleate (Fig. 5A), and there was no difference in the mRNA abundance of CPT1, the rate-limiting enzyme for beta-oxidation (data not shown). Thus, overexpression of ACSL1 did not enhance FA degradation.


Figure 2
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  		FIGURE 2.
Ad-Acsl1 is an ER protein in primary hepatocytes. Primary hepatocytes infected with Ad-Acsl1 for 18 h were fixed and labeled with primary antibodies against the FLAG epitope. Detection of ACSL1-FLAG was attained with either secondary anti-mouse IgG-specific Alexa-568 antibodies (A, red), or secondary anti-mouse IgG-specific Alexa-488 antibodies (B-D, green). A, Ad-Acsl1-infected cells were co-stained with FLAG antibodies (red) and fluorescein isothiocyanate-conjugated concanavalin A (green). Arrows in each panel and inset point to regions of intense co-localization on reticulated endomembranes; B, Ad-Acsl1-infected cells co-stained with FLAG antibodies (green) and the ER membrane protein calnexin (red). Arrows point to areas of intense co-localization (upper cell) and areas with less intensity that show co-localization near the cell periphery (central cell); C, co-staining for FLAG (green) and mitochondria marker VDAC (red) with arrows pointing to VDAC immunopositive structures devoid of Ad-Acsl1; D, co-staining with FLAG antibodies (green) and rhodamine-phalloidin (red) with an arrow pointing to an area on the cell surface that is devoid of Ad-Acsl1. Cells not stained with FLAG antibody are uninfected cells. Bar, 10 µm. Images are representative of experiments repeated at least three times. Cells were imaged by confocal microscopy as 0.5-µm slices with a 63x oil immersion objective at 2x magnification.

 
Ad-Acsl1 Decreased [14C]TAG Secreted into the Medium—Despite similar incorporation of oleate into cellular TAG in Ad-Acsl1 and Ad-GFP-infected hepatocytes, Ad-Acsl1 overexpression decreased the amount of labeled TAG secreted by ~53% (Fig. 5B). In contrast, the incorporation of [14C]oleate into secreted DAG and PL remained unchanged (data not shown). These data suggested that overexpression of ACSL1 might alter the assembly or/and secretion of lipoproteins.

Overexpression of ACSL1 Did Not Increase the Total Amount of FA Metabolized—When ACSL5 is overexpressed in McArdle-RH7777 rat hepatoma cells (10), or when ACSL1 is overexpressed in fibroblasts, the uptake of exogenous FA is enhanced (31), suggesting that FA metabolism facilitates the ingress of FA. To determine whether ACSL1 similarly enhances FA uptake by hepatocytes, we calculated the total amount of 1-14C incorporated into cell and medium lipids and into medium ASM. Ad-Acsl1 did not significantly increase the total amount of FA metabolized at either 100 µM oleate (p = 0.06) or 750 µM oleate (p = 0.09) (Fig. 5C). Thus, in hepatocytes, unlike other cells, exogenous FA uptake was not enhanced by excess ACSL1 activity, perhaps because ACSL1 had a different subcellular location or because there were tissue-specific differences in interacting proteins (31, 32).

ACSL1 Increased Oleate Recycling to TAG and PL during the Chase—Cellular complex lipids undergo dynamic changes via hydrolysis, remodeling, and re-esterification (33, 34). Thus, we wondered whether the absence of enhanced [14C]oleate incorporation into TAG despite a doubling of [14C]DAG was due to increased TAG hydrolysis or to diminished TAG reacylation. To examine these possibilities, we labeled cells with 750 µM [1-14C]oleate for 3 h and followed the fate of labeled complex lipids for 14 h afterward.

In Ad-GFP-infected hepatocytes, 33% of the labeled cellular TAG was lost during the 14-h chase (Fig. 6A). Overexpression of ACSL1 attenuated the loss of label from TAG, resulting in only an 18% decrease, and also helped to retain label present in PL. In contrast, the amount of labeled PL decreased 19% in the Ad-GFP-infected cells. The diminished changes in the amount of 14C-labeled TAG and lack of change in the amount of 14C-labeled PL suggested that Ad-Acsl1 enhanced FA re-esterification of TAG and PL. Because the amount of labeled DAG decreased 27% in Ad-GFP controls during the chase, whereas it decreased 59% in the cells that overexpressed ACSL1 (Fig. 6A), it appeared that ACSL1 maintained label in TAG and PL partly by re-esterifying FA that had been released from DAG.


Figure 3
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  		FIGURE 3.
ACSL1 overexpression increased [1-14C]oleate incorporation into cellular DAG but not TAG. Primary hepatocytes (1.5 x 106 cells/60-mm dish) were infected with Ad-GFP (open bar) or Ad-Acsl1 (filled bar) at 20 m.o.i. After a 21-h infection, cells were incubated with 100 µM or 750 µM [1-14C]oleate for 3 h and harvested. Cellular lipids were extracted and [1-14C]oleate incorporation into neutral lipid species was determined by TLC as described under "Experimental Procedures." [1-14C]Oleate incorporation into cellular DAG (A), TAG (B), CE (C) and E, unesterified FA. D, cell free cholesterol, Ad-GFP, or Ad-Acsl1-infected cells were incubated with 0, 100, or 750 µM oleate for 3 h and free cholesterol was determined. Data are reported as means ± S.E. from a representative experiment performed in triplicate dishes that was repeated four times. Ad-Acsl1 versus Ad-GFP, * p < 0.05; ** p < 0.01.

 


Figure 4
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  		FIGURE 4.
ACSL1 overexpression increased [1-14C]oleate incorporation into cellular PL. Hepatocytes were infected with either Ad-GFP (open bar) or Acsl1 (filled bar) for 21 h, and incubated with 100 µM or 750 µM [1-14C]oleate for 3 h. Cellular lipids were extracted and [1-14C]oleate incorporation into different PL species was determined by TLC. [1-14C]oleate incorporation into cellular PL (A), PE (B), PI (C), and PC (D). Data are reported as means ± S.E. from a representative experiment performed in triplicate dishes that was repeated four times. Ad-Acsl1 versus Ad-GFP; **, p < 0.01; ***, p < 0.001.

 
ACSL1 Decreased the Recycling of Oleate into CE and beta-Oxidation Metabolites—In both the Ad-GFP and Ad-Acsl1 cells during the 14-h chase, the amount of 14C label in CE increased markedly compared with the label present at 3 h (Fig. 6B). However, 64% less label was recycled into CE in Ad-Acsl1 cells. This decrease in [14C]CE was similar to the reduced labeling (70%) observed during the initial 3-h incubation and was consistent with the interpretation that ACSL1 directs acyl-CoAs away from CE synthesis. In contrast, however, to the lack of effect of Ad-Acsl1on[14C]ASM observed during the 3-h pulse (Fig. 5A), Ad-ACSL1-infected cells incorporated 42% less label into ASM during the chase. These data suggested that the 14C label in TAG and PL was retained at the expense of FA oxidation (Fig. 6C).

Overexpressed ACSL1 Increased [14C]Oleate Incorporation into DAG and PL via Both de Novo and Reacylation Pathways—To determine whether overexpressed ACSL1 affected de novo glycerolipid synthesis, we incubated hepatocytes with 250 µM [1,2,3-3H]glycerol in the presence of 750 µM unlabeled oleate for either 3 h or for 3 h followed by a 14-h chase in the absence of labeled glycerol. Similar to the pattern of oleate incorporation during a 3-h incubation, overexpressed ACSL1 increased [3H]glycerol incorporation into DAG and PL 79 and 14%, respectively (Fig. 7A), suggesting that ACSL1 increased de novo synthesis of DAG and PL. Because ACSL1 overexpression increased incorporation into DAG and PL from oleate more than from glycerol, it is likely that reacylation contributed to the increase in labeled DAG and PL. Similar to the [14C]oleate study, [3H]TAG remained unchanged by Ad-Acsl1, again suggesting that ACSL1 overexpression did not increase de novo TAG synthesis despite the increase in [3H]DAG. During the chase, again similar to the effects of ACSL1 on [14C]oleate recycling, more [3H]TAG and [3H]PL and less [3H]DAG remained in the Ad-Acsl1-infected cells (Fig. 7B), confirming that ACSL1 altered lipid recycling in the hepatocytes by retaining labeled TAG and PL and by metabolizing DAG.


Figure 5
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  		FIGURE 5.
ACSL1 overexpression decreased [1-14C]oleate metabolism to medium TAG but not to beta-oxidation. Hepatocytes were seeded and infected with Ad-GFP (open bar) or Ad-Acsl1 (filled bar) as described under "Experimental Procedures." Cells were incubated with 100 µM or 750 µM [1-14C]oleate for 3 h, and the cells and medium were collected and extracted. Total FA metabolized includes cell and medium lipids and ASM. [1-14C]oleate incorporation into medium ASM (A), medium TAG (B), and total metabolized [1-14C]oleic acid (C). Data are shown as means ± S.E. from a representative experiment performed in triplicate dishes and repeated four times. Ad-Acsl1 versus Ad-GFP; **, p < 0.01; ***, p < 0.001.

 


Figure 6
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  		FIGURE 6.
Overexpression of ACSL1 altered [14C]oleate recycling in hepatocytes. Hepatocytes were plated and infected with Ad-GFP (open bar) or Ad-Acsl1 (filled bar) for 21 h. Cells were labeled with 750 µM [1-14C]oleate for 3 h and then were either collected for lipid extraction (pulse) or were washed and incubated with new media containing no FA for 14 h (chase), as described under "Experimental Procedures." The remaining 14C label was analyzed in cellular lipid extracts or medium ASM in chased cells, and compared with pulsed cells. A, remaining label in cellular lipids as a percentage of the label present at 3-h pulse, the label in TAG decreased from 179,114 ± 4,242 dpm to 120,231 ± 5,106 dpm/1.5 x 106 Ad-GFP cells and from 167,514 ± 4,843 dpm to 138,119 ± 2,032 dpm/1.5 x 106 Ad-Acsl1 cells, and the label in DAG decreased from 11,732 ± 588 dpm to 8,577 ± 242 dpm/1.5 x 106 Ad-GFP cells and from 21,740 ± 488 dpm to 8,912 ± 374/1.5 x 106 Ad-Acsl1 cells; B,[14C]oleate incorporation into cellular CE during the 3-h pulse and 14-h chase; C,[14C]oleate incorporation into ASM during the 14-h chase. Data are shown as means ± S.E. from a representative experiment performed in triplicate dishes and that was repeated three times. #, significantly different from pulsed cells, p < 0.01; Ad-Acsl1 versus Ad-GFP; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
ACSL1 Overexpression Increased the Incorporation of FA Derived from de Novo Synthesis into TAG, DAG, and PL—Liver metabolizes both FA imported from the blood and FA synthesized de novo. It has been suggested that FA derived from different sources might have distinct fates (35). We reported that ACSL5 exclusively activates exogenous FA but not FA synthesized endogenously (10). To examine the selectivity of ACSL1, we labeled the adenovirus-infected hepatocytes with 2.5 mM [1-14C]acetate. ACSL1 increased [1-14C]acetate incorporation into DAG and PL 83% and 61%, respectively (Fig. 7C), similar to the effect observed with [14C]oleate incorporation (Fig. 3). In contrast, however, to the [14C]oleate study, overexpressed ACSL1 enhanced [1-14C]acetate incorporation into TAG 13% and did not significantly decrease [14C]acetate incorporation into CE. Lack of effect on [14C]CE may reflect the fact that most of the 14C label in CE was probably derived from de novo synthesized cholesterol rather than from FA synthesis. Thus, it appeared that ACSL1 can activate FA derived from both exogenous and de novo sources and commit them to the synthesis of DAG and PL.


Figure 7
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  		FIGURE 7.
ACSL1 overexpression increased [1,2,3-3H]glycerol incorporation into DAG and PL, and activates FA derived from do novo synthesis. Primary hepatocytes (1.5 x 106 cells/60-mm dish) were infected with Ad-GFP (open bar) or Ad-Acsl1 (filled bar) at 20 m.o.i. for 21 h. Cells were labeled with 250 µM [1,2,3-3H]glycerol in the presence of 750 µM unlabeled oleate (A and B) or 2.5 mM [1-14C]acetate (C). Cells were either collected for lipid extraction after 3 h labeling (pulse) (A and C), or were washed and incubated with new media containing no glycerol nor FA for 14 h (chase) (B), as described under "Experimental Procedures." Remaining 14C label was analyzed in cellular lipid extracts in chased cells and compared with pulsed cells. Data are shown as means ± S.E. from six dishes. #, significantly different from pulsed cells, p < 0.05; Ad-Acsl1 versus Ad-GFP; *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The major findings of this study are that overexpression of ACSL1 altered FA incorporation into specific pathways in rat primary hepatocytes and that these pathways differed from those previously observed after overexpression of ACSL1 in other types of cells. Previous studies in NIH3T3 fibroblasts and PC12 neuronal cells strongly suggest that overexpressed ACSL1 provides acyl-CoAs that are incorporated primarily into TAG (11, 36), and in vivo studies of ACSL1 heart-specific transgenic mice show a marked increase in heart TAG mass (9). In addition, during the differentiation of 3T3-L1 preadipocytes into adipocytes, Acsl1 mRNA increases 160-fold, whereas other Acsl isoforms remain unchanged, concomitant with a 100-fold increase in microsomal ACS specific activity and TAG accumulation (36, 37). Acsl1 mRNA is up-regulated in liver by PPAR{alpha} (18, 20) and in adipose tissue by PPAR{gamma} (38), suggesting that the function of ACSL1 might differ in these tissues.

In contrast to other overexpression studies of ACSL1 (11) and ACSL5 (10), overexpressed ACSL1 in hepatocytes did not increase TAG mass or [14C]oleate incorporation into TAG despite a doubling in [14C]oleate incorporation into DAG. Because less [14C]TAG was hydrolyzed in the Ad-Acsl1-infected cells during the 14-h chase, it appeared that ACSL1 overexpression either diminished the rate of lipid hydrolysis or increased recycling of hydrolyzed [14C]oleate back to TAG. We favor the latter explanation because more [14C]DAG was lost from the Ad-Acsl1-infected cells. Thus, an increase in glycerolipid turnover cannot explain the inconsistency between the increased [14C]DAG without a concomitant increase in [14C]TAG. Incubating cells with [3H]glycerol confirmed the pattern seen with [14C]oleate incorporation. In addition, consistent with the pattern of oleate recycling during the chase, the 3H label increased in TAG and PL, and decreased in DAG, again supporting the hypothesis that ACSL1 increases FA reacylation.

In liver, both ER and MAM are enriched with enzymes of TAG and phospholipid synthesis, including PS, PE, and PC (39, 40). Previous studies identified endogenous ACSL1 in both ER and MAM fractions from rat liver. The present studies in hepatocytes showed co-localization of overexpressed ACSL1 with ER, but not mitochondria or plasma membrane. Although co-localization of ACSL1-FLAG and MAM could not be assessed in current study, the increase in phospholipid synthesis by Ad-ACSL1 is consistent with a location in MAM as well as in ER. Thus, it appears that, instead of being channeled toward TAG synthesis, acyl-CoAs were used primarily to synthesize PL. This is consistent with the previous finding that choline- and ethanolamine-glycerophospholipid masses increased 50% and 15%, respectively, in heart-specific Acsl1 transgenic mice (9). Because less [14C]PL and more [14C]DAG was hydrolyzed in Ad-Acsl1 cells during the chase, ACSL1 may have increased FA incorporation into PL by enhancing FA recycling or by increasing the use of labeled DAG. These data are strikingly different from a study of overexpressed ACSL5 in McArdle-RH7777 rat hepatoma cells in which no increase was observed in either oleate or glycerol incorporation into cellular PL despite an increase in incorporation into DAG (10), again suggesting that ACSL1 and ACSL5 commit FA to different metabolic fates.

The incorporation of [14C]oleate into CE was markedly lower in cells that overexpressed ACSL1, despite unchanged ACAT2 mRNA abundance or cellular content of free cholesterol. Increasing the exogenous oleate concentration to 750 µM did not change this result, suggesting that neither acyl-CoA nor free cholesterol was limiting. Further, even though both Ad-GFP and Ad-Acsl1 cells were able to use [14C]oleate hydrolyzed from labeled glycerolipids to esterify cholesterol during the chase, the Ad-Acsl1 cells continued to incorporate less oleate into CE (Fig. 6B). Thus, we conclude that overexpressed ACSL1 diverted oleate away from cholesterol esterification. This diversion by ACSL1 contrasts with studies of ACSL5 overexpression in rat hepatoma cells, which did not decrease oleate incorporation into CE (10).

Overexpressed ACSL1 did not alter the amount of FA oxidized during the 3-h oleate incubation, but during the 14-h chase 42% less [14C]oleate was released from complex lipids for oxidization. Thus, even though hepatic Acsl1 is a target of PPAR{alpha} (18-20), our data did not indicate that ACSL1 channels FA into the pathway of beta-oxidation. Although PPAR{alpha} agonists up-regulate genes involved in FA oxidation like CPT1 (41), they also up-regulate DGAT activity (42). Treatment of mice with a PPAR{alpha} agonist increased the expression of acetyl-CoA carboxylase and stearoyl-CoA desaturase-1, which are involved in de novo FA and TAG synthesis; this increase was attributed to an increase in the amount of nuclear sterol regulatory element binding protein-1c (17). Thus, PPAR{alpha} regulation of Acsl1 may enhance lipogenesis as well as increase beta-oxidation.

Despite unchanged [14C]oleate incorporation into cellular TAG, Ad-Acsl1 cells secreted less [14C]TAG into the medium, perhaps due to insufficient CE for VLDL synthesis. Although the role of CE in the assembly and secretion of VLDL is controversial (43, 44), CE availability appears to be important, because ACAT inhibitors reduce apoB100 secretion in primary rat hepatocytes and HepG2 cells (45) and overexpression of ACAT1 and ACAT2 stimulates apoB-containing lipoproteins in McArdle-RH7777 cells (44). Additionally, because in rat hepatocytes cytosolic TAG is not incorporated en bloc into the ER for VLDL biogenesis and secretion (46, 47), the amount of cell TAG might not correlate directly with TAG secreted in VLDL. Finally, secretion might be affected by ACSL1-mediated changes in the cellular content of FA and acyl-CoA, which are ligands for nuclear transcription factors like the PPARs (48) and hepatocyte nuclear factor-4{alpha} (49, 50).

Overexpression of several ACSL isoforms increases the uptake of exogenous FA (10, 31, 36). Uptake probably occurs because vectorial acylation and enhanced FA metabolism diminish the rate of efflux of unesterified FA from the cell (51). Most studies have measured the initial rate of FA import within 1-2 min, a time frame that may not represent the physiological uptake of FA that is driven by transporters and metabolic demand (51). We measured the total FA metabolized in cell and medium lipids and in beta-oxidation products during a period of time that takes metabolic demands into account (10, 51). In our study, ACSL1 did not significantly increase the total amount of FA metabolized by hepatocytes. ACSL1 has been reported to interact with FATP1 on the plasma membrane of 3T3-L1 adipocytes (32). Because FATP1 is not present in hepatocytes (52), and because we showed that hepatocyte ACSL1 is not present on the plasma membrane, lack of enhanced FA uptake and metabolism in hepatocytes may reflect the different intracellular location of ACSL1, its interaction with a different FATP isoform, or its association with different downstream enzymes that use acyl-CoAs.

To determine the selectivity of ACSL1 for endogenous versus exogenous FA, we incubated hepatocytes with [14C]acetate, which is used for de novo FA synthesis. In contrast to incubations with [14C]oleate, Ad-Acsl1 increased label incorporation into TAG, as well as DAG and PL, showing enhanced use of de novo synthesized FA for TAG synthesis. It has been suggested that TAG and CE synthesis in hepatocytes requires some FA derived from de novo synthesis (42, 53). Our data suggest that overexpression of ACSL1 channeled exogenous FA into DAG and PL, but not into TAG, in part due to insufficient endogenous FA. However, during the 14-h chase, the required pool of endogenous FA may no longer have been limiting because of the hydrolysis of TAG and PL, so that overexpressed ACSL1 could increase [14C]oleate incorporation into TAG. Differing from the decrease in exogenous oleate used for CE synthesis, ACSL1 overexpression did not diminish [14C]acetate incorporation into CE, suggesting that the 14C label in CE was derived primarily from cholesterol rather than FA.

In summary, consistent with our hypothesis that ACSL1 channels FA toward specific pathways, adenovirus-mediated overexpression of rat Acsl1 in rat primary hepatocytes channeled [14C]oleate toward DAG, PE, PI, and PC synthesis and away from cholesterol esterification. Overexpressed ACSL1 also increased the reacylation of hydrolyzed oleate to TAG and PL but diminished the amount of hydrolyzed oleate used for beta-oxidation. In contrast to its role in adipocytes (54), fibroblasts (31), and heart muscle (9), overexpression of ACSL1 in hepatocytes did not increase incorporation of [14C]oleate into TAG or increase the total amount of FA metabolized. It seems likely that ACSL1 channels FA differently in different tissues, perhaps depending on the subcellular location of ACSL1 or the presence of interacting proteins specific to each cell type. In addition, overexpressed ACSL1 activated both exogenous FA and FA derived from de novo synthesis but channeled the resulting acyl-CoA products into different pathways. Our study suggests that ACSL1 in hepatocytes plays an important role in directing FA into pathways of phospholipid synthesis and away from cholesterol esterification and beta-oxidation.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants DK59935 (to R. A. C.), PO1-DK58398 (to C. B. N.), and P30-DK34987 (to the Center for Gastrointestinal Biology and Disease). 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. Back

1 Present address: Dept. of Food Science and Nutrition, University of Minnesota, St. Paul, MN 55108. Back

2 To whom correspondence should be addressed: Dept. of Nutrition, University of North Carolina, CB# 7461, Chapel Hill, NC 27599. Tel.: 919-966-7213; Fax: 919-966-7216; E-mail: rcoleman{at}unc.edu.

3 The abbreviations used are: ACSL, long chain acyl-CoA synthetase; ACS, acyl-CoA synthetase; ACAT, acyl-CoA:cholesterol acyltransferase; Ad, adenovirus; ASM, acid-soluble metabolites; CE, cholesterol ester; CPT1, carnitine palmitoyltransferase 1; DAG, diacylglycerol; DGAT2, acyl-CoA:diacylglycerol acyltransferase 2; dpm, disintegrations per minute; ER, endoplasmic reticulum; FA, fatty acid; FBS, fetal bovine serum; GFP, green fluorescent protein; MAM, mitochondria-associated membrane; MEM, Minimal essential medium; MEM-DA, MEM plus dexamethasone and nonessential amino acids; m.o.i., multiplicities of infection; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PL, phospholipid; PPAR, peroxisome proliferator-activated receptor; TAG, triacylglycerol; VDAC, voltage-dependent anion channel; VLDL, very low-density lipoprotein; UNC, University of North Carolina. Back


   ACKNOWLEDGMENTS
 
We thank the UNC Cellular Metabolism and Transport Core for hepatocyte preparations, the UNC Michael Hooker Microscopy Facility for assistance with confocal microscopy, and the UNC Gene Expression Core Facility for help with quantitative reverse transcription-PCR. We are grateful to Dr. Dennis Vance (University of Alberta) for anti-PEMT antibodies.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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