AJP - Endo Journal of Applied Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Endocrinol Metab 288: E835-E844, 2005. First published December 14, 2004; doi:10.1152/ajpendo.00300.2004
0193-1849/05 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/5/E835    most recent
00300.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lewin, T. M.
Right arrow Articles by Coleman, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lewin, T. M.
Right arrow Articles by Coleman, R. A.

Mitochondrial glycerol-3-phosphate acyltransferase-1 directs the metabolic fate of exogenous fatty acids in hepatocytes

Tal M. Lewin ,* Shuli Wang,* Cynthia A. Nagle, Cynthia G. Van Horn, and Rosalind A. Coleman

Department of Nutrition, University of North Carolina, Chapel Hill, North Carolina

Submitted 8 July 2004 ; accepted in final form 4 December 2004


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Because excess triacylglycerol (TAG) in nonadipose tissues is closely associated with the development of insulin resistance, interest has increased in the metabolism of long-chain acyl-CoAs toward {beta}-oxidation or the synthesis and storage of TAG. To learn whether a mitochondrial isoform of glycerol-3-phosphate acyltransferase (mtGPAT1) competes with carnitine palmitoyltransferase I (CPT I) for acyl-CoAs and whether it contributes to the formation of TAG, we overexpressed rat mtGPAT1 13-fold in primary hepatocytes obtained from fasted rats. When 100, 250, or 750 µM oleate was present, both TAG mass and the incorporation of [14C]oleate into TAG increased more than twofold in hepatocytes overexpressing mtGPAT1 compared with vector controls. Although the incorporation of [14C]oleate into CO2 and acid-soluble metabolites increased with increasing amounts of oleate in the media, these metabolites were ~40% lower in the Ad-mtGPAT1 infected cells, consistent with competition for acyl-CoAs between CPT I and mtGPAT1. A 50–60% decrease was also observed in [14C]oleate incorporation into cholesteryl ester. With increasing amounts of exogenous oleate, [14C]TAG secretion increased appropriately in vector control-infected hepatocytes, suggesting that the machinery for VLDL-TAG biogenesis and secretion was unaffected. Despite the marked increases in TAG synthesis and storage in the Ad-mtGPAT1 cells, however, the Ad-mtGPAT1 cells secreted the same amount of [14C]TAG as the vector control cells. Thus, in isolated hepatocytes, mtGPAT1 may synthesize a cytosolic pool of TAG that cannot be secreted.

hepatic triacylglycerol; hepatic fatty acid oxidation

PREVIOUSLY BELIEVED TO BE A BENIGN CONDITION, the presence of excess triacylglycerol (TAG) in liver, skeletal muscle, and other nonadipose tissues is now considered a hallmark of insulin resistance. Although TAG itself may not contribute to insulin resistance, the hydrolysis of TAG to diacylglycerol (DAG) and fatty acids may trigger signaling cascades, ceramide production, and acyl-CoA accumulation. Because one or more of these downstream molecules are believed to promote insulin resistance (41), interest is growing in the metabolic pathways that lead to TAG deposition in tissues. In liver, for example, exogenous fatty acid may either be degraded via {beta}-oxidation or enter synthetic pathways that include the esterification of cholesterol and glycerol-3-phosphate to form cholesteryl esters and glycerolipids, respectively. Indirect evidence suggests that channeling of fatty acid toward a pathway of degradation or one of synthesis depends on its initial activation and esterification steps (12, 13). One of these initial steps is catalyzed by glycerol 3-phosphate acyltransferase (GPAT; EC 2.3.1.15 [EC] ), which uses long-chain acyl-CoAs to esterify the sn-1 position of glycerol 3-phosphate to form lysophosphatidic acid. The GPAT esterification step constitutes the committed reaction in the de novo synthesis of TAG and all the acyl-glycerophospholipids.

Three GPAT isoforms have been characterized in mammalian tissues, N-ethylmaleimide (NEM)-sensitive microsomal and mitochondrial (11, 30) isoforms that have not been purified or cloned, and an NEM-resistant mitochondrial isoform (mtGPAT1), which is a member of the gpam family of lipid acyltransferases (11, 12). Hepatic mtGPAT1 is believed to play an important role in hepatic TAG synthesis because its mRNA and protein are upregulated when fasted mice are refed (28) and when insulin is provided to streptozotocin-diabetic mice (40). In contrast, the specific activity of the microsomal GPAT isoform does not appear to change with nutritional alterations but does increase markedly during the differentiation of 3T3-L1 adipocytes and in postnatal liver (8, 10). mtGPAT2 activity is not apparent in normal mouse liver (30).

In hepatocytes, newly synthesized TAG can be either stored in cytosolic droplets or secreted in very-low-density lipoprotein (VLDL) particles after assembly with apolipoprotein B (apoB), phosphatidylcholine, and cholesterol esters (CE). The formation and secretion of VLDL from cultured hepatocytes require ongoing TAG synthesis (17). If TAG is not cotranslationally available for association with newly forming apoB, apoB synthesis pauses, and the partially formed apoB is ubiquinated and then degraded in proteasomes or by nonproteasomal mechanisms (1, 17).

Although numerous studies strongly suggest that the mtGPAT1 isoform initiates the synthesis of TAG, this concept is somewhat surprising, because mtGPAT1 is an intrinsic protein of the outer mitochondrial membrane, whereas the enzymes that catalyze the final steps of TAG synthesis are located in the endoplasmic reticulum (ER) (11). Thus mitochondrially produced lysophosphatidate, the product of mtGPAT1, and/or phosphatidate produced by the subsequent 1-acyl-glycerol-3-phosphate acyltransferase (AGPAT) acylation step must be transported to the ER to complete the synthesis of TAG. We have hypothesized that mtGPAT1, which is located in the outer mitochondrial membrane, competes with CPT I, the rate-limiting step in {beta}-oxidation, for acyl-CoAs (36). If this were true, an increase in mtGPAT1 activity should divert acyl-CoAs away from oxidation and toward TAG synthesis. To test this hypothesis directly, we used an adenovirus construct to overexpress mtGPAT1 in cultured rat hepatocytes. Because mice null for mtGPAT1 have a 15% lower content of plasma TAG, have lower VLDL-TAG, and secrete 30% less TAG from their livers (22), we also predicted that overexpression of mtGPAT1 would cause hepatocytes to secrete more TAG.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. Minimal essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), DMEM with high glucose (DMEM-H), nonessential amino acids (NEAA), HEPES buffer solution, and fetal bovine serum (FBS) were obtained from GIBCO-BRL Life Technologies. Collagen type I was from Collaborative Biomedical Products. Albumin (initial fractionation by heat shock, essentially fatty acid free), oleate, carnitine, and dexamethasone were purchased from Sigma. [1-14C]oleate was obtained from PerkinElmer Life Sciences. DNA restriction endonucleases and ligase for recombinant adenovirus construction were purchased from New England Biolabs. HEK-293 cells were obtained from the American Type Culture Collection. Lipid standards were from Sigma and Avanti Polar Lipids.

Construction of recombinant mtGPAT1-Flag adenovirus. Shuttle vector pAdTrack-CMV, which expresses green fluorescent protein (GFP), was used to produce mtGPAT1 adenoviruses. The primers for amplification of rat mtGPAT1 cDNA were designed to include the COOH-terminal Flag epitope (DYKDDDDK) and specific restriction sites. The upper primer was 5'-AGTATCTA-GAACACATGGAGGAGTCTTCAGTG-3', and lower primer was 5'-GCTCTAGACTTGTCATCGTCGTCCTTGTA-3. The PCR-derived fragment and pAdTrack-CMV were digested with XbaI, ligated, and verified to make certain that the direction of the inserted GPAT-Flag cDNA was correct. The pAdTrack/mtGPAT1-Flag construct was sequenced at the University of North Carolina (UNC) DNA sequencing facility. Expression and activity of pAdTrack/mtGPAT1-Flag were confirmed by transfection into HEK-293 cells, followed by anti-Flag immunoblot and GPAT assay. Generation of recombinant adenovirus by homologous recombination was performed by the UNC Vector Core Facility.

Isolation of hepatocytes. Animal protocols were approved by the UNC Institutional Animal Care and Use Committee. Male Sprague-Dawley rats (200 g) were housed on a 12:12-h light-dark cycle. Rats were fed normally or fasted for 16 h before hepatocyte isolation. Primary hepatocytes were isolated by collagenase perfusion at the UNC Advanced Cell Technologies and Tissue Engineering Core. The cells were isolated and suspended in cold DMEM containing 10% FBS on ice.

Hepatocyte culture and adenovirus infection. Primary rat hepatocytes were seeded at a density of 1.5 x 106 cells/60 mm collagen-coated dish and grown in DMEM-H medium supplemented with 10% (vol/vol) FBS for 5 h in 5% CO2 before infection with adenovirus. Cells were not used if fewer than 70% attached to the dish. The medium was removed from the dish and covered with 1 ml of fresh DMEM-H containing 5 pfu/cell (total 7.5 x 106 virus particles/1.5 x 106 cells) of adenovirus (Ad-GFP or Ad-mtGPAT1). The hepatocytes were incubated with the infection medium for 2 h, after which it was removed, and 2 ml of incubation medium (DMEM-H, 10% FBS, 0.5% BSA, 1 mM carnitine, 10 mM HEPES, pH 7.4, 10 µM dexamethasone, and 10 mM NEAA) was added. Infection was allowed to proceed for an additional 16 h.

Cell labeling and lipid analysis. Bovine serum albumin (BSA)-conjugated oleate was prepared by dissolving Na oleate (C18:1, Sigma Chemical) in 2.5% BSA (essentially fatty acid free) at a concentration of 3.75 mM and heating at 65°C until the Na oleate dissolved completely. Then [1-14C]oleic acid (5 µCi) dissolved in ethanol (PerkinElmer Life and Analytical Sciences) was dried with a stream of N2 gas, resuspended in DMEM-H culture medium, combined with the unlabeled Na oleate solution, and added to DMEM medium to give a final concentration of 100, 250, or 750 µM. Hepatocytes were infected for 2 h as described above, and then 100, 250, or 750 µM [14C]oleic acid (500,000–750,000 dpm in 2 ml) was added together with the incubation medium. Sixteen hours later, the labeling medium was removed and centrifuged to remove floating cells. Media lipids were extracted (6) and concentrated in a SpeedVac concentrator. To obtain cell lipids, hepatocyte cultures were washed with 1 ml of cold 0.9% NaCl containing 10 mg BSA and then with 2 ml of cold phosphate-buffered saline and scraped in 2 ml of cold methanol and 0.5 ml of H2O. Lipids were extracted and concentrated as described above. Neutral lipids were resolved on LKD6 silica plates (Whatman) by thin-layer chromatography using a solvent system consisting of hexane-ethyl ether-acetic acid (80:20:2, vol/vol/vol). All samples were chromatographed in parallel with pure lipid standards. The 14C-labeled lipids were detected and quantified with a Bioscan 200 Image System.

Quantification of lipids. Unlabeled oleate was conjugated with BSA as described above. Hepatocytes were infected for 2 h as described above, and then 100, 250, or 750 µM of unlabeled oleate was added together with incubation medium. Medium and cell lipids were extracted as described above. TAG mass was determined using an enzymatic colorimetric method (Stanbio Laboratory, Boerne, TX) following the manufacturer's instructions except for the sample preparation, in which 100 µl of total cell lipids or 200 µl of media lipids were dried in a SpeedVac concentrator and dissolved in 30 µl of isopropyl alcohol containing 1% Triton X-100. Phosphatidylcholine and sphingomyelin mass were determined by HPLC (27). For cholesterol content, lipids were extracted (18) and subjected to alkaline hydrolysis in 10% KOH in methanol for 48 h at room temperature. Free and total cholesterol were determined using enzymatic colorimetric assays (Free Cholesterol E and Cholesterol CII, Wako Chemical). CE was determined by subtracting free cholesterol from total cholesterol.

{beta}-Oxidation. Hepatocytes were infected and labeled with 100, 250, or 750 µM [14C]oleic acid (500,000–750,000 dpm in 2 ml) as described above. The labeling medium was removed 16 h after label was added and centrifuged to remove floating cells. [14C]oleate oxidized to CO2 and acid-soluble metabolites (ASM) was measured (36). Although we did not use closed flasks for our CO2 measurements, the percentage of [14C]CO2 that we measured per total [14C]oleate oxidized was similar to that in other studies that used closed flasks (3, 16). Briefly, CO2 was driven from 1 ml of medium by adding 200 µl of 70% perchloric acid and trapped on a suspended filter wick (Kontes) saturated with NaOH. Wicks were placed in Cytoscint (ICN Biochemicals) and subjected to scintillation counting to determine the amount of [14C]CO2 generated. The acidified medium was centrifuged in a microfuge for 10 min twice to remove particulate matter. Two hundred microliters of the supernatant were added to Ecolite (ICN) and subjected to scintillation counting to determine the amount of 14C-labeled ASM generated.

Isolation of subcellular fractions. Twelve 100-mm dishes (4 x 106 hepatocytes/plate) were infected with 5 pfu/cell Ad-mtGPAT1 or Ad-GFP for 18 h. Cells were washed twice with ice-cold PBS, scraped into 1.5 ml/dish isolation medium (250 mM mannitol, 5 mM HEPES pH 7.4, 0.5 mM EGTA, 0.1% BSA), and homogenized with 10 up-and-down strokes with a Teflon-glass motor-driven homogenizer. Debris and nuclei were removed by centrifugation at 600 g for 5 min. The supernatant was centrifuged at 10,300 g for 10 min to collect the crude mitochondrial pellet. This pellet was resuspended by homogenizing in 1.2 ml of isolation medium and then layered over 30% (vol/vol) Percoll in isolation medium (20 ml). The Percoll gradient was allowed to form by centrifugation at 95,000 g for 30 min. The supernatant fraction over the crude mitochondrial pellet was centrifuged at 95,000 g for 30 min to collect microsomes. Four membrane-containing fractions (3–4 ml each) were collected from the Percoll gradient (Percoll fractions 1–3 and mitochondria), diluted in isolation medium, and centrifuged at 10,000 g for 10 min. The pellets were suspended in 10 ml of medium I (250 mM sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA, and 1 mM DTT) and recentrifuged. The Percoll fractions 1–3, mitochondrial, and microsomal pellets were resuspended by homogenization in medium I and stored in 100-µl aliquots at –80°C. The supernatant fraction over the microsomal pellet was saved as the cytosolic fraction. Purity of the subcellular fractions was established by measuring the activity of marker enzymes, NADH cytochrome c reductase (14) and cytochrome c oxidase (Cytochrome C Oxidase Kit, Sigma), for ER and mitochondria, respectively.

Isolation of total membrane fractions. Uninfected, Ad-GFP infected, or Ad-mtGPAT1 infected hepatocytes were washed with cold PBS, scraped into medium I, and homogenized with 10 up-and-down strokes in a Teflon-glass motor-driven homogenizer. The total membrane fraction was obtained by centrifuging at 100,000 g for 1 h. The total membrane pellet was rehomogenized in medium I and stored in 100-µl aliquots at –80°C for enzyme assay.

Immunoblotting. Proteins (60 µg) from the total membrane fraction were separated by electrophoresis on an 8% polyacrylamide gel containing 1% SDS, transferred to a PVDF membrane (Bio-Rad), and incubated with antibody against the Flag epitope (M2 anti-flag monoclonal antibody, Sigma). For chemiluminescent detection, the immunoreactive bands were visualized by incubating the membrane with horseradish peroxidase-conjugated goat anti-mouse IgG and PicoWest reagents (Pierce). For subcellular fractions, 20 µg of subcellular and Percoll fractions were immunoblotted for the Flag epitope or voltage-dependent ion channel (Anti-VDAC1, Abcam), as described above.

Enzyme assays. sn-[2-3H]glycerol-3-phosphate was synthesized enzymatically from [2-3H]glycerol (1 mCi/ml) and purified and assayed as described previously (7). GPAT specific activity was assayed at room temperature in a 200-µl mixture containing 75 mM Tris·HCl, pH 7.5, 4 mM MgCl2, 1 mg/ml BSA (essentially fatty acid free), 1 mM DTT, 8 mM NaF, 800 µM [3H]glycerol 3-phosphate, and 80 µM palmitoyl-CoA (10). The reaction was initiated by adding 5–20 µg of total membrane protein to the assay mix after incubation for 15 min on ice in the absence or presence of 1 mM NEM. mtGPAT1 activity is calculated as the activity that is uninhibited by NEM.

[14C]oleoyl-CoA was synthesized enzymatically from [1-14C]oleate (0.1 mCi/ml) (45). ACAT specific activity was assayed in a 200-µl mixture containing 50 µg of total membrane protein, medium I, and 1.67 µg/ml BSA, as described previously (42). The protein mixture was preincubated at 37°C for 5 min in the absence, or 50 min in the presence, of 8 µg of cholesterol (phosphatidylcholine-cholesterol, 8:1 by weight). The assay was started by adding 25 µM [14C]oleoyl-CoA (38,000 dpm/nmol). After incubation at 37°C for 6 min, the reaction was stopped by adding 3 ml of chloroform-methanol (2:1), followed by 1 ml of H2O for Folch lipid extraction (18). The organic phase (2 ml) was dried down in a SpeedVac concentrator, and the lipids were resuspended in 50 µl of 0.5 mg/ml cholesteryl oleate in chloroform. The entire sample was spotted onto an LK6D silica gel plate (Whatman), and neutral lipids were resolved as described above. The 14C-labeled CE was detected and quantified using a Bioscan Image System.

Other methods. Protein concentrations were determined by the bicinchoninic acid method (Pierce) using BSA as the standard. Data are presented as means ± SD. Significant differences between the Ad-mtGPAT1 infected hepatocytes and Ad-GFP-infected controls were analyzed by two-tailed Student's t-test.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adenoviral mediated overexpression of mtGPAT1 in hepatocytes increases NEM-resistant GPAT activity. Mitochondrial GPAT activity comprises 30–50% of the total GPAT activity measured in rat liver (11), but its contribution to the synthesis of cellular phospholipid and TAG and to VLDL-TAG is not known. We infected primary rat hepatocytes with adenovirus containing GFP (Ad-GFP) or with adenovirus containing both GFP and mtGPAT1-Flag (Ad-mtGPAT1). Uninfected hepatocytes provided an additional control. NEM-resistant GPAT specific activity (mtGPAT1) began to increase 12 h after infection with Ad-mtGPAT1 and was maximal by 18 h (Fig. 1A). At 18 h, infection with Ad-mtGPAT1 increased NEM-resistant specific activity 13-fold compared with uninfected or Ad-GFP controls (Fig. 1B). No change was observed in microsomal (NEM-sensitive) GPAT activity. To verify that overexpressed mtGPAT1-Flag was targeted to mitochondria, we obtained microsomal and mitochondrial fractions from Ad-GPAT infected hepatocytes (Table 1). Immunoblotting of these fractions with antibody against the Flag epitope revealed that mtGPAT1 was present primarily in mitochondria but was also present in an ER fraction (Fig. 1C). Although little of the mitochondrial marker enzyme cytochrome oxidase was present in the microsomal fraction (Table 1), the presence of VDAC, an outer mitochondrial membrane marker, in the microsomal fraction (Fig. 1C) clearly indicates that the microsomal fraction was contaminated with broken mitochondrial outer membrane. VDAC appears to be equally distributed in the microsomal and mitochondrial fractions, consistent with the distribution of NEM-resistant GPAT activity (total activity: microsomal, 3.47 nmol/min; mitochondria, 5.00 nmol/min). Although not conclusive, these data suggest that overexpressed mtGPAT1-Flag is properly localized to the mitochondrial outer membrane.



View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1. Adenvirus-infected mitochondrial glycerol-3-phosphate acyltransferase-1 (Ad-mtGPAT1) increases N-ethylmaleimide (NEM)-resistant GPAT specific activity (S.A.) in hepatocytes. Hepatocytes from fasted rats were plated. After 5 h, medium was replaced with new medium alone (uninfected) or medium containing 5 pfu/cell adenovirus containing green fluorescent protein (Ad-GFP) or Ad-mtGPAT1. A: at indicated time points, cells were scraped from the dish and NEM-resistant GPAT activity was assayed in total membrane preparations. Data are averages of triplicates. B: 18 h after adenovirus infection, cells were scraped, homogenized, and centrifuged to obtain total membranes, and GPAT activity was measured in the presence (mitochondrial) and absence (total) of 1 mM NEM. Microsomal activity is calculated as total minus NEM-resistant activity. Data are from 8 separate experiments and expressed as means ± SD. C: subcellular fractions were isolated from Ad-mtGPAT1-infected hepatocytes, as described in EXPERIMENTAL PROCEDURES. Cytosol (Cyt, 20 µg), microsomes (Mcs), mitochondria (Mito), and nuclei/debris (Debris) were immunoblotted for the Flag epitope or for voltage-gated ion channel (VDAC). Positions of molecular mass markers are indicated on right. Molecular mass of mtGPAT1 was ~90 kDa.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Activities of endoplasmic reticulum and mitochondrial marker enzymes

 
Overexpression of mtGPAT1 decreases fatty acid oxidation. Because both mtGPAT1 and CPT I are located on the outer mitochondrial membrane, it seemed that mtGPAT1 might play a significant role in liver in directing the fate of exogenous fatty acids toward glycerolipid synthesis and away from fatty acid oxidation. To test this hypothesis, we used hepatocytes from fasted rats to determine whether increasing mtGPAT1 activity would affect their high oxidation rates. When primary hepatocytes from fasted rats were either uninfected or infected with Ad-GFP or Ad-mtGPAT1 and incubated with [14C]oleate, a 13-fold overexpression of mtGPAT1 resulted in decreased oxidation of [14C]oleate to ASM and CO2. With 100, 250, or 750 µM [14C]oleate, incorporation of label into ASM decreased 95, 50, and 53%, respectively, compared with controls (Fig. 2B). Although ASM is considered a more accurate measure of {beta}-oxidation (47), we also measured [14C]oleate oxidation to CO2. At 100, 250, or 750 µM [14C]oleate, labeled CO2 decreased 72, 60, and 40%, respectively (Fig. 2A). Similar results were obtained for hepatocytes from fed rats incubated with either 250 or 750 µM oleate; at each oleate concentration, ASM and CO2 each decreased ~60% (data not shown). As previously reported (34), hepatocytes obtained from fasting rats incorporate less [14C]oleate into TAG and more into ASM than do hepatocytes from fed rats. When hepatocytes from fasted rats were incubated with 250 µM oleate, the ratio of [14C]oleate incorporation into TAG:ASM was 1.0, whereas the ratio in hepatocytes from fed rats was 2.9. These data show that, although we incubated the fasted hepatocytes in 10% serum and 25 mM glucose, the resulting metabolic profile was similar to that reported for fasted hepatocytes that had been incubated without serum and in low glucose (34).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 2. Ad-mtGPAT1 overexpression decreases [14C]oleate oxidation. Hepatocytes were treated as described in Fig. 1 and EXPERIMENTAL PROCEDURES. At 2 h after infection, medium was replaced with new medium containing [14C]oleate as indicated. After 16 h, medium was collected, and oxidation of [14C]oleate to CO2 (A) or acid-soluble metabolites (ASM; B) was measured as described in EXPERIMENTAL PROCEDURES. Data are averages from 2 experiments, each performed in triplicate.

 
The present studies show that, as a percentage of total oleate incorporation, CO2 plus ASM increased from 7% at 100 µM oleate to 21% at 250 µM oleate in control Ad-GFP-infected cells (Fig. 3). Although with increasing exogenous fatty acid one expects an increase in fatty acid oxidation because of decreased malonyl-CoA inhibition of CPT I, the percentage of [14C]oleate oxidized to CO2 plus ASM is the same with both 250 and 750 µM oleate. This result is similar to our previous observation that conversion of [14C]oleate to {beta}-oxidation products is linear from 0 to 250 µM oleate but reaches a plateau at higher concentrations (34). Consistent with these findings is our result that overexpressing mtGPAT-1 inhibits 96% of the incorporation of [14C]oleate into CO2 plus ASM at 100 µM oleate but inhibits only 66% at 250 or 750 µM oleate (Fig. 3). This reduction in mtGPAT-1-mediated inhibition of {beta}-oxidation would be expected because of the increased competition from CPT I at higher fatty acid concentrations. Despite the increase in labeled oxidation products that depends on exogenous fatty acid, the relative percentage of oxidation at all oleate concentrations decreased with the addition of mtGPAT1 (Figs. 2 and 3). These results suggest that mtGPAT1 competes with CPT I to direct exogenous fatty acids away from {beta}-oxidation.



View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3. Ad-mtGPAT1 alters incorporation of fatty acid into various metabolites. Hepatocytes were treated as described in the legend for Fig. 2 and incubated with [14C]oleate as indicated. Cell and media lipids and media CO2 and ASM were analyzed as described in EXPERIMENTAL PROCEDURES and are graphed as a percent total nmol 14C label incorporated. PL, phospholipids; CE, cholesterol ester. Totals for Ad-GFP at 100, 250, or 750 µM oleate were 46, 91, and 433 nmol, respectively. Totals for Ad-mtGPAT1 at 100, 250, or 750 µM oleate were 76, 127, and 656 nmol, respectively. Data represent the average of 2–3 independent experiments performed in triplicate.

 
Overexpression of mtGPAT1 increases cell, but not media, TAG. We previously showed in Chinese hamster ovary (CHO) cells that a 3.8-fold increase in mtGPAT1 activity increases TAG content 2.7-fold and increases [14C]oleate incorporation into TAG 3.4-fold (25). In the Ad-GPAT1-infected hepatocytes, a 13-fold increase in GPAT activity resulted in 2.4-, 3-, and 2.4-fold increases in TAG mass at 100, 250, and 750 µM oleate, respectively (Fig. 4A). When hepatocytes prepared from fasted rats were incubated with [14C]oleate, [14C]TAG in uninfected and vector control cells increased sixfold with 750 µM oleate compared with 100 µM oleate, and infection with Ad-mtGPAT1 further increased [14C]oleate incorporation into cellular TAG 2.4- to 2.7-fold compared with the Ad-GFP controls at each fatty acid concentration (Fig. 4B). As expected, the pattern for [14C]oleate incorporation into cellular DAG paralleled that for TAG, although DAG values were always ~10% of TAG values (Fig. 4C). As a percentage of total [14C]oleate incorporation, labeled TAG plus DAG increased from 56% in Ad-GFP cells to 69% in Ad-GPAT cells with 250 µM oleate (Fig. 3). Similar increases were observed with the 750 µM oleate incubation; labeled TAG plus DAG increased from 66% in Ad-GFP cells to 82% in Ad-GPAT cells (Fig. 3). These data support the hypothesis that mtGPAT1 enhances the incorporation of exogenous fatty acid into TAG, even in hepatocytes from fasted rats.



View larger version (25K):
[in this window]
[in a new window]
  		Fig. 4. Ad-mtGPAT1 increases incorporation of [14C]oleate into cell but not media triacylglycerol (TAG). Hepatocytes were treated as described in the legend for Fig. 2 and incubated with 100, 250, or 750 µM [14C]oleate as indicated. A: cell TAG mass was measured by colorimetric assay. [14C]oleate incorporated into cell TAG (B), cell diacylglycerol (DAG; C), media TAG (D), and cell PL (E) was determined as described in EXPERIMENTAL PROCEDURES. Data for B–D are means of experiments performed 3 times in triplicate. Data for A at 250 and 750 µM oleate and for E at 100 and 250 µM oleate are means ± SD of experiments performed 3 times in triplicate. Data for A at 100 µM oleate and for E at 750 µM oleate are averages of experiments performed 2 times in triplicate. *P ≤ 0.05, **P < 0.01, ***P = 0.005.

 
In hepatocytes, newly synthesized TAG can be stored in cytosolic droplets or assembled together with apoB, phosphatidylcholine, and CEs to form VLDL particles. Because it remains unclear whether newly synthesized TAG or TAG previously stored in cytosolic droplets is preferentially secreted from liver (20), we determined whether an increase in TAG synthesis would be reflected by an increase in secreted TAG. The effect of increased availability of exogenous oleate was apparent in the 7.9- and 37.6-fold increases in labeled media TAG secreted from cells incubated with 250 or 750 µM oleate compared with 100 µM oleate (Fig. 4D). However, although overexpression of Ad-mtGPAT1 increased [14C]oleate incorporation into cell TAG 2.4- to 2.7-fold, incorporation into media TAG was identical for Ad-mtGPAT1 and control cells. In hepatocytes from fed rats, overexpression of GPAT did not increase media TAG more than the vector control, although the total incorporation of oleate into cell and media TAG increased two- to fourfold compared with hepatocytes from fasted rats (data not shown), reflecting greater synthesis of TAG after a meal. Because exogenous fatty acids stimulate the formation and secretion of apoB by primary mouse hepatocytes (31), we considered the possibility that either the oleate concentration or the cellular machinery required to assemble and secrete VLDL was limiting. It was clear, however, in the two controls that sufficient oleate was present: both cell and media [14C]TAG increased in direct proportion to the exogenous oleate concentration, whereas overexpression of mtGPAT increased label incorporation only into cellular [14C]TAG. Furthermore, VLDL assembly and secretion appeared sufficient: 750 µM oleate led to fivefold more media [14C]TAG than 250 µM oleate (Fig. 4D), demonstrating that, when additional exogenous fatty acid was available, Ad-mtGPAT1 infected cells could secrete more TAG. In fact, at 250 µM oleate, the amount of [14C]TAG present in control cell and medium were each ~50 nmol/1.5 x 106 cells and at 750 µM oleate, each was ~120 nmol/1.5 x 106 cells. In contrast, at 250 and 750 µM oleate, the Ad-mtGPAT1 cell TAG was ~80 and 400 nmol, respectively, and should have resulted in more secreted [14C]TAG. It appears that, despite an unimpaired ability to assemble and secrete VLDL, the excess synthesis of TAG initiated by mtGPAT1 could not be secreted.

In Ad-GPAT-infected hepatocytes [14C]oleate incorporation into cellular phospholipids increased 30–60% compared with vector control cells (Fig. 4E) but did not change as a percentage of total label incorporation (Fig. 3). Phosphatidylcholine and sphingomyelin mass did not change (Table 2), suggesting that overexpression of mtGPAT1 did not alter the relative amount of phospholipid in cells and that impaired synthesis of phosphatidylcholine did not contribute to the diminished secretion of VLDL-TAG by the mtGPAT1 cells (50).


View this table:
[in this window]
[in a new window]
  		Table 2. Hepatocyte metabolites

 
Overexpression of mtGPAT1 decreases CE synthesis. When the amount of exogenous [14C]oleate increased from 250 to 750 µM, incorporation into CE increased 2.5-fold in vector control cells. A 13-fold overexpression of mtGPAT1, however, resulted in a marked decrease in the incorporation of [14C]oleate into cellular CE. At 100, 250, and 750 µM oleate, the amount of [14C]CE in the cells decreased 84, 60, and 30%, respectively, compared with controls (Fig. 5A). Similarly, although increasing exogenous oleate raised the amount of secreted [14C]CE proportionally, the presence of Ad-mtGPAT1 severely blocked [14C]oleate incorporation into media CE at 100 µM oleate and diminished it 50–60% at concentrations of 250 and 750 µM oleate (Fig. 5B). With hepatocytes obtained from fed rats, similar decreases in [14C]oleate incorporation into cell and media CE were observed at 250 and 750 µM (data not shown). Because the decrease in label incorporation into cell CE diminished as the exogenous oleate concentration increased, it appeared that oleate might be limiting for CE secretion, possibly because mtGPAT1 diverted fatty acyl-CoA away from ACAT. Arguing against limiting oleate, however, is the fact that media [14C]CE as a percentage of total [14C]oleate incorporation were essentially identical (13 and 19%) at both 250 and 750 µM oleate (Fig. 3).



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 5. Ad-GPAT decreases incorporation of [14C]oleate into cell and media CE. Hepatocytes were treated as described in Fig. 2 and incubated with 100, 250, or 750 µM [14C]oleate as indicated. [14C]oleate incorporated into cellular CE (A) and media CE (B) was determined as described in EXPERIMENTAL PROCEDURES. Data are averages of experiments performed in triplicate, at least twice. Where error bars are shown, data are means ± SD of experiments performed 3 times in triplicate. *P < 0.05.

 
To understand why CE synthesis was diminished, we measured ACAT activity in total membrane fractions. ACAT activity depends on the availability of both the amount of labeled oleate and the amount of cholesterol substrate present in the ER. In either the absence or the presence of cholesterol added to the assay mixture, ACAT activity in Ad-mtGPAT1-infected hepatocytes was unchanged compared with the two controls (Fig. 6), and the amount of free cholesterol in the cell was identical in uninfected (data not shown), Ad-GFP, and Ad-mtGPAT1 infected cells (Table 2). As would be expected, hydroxymethylglutaryl (HMG)-CoA reductase specific activity was similar (62–67 pmol·min–1·mg–1 protein) in membranes from all treatments. If the cholesterol content in the ER had decreased, HMG-CoA reductase specific activity should have increased due to increased nuclear content of sterol regulatory element-binding protein-2 (SREBP-2) and the resulting upregulation of genes involved in the pathway of cholesterol synthesis.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 6. Ad-GPAT overexpression does not affect acyl-CoA:cholesterol acyltransferase (ACAT) activity. Hepatocytes were treated as described in Fig. 2 and incubated with 250 or 750 µM [14C]oleate as indicated. Total membrane fractions were isolated and then assayed for ACAT activity in the absence and presence of 8 µg of cholesterol. Data without error bars are averages of experiments performed in triplicate, at least twice. Where error bars are shown, data are means ± SD of experiments performed 3 times in triplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
GPAT activity in cells is comprised of at least three isoforms encoded by different genes (11, 30). The independent functions of these isoforms have remained unclear, but studies of mtGPAT1 mRNA under different physiological conditions suggest that mtGPAT1 contributes specifically to TAG synthesis. For example, liver mtGPAT1 mRNA abundance is downregulated under conditions where TAG synthesis declines, as in fasting and streptozotocin-diabetes, whereas mRNA expression increases 19-fold 6 h after streptozotocin-diabetic mice are injected with insulin (40). Furthermore, when mice are fasted for 24 h and then refed a high-carbohydrate, fat-free diet for 8 h, mtGPAT1 mRNA increases 20-fold, and this increase is blocked 70% by injecting the animals with dibutyryl-cAMP (40). mtGPAT1 is also upregulated by SREBP-1c, a powerful inducer of lipogenic genes (15). Livers from mtGPAT1 null mice contain 37% less TAG than livers from wild-type mice, consistent with the observation that the mitochondrial isoform comprises 30–50% of total hepatic GPAT activity (22). Acutely, mtGPAT1 specific activity is inhibited 40% by AMP-activated protein kinase (36), a kinase that is active under conditions when cellular energy stores are low and synthetic pathways such as cholesterol and TAG synthesis are downregulated (26). In contrast to the responsiveness of mtGPAT1 to lipogenic stimuli, the microsomal GPAT activity appears to change minimally, and mtGPAT2, the third isoform, is not normally expressed in liver (30).

We (25) have shown that a 3.8-fold increase in mtGPAT1 activity in CHO cells increases TAG content 2.7-fold and [14C]oleate incorporation into TAG 3.4-fold. Although that study strongly suggested that mtGPAT1 directs fatty acid toward TAG synthesis, CHO cells provide limited information because their oxidation rate is low and exogenous fatty acids are primarily metabolized to glycerolipids. Therefore, to determine whether mtGPAT1 would alter pathways of fatty acid oxidation and TAG secretion, we overexpressed mtGPAT in primary hepatocytes obtained from fasted rats to maximize the cells' oxidative capacity.

Liver from mtGPAT1 null mice contains 40% less TAG mass than that from wild-type mice (22), whereas TAG mass and label incorporation into TAG increase in CHO cells when mtGPAT1 is stably overexpressed (25). Thus we expected that overexpression of mtGPAT1 in rat hepatocytes would similarly result in an increase in TAG mass and in the incorporation of labeled fatty acid into TAG. These increases occurred as predicted. In addition, we wanted to determine whether high mtGPAT1 activity would decrease {beta}-oxidation. We found that label incorporation into CO2 and ASM decreased by at least 40%. When mtGPAT1 was overexpressed, decreases occurred at each fatty acid concentration provided to hepatocytes obtained from both fasted and fed animals. Thus neither the availability of fatty acid nor the physiological attributes of the cells played a determining role, and the percent decrease in CO2 plus ASM was identical at 250 and 750 µM oleate, consistent with the hypothesis that the presence of mtGPAT1 activity on the outer mitochondrial membrane competes with CPT I for acyl-CoAs and diverts long-chain acyl-CoAs toward TAG synthesis and away from {beta}-oxidation.

Although a decrease in ASM production in rat hepatocytes that overexpress mtGPAT1 has been recently reported (32), our detailed study reveals additional novel findings. Measuring the effect of overexpressed mtGPAT1 at three different physiological concentrations of exogenous fatty acids, we found that mtGPAT1 overexpression also markedly increased the incorporation of [14C]oleate into TAG. The incorporation of [14C]oleate into TAG was always more than sixfold higher than the incorporation into DAG. Surprisingly, the other study, in which murine mtGPAT1 was overexpressed, showed an increase in [3H]palmitate incorporation into DAG but not TAG unless the cells were preloaded with 300 µM 18:1–16:0 (1:1) (32). It is likely that, in the absence of 18:1, either dipalmitoylglycerol was formed and was a poor substrate for diacylglycerol acyltransferase (9) or that the exogenously provided 16:0 caused the hepatocytes to undergo apoptosis (33, 38).

Our study also documents and examines, for the first time, the lack of effect of mtGPAT1 overexpression on TAG secretion from hepatocytes. Despite greater than 2.4-fold increases in TAG mass and [14C]oleate incorporation into TAG, the amount of [14C]TAG secreted was unaffected by mtGPAT overexpression. Studies in cultured hepatoma cells show that exogenous fatty acid promotes the amount of VLDL-TAG secreted (17) and that inhibiting the de novo synthesis of TAG with triacsin (48), an inhibitor of acyl-CoA synthetase, long-chain (ACSL)1 and ACSL4 (29), or with troglitazone (19), an inhibitor of ACSL4 (29), decreases both TAG synthesis and apoB secretion. Although the initiation of apoB synthesis is constitutive, apoB undergoes both proteasomal and nonproteasomal degradation if TAG is not synthesized concomitantly. Because the rate of TAG synthesis critically affects the production of apoB-containing lipoproteins, the increase in TAG synthesis in the Ad-mtGPAT1 hepatocytes should have increased their secretion of VLDL-TAG. The discrepancy cannot be explained by a defect in the machinery for VLDL assembly and secretion, because both control and Ad-mtGPAT1 hepatocytes incubated with 750 µM oleate increased TAG secretion appropriately (Fig. 4D). Nor can the lack of increased TAG secretion be attributed to a deficiency of phosphatidylcholine (Table 2) (50).

Contradictory data exist regarding whether a decrease in CE interferes with VLDL secretion. In HepG2 cells, inhibition of CE synthesis and VLDL-CE content variably inhibit VLDL-stimulated or oleate-stimulated apoB secretion (5, 21, 48). Conversely, increasing CE synthesis and mass by overexpression of HMG-CoA reductase (48) or treatment with sphingomyelinase (48) does not effect apoB secretion. Thus neither diminishing nor overproducing cellular CE consistently alters lipoprotein secretion from HepG2 cells. Studies that do show a relationship between CE production and lipoprotein secretion (37, 43, 46) suggest that CE content or production must be higher than basal for subsequent inhibition to show an effect.

mtGPAT1 overexpression diminished the incorporation of [14C]oleate into CE, but when the amount of exogenous oleate was increased, the decrease in CE was partially ameliorated (Fig. 5). These data suggest that overexpressed mtGPAT1 might be diverting oleate away from cholesterol esterification but that the diversion can be overcome by increasing the amount of acyl-CoA available. In our experiments, it is unlikely that the decrease observed in CE synthesis in the Ad-mtGPAT1 hepatocytes diminished the ability of the cells to secrete more TAG. In all treatment groups, TAG secretion was proportional to the amount of exogenous oleate provided, despite the fact that incorporation of [14C]oleate into CE decreased only in the Ad-mtGPAT1 cells.

Because VLDL assembly was adequate, the rate of TAG synthesis was high, cellular phosphatidylcholine content was sufficient, and CE was not limiting, how can we explain the lack of mtGPAT1 effect on secretion of labeled TAG? A likely possibility is that the glycerolipid synthesis initiated by mtGPAT1 produces TAG that cannot be secreted and, instead, becomes sequestered in lipid droplets. Studies in Hep G2 cells suggest that two TAG pools exist, a microsomal pool that is coupled to secretion and a cytosolic pool that is not (49). Several other studies suggest the presence of separate and functionally different intracellular lipid pools. In Neutral Lipid Storage Disease, for example, hydrolyzed DAG intermediates are available for TAG, but not phospholipid, biosynthesis (24). Furthermore, the human deficiency of AGPAT2 leads to a severe congenital lipodystrophy (2), and apparently none of the other five putative AGPAT isoforms can alter the excess of the lysophosphatidic acid substrate or the deficiency of the phosphatidic acid product that contributes to the pathophysiology. Separate pools of cardiac TAG synthesized by distinct diacylglycerol acyltransferase (DGAT) activities have also been proposed (44). A final example of separate phospholipid pools is that of a temperature-sensitive mutation in the cytidine diphosphocholine pathway in CHO cells that cannot be complemented by overexpression of an enzyme that contributes to an alternate phosphatidylcholine-synthetic pathway (23).

Overexpression of mtGPAT1 leads to both an increase in cell TAG content and an increase in the entry of fatty acid as measured by the total amount of 14C label in cellular metabolites. With a 13-fold overexpression of mtGPAT1, at each concentration of oleate, the total 14C-labeled metabolites (cell plus medium) increased 40–50% compared with the vector control. Smaller changes were observed with stable mtGPAT1 overexpression in CHO cells, where a 3.8-fold increase in mtGPAT specific activity resulted in a 16% increase in total labeled glycerolipids (25). Fatty acid entry into cellular lipids also increases with overexpression of other acyltransferases, such as AGPAT1 activity in 3T3-L1 adipocytes and C2C12 myotubes (39), and DGAT-1 in 3T3-L1 adipocytes (51). The only exception to this pattern of increased fatty acid uptake is a study in which stable overexpression of DGAT-1 activity in SV40-transformed human lung fibroblasts caused a marked decrease in cell proliferation and thus phospholipid synthesis (4). Taken as a whole, observations on the overexpression of three different acyltransferases in the Kennedy pathway suggest that metabolism, rather than transport, is the major factor limiting fatty acid entry into cells.

Altered fatty acid metabolism plays a significant role in the development of obesity and type 2 diabetes. We have shown that overexpressing mtGPAT1 decreases fatty acid oxidation and increases storage of fatty acid as TAG. A similar hepatic diversion of fatty acid toward TAG storage is observed in obesity, and our data suggest that dysregulation at the mitochondrial outer membrane may be significant in controlling fatty acid partitioning. Nonadipose TAG accumulation is also a hallmark of insulin resistance. Although beyond the scope of these studies, our model of hepatic TAG accumulation may be useful in studying TAG-associated hepatic insulin resistance.


   GRANTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-59935 (R. A. Coleman), DK-59931 (T. M. Lewin), and F32-DK-61190 (C. G. Van Horn) from the National Institutes of Health, and by Grant 230323N from the American Heart Association (T. M. Lewin), the Center for Gastrointestinal Biology and Disease, Vector and ACT Cores (P30-DK-34987).


   ACKNOWLEDGMENTS
 
We thank Dr. S. K. Krisans (San Diego State University) for the measurements of HMG-CoA reductase, and Mei-Heng Mar for the choline metabolite measurements.

Current address of C. G. Van Horn: Department of Biochemistry, Rm. 239 Nutrition Research Center, Wake Forest University Health Sciences Medical Center Blvd, Winston-Salem, NC 27157


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. A. Coleman, CB# 7461, Univ. of North Carolina, Chapel Hill, NC 27599 (E-mail: rcoleman{at}unc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* These authors contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adeli K, Taghibiglou C, Van Iderstine SC, and Lewis GF. Mechanisms of hepatic very low-density lipoprotein overproduction in insulin resistance. Trends Cardiovasc Med 11: 170–176, 2001.[CrossRef][Web of Science][Medline]
  2. Agarwal AK, Arioglu E, De Almeida S, Akkoc N, Taylor SI, Bowcock AM, Barnes RI, and Garg A. AGPAT2 is mutated in congenital generalized lipodystrophy linked to chromosome 9q34. Nat Genet 31: 21–23, 2002.[CrossRef][Web of Science][Medline]
  3. Agius L, Chowdhury MH, and Alberti KG. Regulation of ketogenesis, gluconeogenesis and the mitochondrial redox state by dexamethasone in hepatocyte monolayer cultures. Biochem J 239: 593–601, 1986.[Web of Science][Medline]
  4. Bagnato C and Igal RA. Overexpression of diacylglycerol acyltransferase-1 reduces phospholipid synthesis, proliferation, and invasiveness in simian virus 40-transformed human lung fibroblasts. J Biol Chem 278: 52203–52211, 2003.[Abstract/Free Full Text]
  5. Benoist F and Grand-Perret T. ApoB-100 secretion by HepG2 cells is regulated by the rate of triglyceride biosynthesis but not by intracellular lipid pools. Arterioscler Thromb Vasc Biol 16: 1229–1235, 1996.[Abstract/Free Full Text]
  6. Bligh EG and Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959.
  7. Chang Y-Y and Kennedy EP. Biosynthesis of phosphatidyl glycerophosphate in Escherichia coli. J Lipid Res 8: 447–455, 1967.[Abstract]
  8. Coleman RA and Bell RM. Selective changes in enzymes of the sn-glycerol-3-phosphate and dihydroxyacetone-phosphate pathways of triacylglycerol biosynthesis during differentiation of 3T3-L1 pre-adipocytes. J Biol Chem 255: 7681–7687, 1980.[Abstract/Free Full Text]
  9. Coleman RA and Bell RM. Triacylglycerol synthesis in isolated fat cells: studies on the microsomal diacylglycerol acyltransferase activity using ethanol-dispersed diacylglycerol. J Biol Chem 251: 4537–4543, 1976.[Abstract/Free Full Text]
  10. Coleman RA and Haynes EB. Selective changes in microsomal enzymes of triacylglycerol and phosphatidylcholine synthesis in fetal and postnatal rat liver: induction of microsomal sn-glycerol 3-P and dihydroxyacetone-P acyltransferase activities. J Biol Chem 258: 450–465, 1983.[Free Full Text]
  11. Coleman RA and Lee DP. Enzymes of triacylglycerol synthesis and their regulation. Prog Lipid Res 43: 134–176, 2004.[CrossRef][Web of Science][Medline]
  12. Coleman RA, Lewin TM, and Muoio DM. Physiological and nutritional regulation of enzymes of triacylglycerol synthesis. Ann Rev Nutr 20: 77–103, 2000.[CrossRef][Web of Science][Medline]
  13. Coleman RA, Lewin TM, Van Horn CG, and Gonzalez-Baró MR. Do acyl-CoA synthetases regulate fatty acid entry into synthetic versus degradative pathways? J Nutr 132: 2123–2126, 2002.[Abstract/Free Full Text]
  14. Dallner G, Siekevitz P, and Palade GE. Biogenesis of endoplasmic reticulum membranes. II. Synthesis of constitutive microsomal enzymes in developing rat hepatocyte. J Cell Biol 30: 97–117, 1966.[Abstract/Free Full Text]
  15. Ericsson J, Jackson SM, Kim JB, Spiegelman BM, and Edwards PA. Identification of glycerol-3-phosphate acyltransferase as an adipocyte determination and differentiation factor 1-and sterol regulatory element-binding protein-responsive gene. J Biol Chem 272: 7298–7305, 1997.[Abstract/Free Full Text]
  16. Ferre P, Satabin P, Decaux JF, Escriva F, and Girard J. Development and regulation of ketogenesis in hepatocytes isolated from newborn rats. Biochem J 214: 937–942, 1983.[Web of Science][Medline]
  17. Fisher EA and Ginsberg HN. Complexity in the secretory pathway: the assembly and secretion of apolipoprotein B-containing lipoproteins. J Biol Chem 277: 17377–17380, 2002.[Free Full Text]
  18. Folch J, Lees M, and Sloane-Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 226: 497–509, 1957.[Free Full Text]
  19. Fulgencio JP, Kohl C, Girard J, and Pegorier JP. Troglitazone inhibits fatty acid oxidation and esterification, and gluconeogenesis in isolated hepatocytes from starved rats. Diabetes 45: 1556–1562, 1996.[Abstract]
  20. Gibbons GF, Islam K, and Pease RJ. Mobilisation of triacylglycerol stores. Biochim Biophys Acta 1483: 37–57, 2000.[Medline]
  21. Graham A, Wood JL, and Russell LJ. Cholesterol esterification is not essential for secretion of lipoprotein components by HepG2 cells. Biochim Biophys Acta 1302: 46–54, 1996.[Medline]
  22. Hammond LE, Gallagher PA, Wang S, Posey-Marcos E, Hiller S, Kluckman K, Maeda N, and Coleman RA. Mitochondrial glycerol-3-phosphate acyltransferase deficient mice have reduced hepatic triacylglycerol content, decreased VLDL, and altered fatty acid composition. Mol Cell Biol 22: 8204–8214, 2002.[Abstract/Free Full Text]
  23. Houweling M, Cui Z, and Vance DE. Expression of phosphatidylethanolamine N-methyltransferase-2 cannot compensate for an impaired CDP-choline pathway in mutant Chinese hamster ovary cells. J Biol Chem 270: 16277–16282, 1995.[Abstract/Free Full Text]
  24. Igal RA and Coleman RA. Acylglycerol recycling from triacylglycerol to phospholipid, not lipase activity, is defective in Neutral Lipid Storage Disease fibroblasts. J Biol Chem 271: 16644–16651, 1996.[Abstract/Free Full Text]
  25. Igal RA, Wang S, Gonzalez-Baró M, and Coleman RA. Mitochondrial glycerol phosphate acyltransferase directs incorporation of exogenous fatty acids into triacylglycerol. J Biol Chem 276: 42205–42212, 2001.[Abstract/Free Full Text]
  26. Kemp BE, Mitchelhill KI, Stapleton D, Michell BJ, Chen Z-P, and Witters LA. Dealing with energy demand: the AMP-activated protein kinase. Trends Biochem Sci 24: 22–25, 1999.[CrossRef][Web of Science][Medline]
  27. Koc H, Mar MH, Ranasinghe A, Swenberg JA, and Zeisel SH. Quantitation of choline and its metabolites in tissues and foods by liquid chromatography/electrospray ionization-isotope dilution mass spectrometry. Anal Chem 74: 4734–4740, 2002.[Medline]
  28. Lewin TM, Granger DA, Kim J-H, and Coleman RA. Regulation of mitochondrial sn-glycerol-3-phophate acyltransferase activity: response to feeding status is unique in various rat tissues and is discordant with protein expression. Arch Biochem Biophys 396: 119–127, 2001.[CrossRef][Web of Science][Medline]
  29. Lewin TM, Kim J-H, Granger DA, Vance JE, and Coleman RA. Acyl-CoA synthetase isoforms 1, 4, and 5 are present in different subcellular membranes in rat liver and can be inhibited independently. J Biol Chem 276: 24674–24679, 2001.[Abstract/Free Full Text]
  30. Lewin TM, Schwerbrock NMJ, Lee DP, and Coleman RA. Identification of a new glycerol-3-phosphate acyltransferase isoenzyme, mtGPAT2, in mitochondria. J Biol Chem 279: 13488–13495, 2004.[Abstract/Free Full Text]
  31. Liang J-S, Distler O, Cooper DA, Jamil H, Deckelbaum RJ, Ginsberg HN, and Sturley SL. HIV protease inhibitors protect apolipoprotein B from degradation by the proteasome: a potential mechanism for protease inhibitor-induced hyperlipidemia. Nat Med 7: 1327–1331, 2001.[CrossRef][Web of Science][Medline]
  32. Lindén D, William-Olsson L, Rhedin M, Asztély A-K, Clapham JC, and Schreyer S. Overexpression of mitochondrial glycerol-3-phosphate acyltransferase in rat hepatocycytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis. J Lipid Res 45: 1279–1288, 2004.[Abstract/Free Full Text]
  33. Listenberger LL, Han X, Lewis SE, Cases S, Farese RV Jr, Ory DS, and Schaffer JE. Triglyceride accumulation protects against fatty acid-induced lipotoxicity. Proc Natl Acad Sci USA 100: 3077–3082, 2003.[Abstract/Free Full Text]
  34. Muoio DM, Lewin TM, Weidmar P, and Coleman RA. Acyl-CoAs are functionally channeled in liver: potential role of acyl-CoA synthetase. Am J Physiol Endocrinol Metab 279: E1366–E1373, 2000.[Abstract/Free Full Text]
  35. Muoio DM, Seefield K, Witters L, and Coleman RA. AMP-activated kinase (AMPK) reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is novel target. Biochem J 338: 783–791, 1999.
  36. Musanti R, Giorgini L, Lovisolo PP, Pirillo A, Chiari A, and Ghiselli G. Inhibition of acyl-CoA: cholesterol acyltransferase decreases apolipoprotein B-100-containing lipoprotein secretion from HepG2 cells. J Lipid Res 37: 1–14, 1996.[Abstract]
  37. Ostrander DB, Sparagna GC, Amoscato AA, McMillin JB, and Dowhan W. Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis. J Biol Chem 276: 38061–38067, 2001.[Abstract/Free Full Text]
  38. Ruan H and Pownall HJ. Effect of 1-acyl-glycerol-3-phosphate acyltransferase over-expression on cellular energy trafficking (Abstract). Diabetes 48: A258, 1999.
  39. Shin D-H, Paulauskis JD, Moustaid N, and Sul HS. Transcriptional regulation of p90 with sequence homology to Escherichia coli glycerol-3-phosphate acyltransferase. J Biol Chem 266: 23834–23839, 1991.[Abstract/Free Full Text]
  40. Shulman GI. Cellular mechanisms of insulin resistance. J Clin Invest 106: 171–176, 2000.[Web of Science][Medline]
  41. Smith JL, Lear SR, and Erickson SK. Developmental expression of elements of hepatic cholesterol metabolism in the rat. J Lipid Res 36: 641–652, 1995.[Abstract]
  42. Spady DK, Willard MN, and Meidell RS. Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster. J Biol Chem 275: 27005–27012, 2000.[Abstract/Free Full Text]
  43. Swanton EM and Saggerson ED. Effects of adrenaline on triacylglycerol synthesis and turnover in ventricular myocytes from adult rats. Biochem J 328: 913–922, 1997.
  44. Taylor DC, Weber N, Hogge LR, and Underhill EW. A simple enzymatic method for the preparation of radiolabeled erucoyl-CoA and other long-chain fatty acyl-CoAs and their characterization by mass spectrometry. Anal Biochem 184: 311–316, 1990.[CrossRef][Web of Science][Medline]
  45. Thompson GR, Naoumova RP, and Watts GF. Role of cholesterol in regulating apolipoprotein B secretion by the liver. J Lipid Res 37: 439–447, 1996.[Abstract]
  46. Veerkamp JH, van Moerkerk TB, Glatz JF, Zuurveld JG, Jacobs AE, and Wagenmakers AJ. 14CO2 production is no adequate measure of [14C]fatty acid oxidation. Biochem Med Metab Biol 35: 248–259, 1986.[CrossRef][Web of Science][Medline]
  47. Wu X, Sakata N, Lui E, and Ginsberg HN. Evidence for a lack of regulation of the assembly and secretion of apolipoprotein B-containing lipoprotein from HepG2 cells by cholesteryl ester. J Biol Chem 269: 12375–12382, 1994.[Abstract/Free Full Text]
  48. Wu X, Shang A, Jiang H, and Ginsberg HN. Low rates of apoB secretion from HepG2 cells result from reduced delivery of newly synthesized triglyceride to a "secretion-coupled" pool. J Lipid Res 37: 1198–1206, 1996.[Abstract]
  49. Yao ZM and Vance DE. The active synthesis of phosphatidylcholine is required for very low density lipoprotein secretion from rat hepatocytes. J Biol Chem 263: 2998–3004, 1988.[Abstract/Free Full Text]
  50. Yu Y-H, Zhang Y, Oelkers P, Sturley SL, Rader DJ, and Ginsberg HN. Posttranscriptional control of the expression and function of diacylglycerol acyltransferase-1 in mouse adipocytes. J Biol Chem 277: 50876–50884, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
W. T. Festuccia, P.-G. Blanchard, V. Turcotte, M. Laplante, M. Sariahmetoglu, D. N. Brindley, and Y. Deshaies
Depot-specific effects of the PPAR{gamma} agonist rosiglitazone on adipose tissue glucose uptake and metabolism
J. Lipid Res., June 1, 2009; 50(6): 1185 - 1194.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
K. Takeuchi and K. Reue
Biochemistry, physiology, and genetics of GPAT, AGPAT, and lipin enzymes in triglyceride synthesis
Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1195 - E1209.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
W. T. Festuccia, P.-G. Blanchard, V. Turcotte, M. Laplante, M. Sariahmetoglu, D. N. Brindley, D. Richard, and Y. Deshaies
The PPAR{gamma} agonist rosiglitazone enhances rat brown adipose tissue lipogenesis from glucose without altering glucose uptake
Am J Physiol Regulatory Integrative Comp Physiol, May 1, 2009; 296(5): R1327 - R1335.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
L. W. Collison, E. J. Murphy, and C. A. Jolly
Glycerol-3-phosphate acyltransferase-1 regulates murine T-lymphocyte proliferation and cytokine production
Am J Physiol Cell Physiol, December 1, 2008; 295(6): C1543 - C1549.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
R. E. Gimeno and J. Cao
Thematic Review Series: Glycerolipids. Mammalian glycerol-3-phosphate acyltransferases: new genes for an old activity
J. Lipid Res., October 1, 2008; 49(10): 2079 - 2088.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Gastrointest. Liver Physiol.Home page
M. R. Gonzalez-Baro, T. M. Lewin, and R. A. Coleman
Regulation of Triglyceride Metabolism II. Function of mitochondrial GPAT1 in the regulation of triacylglycerol biosynthesis and insulin action
Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1195 - G1199.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. O. Li, D. G. Mashek, J. An, S. D. Doughman, C. B. Newgard, and R. A. Coleman
Overexpression of Rat Long Chain Acyl-CoA Synthetase 1 Alters Fatty Acid Metabolism in Rat Primary Hepatocytes
J. Biol. Chem., December 1, 2006; 281(48): 37246 - 37255.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
P. J. Raubenheimer, M. J. Nyirenda, and B. R. Walker
A Choline-Deficient Diet Exacerbates Fatty Liver but Attenuates Insulin Resistance and Glucose Intolerance in Mice Fed a High-Fat Diet.
Diabetes, July 1, 2006; 55(7): 2015 - 2020.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
288/5/E835    most recent
00300.2004v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (13)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lewin, T. M.
Right arrow Articles by Coleman, R. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lewin, T. M.
Right arrow Articles by Coleman, R. A.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.