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* AstraZeneca R&D, Mölndal, Sweden
Wallenberg Laboratory for Cardiovascular Research, Göteborg University, Sweden
Published, JLR Papers in Press, April 21, 2004. DOI 10.1194/jlr.M400010-JLR200
1 To whom correspondence should be addressed. e-mail: sandra.schreyer{at}astrazeneca.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis.
Abbreviations: CHO, Chinese hamster ovary; CPT-1, carnitine palmitoyl transferase-1; ER, endoplasmic reticulum; GPAT, glycerol-3-phosphate acyltransferase; MOI, multiplicity of infection; wt, wild type
Supplementary key words glycerol-3-phosphate acyltransferase • obesity • triglyceride
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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10% of the total activity, but in the liver, mtGPAT comprises up to 50% of the total activity (2). Activity between mtGPAT and microsomal GPAT is differentiated by their selective sensitivity to sulfhydryl group reactive reagents such as N-ethylmaleimide (NEM). These reagents inhibit microsomal GPAT, whereas mtGPAT activity is resistant to inhibition (3, 4). Analysis of mtGPAT-deficient mice (mtGPAT–/–) clearly established that two separate genes encode these activities, because microsomal GPAT activity was normal in the mtGPAT–/– mice (5). MtGPAT has a preference for saturated fatty acyl-CoAs, especially palmitoyl-CoA, whereas microsomal GPAT does not show such substrate preferences (1, 6, 7). Because phosphatidic acid is the predominant intermediate for both phospholipid and TG synthesis, it may be that these two GPAT isoforms are involved with regulating both the amount and the fatty acid saturation of de novo synthesized phospholipids, diacylglycerols (DGs), and TGs (1). Recent studies suggest that mtGPAT is involved in both obesity and dyslipidemia associated with obesity. MtGPAT–/– mice have reduced body weight and total adipose tissue weight and lower hepatic TG content, as compared with wild-type (wt) mice (5). In obese Zucker (fa/fa) rats, total GPAT activity is elevated in adipose tissue and intestine, when compared with lean rats (8), indicating that mtGPAT is induced in response to obesity. Overexpressing mtGPAT in Chinese hamster ovary (CHO) cells increases the incorporation of labeled fatty acids into TGs (9). The promotor of mtGPAT contains both sterol- and carbohydrate-responsive elements (10, 11), and mtGPAT mRNA level is upregulated by a high-carbohydrate, fat-free diet and by insulin administration to streptozotocin-diabetic mice (12). Taken together, these results show that mtGPAT activity is upregulated in tissues from obese animals or from animals fed high-carbohydrate diets and that increased activity may promote TG synthesis.
In this study, we investigated whether overexpressing mtGPAT in hepatocytes alters fatty acid metabolism and whether obesity influences hepatic mtGPAT activity. Primary rat hepatocytes were infected with control (ZsGreen) adenovirus or viruses expressing murine mtGPAT. Overexpressing mtGPAT resulted in a sharp reduction in fatty acid oxidation and a significant increase in glycerolipid synthesis. In addition, an invariant aspartic acid residue was changed to glycine [D235G] in the presumed active site within the N terminus of the enzyme (13, 14) by site-directed mutagenesis, and the activity of this enzyme was evaluated in infected cells. No activity was observed with this mutant mtGPAT, demonstrating that this residue is critical for activity. Finally, mtGPAT activity was measured in livers from lean and obese rodents and shown to be increased >2-fold in obese animals. These results support the hypothesis that increased hepatic mtGPAT activity associated with obesity contributes to increased lipid synthesis and inhibition of fatty oxidation, responses that would promote obesity itself as well as lipid disorders associated with obesity.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Animal experimental procedures
Female Sprague-Dawley rats, 200 g, were obtained from Harlan Laboratories (Horst, The Netherlands). Six-week-old female C57BL/6, C57BL/6-ob/ob (referred to as ob/ob), and C57BL/6J-lean/? (referred to as lean/?) mice were obtained from Taconic M and B Laboratory (Ry, Denmark). Animals were maintained under standardized conditions of temperature (21–22°C) and humidity (40–60%), with lights on between 6 AM and 6 PM. The animals had free access to water and regular chow diet containing 12% fat, 62% carbohydrates, and 26% protein with a total energy content of 3.0 Kcal/g (R3 diet, Lactamin AB, Stockholm, Sweden). C57BL/6 mice were fed either the chow diet or a high-fat/high-sucrose (HFHS) diet (diet F1850, Bioserve, Frenchtown, NJ) for 12 weeks. The HFHS diet contained 36% fat (lard), 37% carbohydrate (mainly sucrose), and 20% protein, with a total energy content of 5.5 Kcal/g. Caloric content of the HFHS diet included 58% from fat and 27% from carbohydrate. Ob/ob and lean/? mice were analyzed at 12 weeks of age. Mice were fasted for 3 h and then anesthetized with isoflurane, and blood was collected by cardiac puncture. The livers were removed, immediately frozen in liquid nitrogen, and stored at –70°C until analysis. All animal experiments were conducted in accordance with accepted standards of humane animal care and approved by the Ethics Committee of Göteborg University.
Cloning and construction of mtGPAT-FLAG and transfection of CHO cells
A murine mtGPAT open reading frame having an FLAG epitope (DYKDDDDK) at the C terminus was cloned by polymerase chain reaction (PCR) amplification from a mouse liver cDNA library. Two PCR fragments were amplified and cloned into a pBluescript II SK vector using 5'-Sal I, 3'-Not I, and an internal EcoRI site (nt 1,582 from ATG). The first 1,650 bp PCR fragment was amplified using the oligonucleotides 5'-GTACCGTCGACCACCATGGAGGAGTCTTCAGTG-3' (5'Sal I) and 5'-GTGACACAGTTCCCCAGAAGCTG-3' (3'EcoRI). The second 978 bp fragment containing the FLAG coding sequence was amplified using the oligonucleotides 5'-GGAAGTCCTAGCTCGCGATTTCG-3' (5'EcoRI) and 5'-TCGAGCGGCCGCCTACTTGTCATCATCATCCTTGTAGTCCAGCACCACAAAACTCAG-3' (3'-Not I-FLAG). The sequences were confirmed by sequencing using the M13 forward/reverse primers in addition to four internal primers. The GPAT construct was subcloned into the mammalian expression vector pcDNA 3.1/Hygro using Apa I and Not I restriction enzymes. CHO cells were cultured in DMEM with 10% heat-inactivated FBS at 37°C, 5% CO2, and 100% humidity. The cells were seeded in 10 cm culture dishes, cultured to 60–80% confluence, and transiently transfected with empty pcDNA3.1 or pcDNA3.1-mtGPAT using lipofectamine in serum-free DMEM according to the Invitrogen manual. After 4 h, 10% FBS was added, and the cells were harvested after 24 h and assayed for mtGPAT protein expression and activity.
Site-directed mutagenesis and production of recombinant adenoviruses
A D235G mtGPAT mutant construct was generated using a PCR-based site-directed mutagenesis kit (Quickchange) and the oligonucleotides 5'-GCACAGATCCCACATTGGCTACCTGTTGCTCACC-3' (D235G forward) and 5'-GGTGAGCAACAGGTAGCCAATGTGGGATCTGTGC-3' (D235G reverse) according to the manufacturer's instructions (mutated base in boldface type). The full-length sequence was confirmed by sequencing using the same primers described before and recloned into pBluescript II SK. To verify that constructs remained in-frame, a coupled in vitro transcription-translation assay was performed with a TNT rabbit reticulocyte lysate system. Reactions were performed with [35S]methionine and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography. Both mitochondrial GPAT, referred to as mtGPATwt, and mutated mtGPAT, referred to as mtGPATdg, were subcloned into pShuttle-CMV using Sal I and Not I restriction enzymes and resequenced. Recombinant adenoviruses were produced by Escherichia coli-mediated recombination of cotransformed adenoviral backbone plasmid pAdEasy-1 and linearized pShuttle-mtGPATwt and mtGPATdg constructs. Positive clones were selected and confirmed by Pac I digestion. Correct clones were amplified following transformation of XL 10-Gold cells, linearized with Pac I, and purified using a Strataprep PCR purification kit. The packaging cell line AD-293 was grown in DMEM with 10% FBS supplemented with 100,000 IU/l penicillin and 100 mg/l streptomycin. Cells were seeded in 25 cm2 culture flasks, cultured to 60–80% confluence, and transfected with adenoviral constructs for mtGPATwt, mtGPAT D235G, or ZsGreen using lipofectamine in DMEM without serum. After four h incubation, 10% FBS was added, and the cells were cultured for 10–14 days before harvesting. Viruses were harvested by repeated freeze/thaw cycles in 10 mM Tris, pH 8.0, centrifuged, and used for further amplification of viral stocks. Virus titer was determined using a quantitative real-time PCR (RT-PCR) method (15).
Isolation and culture of primary rat hepatocytes
Hepatocytes were prepared by a nonrecirculating collagenase perfusion (37°C and 95% O2) through the portal vein of 200–300 g female Sprague-Dawley rats anesthetized by isoflurane inhalation. The livers were first perfused with HBSS medium supplemented with 0.6 mM EGTA, 20 mM HEPES, and 10 mM Na HCO3. This solution was followed by WE medium supplemented with 50,000 IU/l penicillin, 50 mg/l streptomycin, 0.28 mM sodium ascorbate, 0.1 µM sodium selenite, and 400 mg/l collagenase. Cells were filtered through 250 µm and 100 µm pore size mesh nylon filters and washed three times in WE medium supplemented as described above but without collagenase and set to contain 28 mM glucose, 3 nM insulin, and 1 nM dexamethasone. The cells were cultured (200,000 cells/cm2) in the same medium as that used for washing the cells supplemented with 10% FBS. Cells were plated for 4 h and incubated with recombinant adenoviruses for 2 h in serum-free medium. Serum-containing medium was then added without removing the viruses. Media were changed at 23 h to remove virus. For cells cultured under high-fat conditions, the media for the following 24 h also contained 150 µM oleate plus 150 µM palmitate conjugated to 0.5% BSA. In some experiments, hepatocytes were cultured on laminin-rich matrigel in serum-free medium. Similar results were obtained as compared with cells cultured on plastic, and only results from those cultured on plastic are presented here.
Mitochondrial GPAT protein expression
Total cellular proteins were isolated by homogenization in 1% Triton X-100 supplemented with Complete protease inhibitors. Mitochondrial proteins were isolated in sucrose solution containing Complete protease inhibitors as described for the GPAT assay. Protein (30 µg) was separated in 4–12% NuPage gels and transferred to immobilon P membranes, which were probed with polyclonal rabbit anti-mtGPAT antibodies directed against the murine amino acid sequence 312–326 (AgriSera, Vännäs, Sweden) or anti-FLAG M2 antibodies. The membranes were washed and incubated with horseradish peroxidase-linked donkey anti-rabbit Ig and developed using Super signal West Pico chemiluminescent substrate.
GPAT activity
Liver samples were homogenized in sucrose solution (250 mM sucrose, 10 mM Tris, pH 7.4, 1 mM EDTA, and 1 mM DTT), and nuclei were removed by centrifuging at 600 g for 15 min at 4°C. Supernatants were then spun at 8,000 g for 15 min at 4°C to pellet crude mitochondria, then at 100,000 g for 1 h at 4°C to pellet microsomes. Mitochondria and microsomal fractions were then tested for GPAT activity in the presence and absence of 2 mM NEM. Subsequent analysis of these fractions demonstrated that the mitochondrial fraction still contained significant amounts of ER protein. Thus, the NEM-resistant activity for mitochondrial and microsomal pellets was pooled and assigned as mitochondria activity (Table 2). Similarly, the NEM-sensitive activity for both pellets was pooled and assigned as microsomal activity. For assays with cultured hepatocytes, cells were rinsed in PBS, lysed in sucrose solution, and centrifuged to pellet nuclei; then the supernatant was centrifuged at 100,000 g to obtain both microsomes and mitochondria. Pellets were then tested for NEM-sensitive and NEM-resistant GPAT activity. The use of NEM sensitivity on a single high-speed spin pellet has been previously demonstrated to effectively differentiate mitochondrial from ER-associated GPAT activity (4, 9, 16–18).
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ß-oxidation
Cells were incubated in control medium or infected with virus as described earlier. The medium was then changed to serum-free WE medium containing 3 nM insulin, 1 nM dexamethasone, and either 300 µM palmitic acid plus 1 µCi [9,10(n)-3H]palmitic acid/ml or 300 µM oleic acid containing 1 µCi [9,10(n)-3H]oleic acid/ml oleic acid. Fatty acids were complexed to 0.5% fatty acid-free BSA. After the given incubation period at 37°C, the medium was removed and mixed with concentrated perchloric acid (0.5% final volume) plus BSA (2% final volume) to precipitate the labeled fatty acids. Samples were vortexed and centrifuged (5,000 rpm for 5 min), and a second round of BSA-mediated fatty acid precipitation was performed. Radioactivity was determined in the supernatant to measure water-soluble ß-oxidation products.
Lipid biosynthesis
Hepatocytes were incubated with radioactive palmitate or oleate as described above. Cells were rinsed with PBS and scraped from the plates using PBS containing 1% Triton X-100. Lipids were extracted from either the cellular homogenate or the media in methanol-chloroform (1:2) (20) followed by a second chloroform extraction, and samples were subjected to thin-layer chromatography for separation. To separate neutral lipids, hexane-diethyl ether-acetic acid (80:20:1) or petroleum ether-diethyl ether-acetic acid (70:30:1) was used, whereas chloroform-methanol-acetic acid-water (25:15:4:2) was used to separate polar lipids. Lipids were visualized with iodine vapor and compared with known standards. The radioactivity incorporated into lipids was determined by scraping corresponding bands into scintillation vials and counting radioactivity.
RNA quantification
Mouse GPAT was quantified from liver samples using RT-PCR. RNA was isolated from liver samples using Trizol (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. cDNA was then synthesized using the Invitrogen Superscript synthesis system. Samples were quantified for mouse GPAT mRNA with the following primers, obtained from Applied Biosystems UK (Cheshire, UK): GPAT forward, 5'-GGCTTGAGAGCTGCCACCT-3'; probe, VIC 5'-TAGTGCGGCCAGTGACTGCTTCAAGA-3' TAMRA; GPAT reverse, 5'-AGCCCTTTAGCTGTTGGGAAG-3'.
Samples were quantified using an Applied Biosystems 7700 sequence detector (Foster City, CA) and corrected to the expression of the endogenous control, acidic ribosomal phosphoprotein P0 (36B4) using the following primers: 36B4 forward, 5'-GAGGAATCAGATGAGGATATGGGA-3'; probe, VIC 5'-TCGGTCTCTTCGACTAATCCCGCCAA-3' TAMRA; and 36B4 reverse, 5'-AAGCAGGCTGACTTGGTTGC-3'.
Total lipid mass measurements
Following extraction of lipids (20), samples were dissolved with a mix of solutions A and B (see below) at a ratio of 9:1. The mobile phase was created by a combination of three solvent mixtures: A) heptane-tetrahydrofuran (99:1, v/v); B) acetone-dichloromethane (2:1; v/v); and C) isopropanol-water (85:15, v/v) containing 7.5 mM acetic acid and 7.5 mM ethanolamine. Samples were separated on an HPLC system using a Waters spherisorb, 5 µm silica column (4.6 x 100 mm, no. 830112, Sorbent AB, Sweden). Lipids were detected with a PL-ELS 1000 Evaporative Light Scattering Detector from Polymer Laboratories, and HPLC Mix 41 was used as the standard.
Statistics
Values are expressed as mean ± SEM. All experiments were repeated two to four times using different rat hepatocyte perfusions, and representative experiments are presented. Comparisons between means were made by ANOVA. ANOVA was followed, when appropriate, by Tukey's posthoc test to do pair-wise comparisons between all groups. Student's t-test was used to evaluate pairwise comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Along with overexpressing wt mtGPAT protein, we aimed to identify amino acid domains of mtGPAT critical for activity. Studies in bacteria have demonstrated that aspartic acid D311 is critical for activity (13, 14). Because the homology in this region is conserved between several species, a corresponding mutation was introduced in the murine mtGPAT construct, changing aspartic acid 235 to a glycine residue (D235G). The mtGPAT protein level was then compared in cells infected with 20 multiplicity of infection (MOI) of either AdmtGPATwt or the mutant protein, AdmtGPATdg. Results show that AdmtGPATdg protein was expressed, although expression levels were slightly lower than those observed in AdmtGPATwt-infected cells (Fig. 2B). When cells were infected with a five-times-higher titer (100 MOI; AdmtGPATdg-ht), a much higher protein expression than 20 MOI of either AdmtGPATwt or AdmtGPATdg was observed (Fig. 2B). Despite showing high protein expression, AdmtGPATdg had no effect on GPAT activity, even when administered at the high titer (Fig. 2C). Thus, this single amino acid substitution gives rise to an inactive enzyme.
Effect of overexpressing mtGPAT on ß-oxidation
To study the effect of overexpression of mtGPAT on fatty acid oxidation, infected hepatocytes were incubated for 1 h with [3H]palmitic acid, and the water-soluble ß-oxidation products were assayed in the media (Fig. 3A)
. Hepatocytes infected with AdmtGPATwt had an 81% reduction in palmitate oxidation, as compared with AdZsGreen-infected cells (P < 0.05). After a 2 h pulse, we observed a similar reduction in palmitate oxidation (AdZsGreen: 14.6 ± 1.0 nmol/mg; AdmtGPATwt: 2.9 ± 0.8 nmol/mg; P < 0.001), and dramatic differences continued through 4 h. Hepatocytes infected with AdmtGPATdg showed no difference in oxidation, as compared with AdZsGreen-infected cells, again demonstrating that this mutation results in an inactive enzyme (Fig. 3A). Finally, infected hepatocytes were lipid loaded by incubating them for 24 h with 300 µM oleic acid-palmitic acid (1:1) and then tested for fatty acid oxidation using either [3H]oleic acid or [3H]palmitic acid. As shown in Fig. 3B, lipid-loaded hepatocytes infected with AdmtGPATwt had decreased oleate oxidation, as compared with AdZsGreen-infected cells and the difference was maintained during all time-points examined. Similar results were observed for cells pulsed with [3H]palmitic acid (7.8 ± 0.3 nmol/mg/hr for AdZsGreen vs. 1.3 ± 0.2 nmol/mg/hr for AdmtGPATwt; P < 0.0001). Results demonstrate that under both low and high fatty acid concentrations and regardless of the radiolabeled fatty acid tracer utilized, overexpression of mtGPAT impairs fatty acid oxidation.
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20 nmol/mg/hr. These results suggest that the radiolabeled DG is being directed more toward phospholipid synthesis than TG synthesis. The intracellular cholesteryl ester radioactivity was low and did not differ between AdmtGPATwt- and AdZsGreen-infected cells (data not shown).
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To ascertain whether AdmtGPATwt-infected hepatocytes have increased lipid accumulation, intracellular total lipid mass was measured in cells cultured for 24 h with or without 300 µM oleic acid/palmitic acid (Table 1). Lipid loading of the cells resulted in a 1.7–2.3-fold increase in intracellular DG and TG. Under normal culture media (non-lipid loading), AdmtGPATwt-infected hepatocytes had elevated levels of DG but not TG, as compared with AdZsGreen-infected cells (Table 1). Following lipid loading of the cells, both intracellular DG and TG levels were significantly increased in response to mtGPAT overexpression (Table 1). The intracellular phospholipid and cholesteryl ester total mass was not influenced by mtGPAT overexpression (data not shown). Thus, results were in good overall agreement with the results obtained for measuring de novo synthesis of these lipids.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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In states of nutrient excess in which there are high circulating insulin and low glucagon levels, hepatic de novo lipogenesis is upregulated while fatty acid oxidation is reduced (22–24). Activation of lipogenic pathways and suppression of fatty acid oxidation are also observed in obesity (25, 26). Insulin treatment or carbohydrate-rich diets upregulate hepatic mtGPAT activity in vivo (12). In this study, we show that obesity also increases hepatic mtGPAT activity. Similar to mtGPAT, acetyl-CoA carboxylase is upregulated in response to carbohydrate-rich diets (27) and in the obese state (28). Increased acetyl-CoA carboxylase activity increases malonyl-CoA concentrations; this increase, in turn, inhibits carnitine palmitoyl transferase-1 (CPT-1), leading to reduced fatty acid oxidation (29, 30). Both CPT-1 and mtGPAT are located on the mitochondrial outer membrane (31–33), suggesting that they compete for the same pool of fatty acids for utilization in mitochondrial oxidation or glycerolipid synthesis, respectively. Our results demonstrate that overexpression of mtGPAT decreases fatty acid oxidation, supporting the concept that mtGPAT can compete fatty acids away from CPT-1. Therefore, decreased hepatic ß-oxidation associated with obesity may result from a combination of reduced CPT-1 activity and selective partitioning of fatty acids away from CPT-1 and into glycerolipid synthesis via mtGPAT. Whether this occurs in tissues other than liver remains to be determined.
In this study, overexpression of mtGPAT increased DG, TG, and phospholipid biosynthesis. However, the increase in TG synthesis was seen only in cells provided with additional fatty acids. This suggests that triglyceride synthesis is not necessarily the default endpoint when glycerolipid synthesis is promoted. Indeed, our results suggest that PC synthesis is the primary default pathway induced in response to mtGPAT overexpression. In HEK 293 cells transiently transfected with mtGPAT, increased incorporation of labeled oleate into both TG and phospholipids is observed (9). However, when transfections were done in CHO cells, TG biosynthesis increased, whereas the PC biosynthesis was decreased (9). Differences may reflect the altered capabilities of nonhepatic cell lines to respond to increased lipid synthesis. Studying the overexpression of mtGPAT in hepatocytes, a cell whose major function is to regulate the synthesis and secretion of these lipids, more clearly defines the actual role of mtGPAT in lipid partitioning.
Surprisingly, increased glycerolipid biosynthesis was seen in response to mtGPAT overexpression when using either palmitic acid or oleic acid as radioactive tracers, indicating that mtGPAT can utilize both saturated and monounsaturated fatty acids. Numerous studies have demonstrated that mtGPAT prefers palmitic acid over other saturated or unsaturated fatty acids (1, 6, 7). Our findings suggest that this preference may not be reflected in the intact cell. Indeed, it has recently been shown that mtGPAT substrate preference depends partially on fatty acid binding protein availability (34). In that study, mtGPAT substrate preference depended on the availability of albumin or acyl-CoA binding protein in the assay media. Thus, the cellular fatty acid/fatty acid binding protein concentrations may influence mtGPAT activity more than does fatty acid composition.
There is growing evidence that GPAT activity is upregulated in multiple tissues from obese animals. In obese Zucker (fa/fa) rats, total GPAT activity is elevated in adipose tissue and intestine (8) and in pancreatic ß-cells (35), as compared with lean rats. We show that mtGPAT activity is upregulated in livers from two different mouse models of obesity. In the HFHS diet-induced obese mice, mtGPAT activity was increased but protein levels were unchanged. A discordance between mtGPAT activity and protein expression in rat tissues in response to dietary treatment has been reported (21), suggesting that mtGPAT activity may be regulated posttranslationally. However, in livers of severely obese (ob/ob) mice, mtGPAT mRNA, protein, and activity were all increased. Possible explanations for this are that a threshold of obesity is required before mtGPAT mRNA levels are increased or that leptin deficiency upregulates the expression of this gene.
The extent of increased mtGPAT activity we observed in mtGPAT-overexpressing hepatocytes was comparable with the in vivo induction in response to obesity. This suggests that results obtained from the in vitro studies are metabolically consistent with what may be occurring in vivo. Thus, increased mtGPAT activity associated with obesity likely contributes to increased fat synthesis and deposition, a process that would further contribute to obesity.
Alignment of various membrane-bound glycerolipid acyltransferases from different species and mutagenesis studies of bacterial GPAT (13, 14) and murine mtGPAT (36) have demonstrated that a highly conserved region in the N terminus (aa 224-318) is critically important for GPAT activity. Using site-directed mutagenesis of bacterial GPAT, an invariant aspartic acid residue in the N terminus was shown to be of importance for GPAT activity (13, 14). In this study, the corresponding and conserved murine aspartic acid D235 was found to be critical for mtGPAT function in rat hepatocytes. Thus, in addition to a highly conserved arginine residue, R318 (36), D235 is required for murine mtGPAT activity. These findings help define domains of mtGPAT that may be targets for inhibition to reduce cellular mtGPAT activity.
The results presented here help identify mtGPAT as a major regulator of fatty acid partitioning in hepatocytes and suggest that this enzyme may be involved in promoting lipid synthesis. The upregulation of mtGPAT in livers from obese rodents is consistent with the involvement of mtGPAT in fatty liver and may contribute to hyperlipidemia in states of nutrient excess. Elevated hepatic mtGPAT associated with obesity may contribute to increased lipid synthesis and inhibition of fatty oxidation, responses that would drive obesity itself and lipid disorders associated with obesity. These findings suggest that inhibiting mtGPAT may be beneficial in reducing hepatic lipid accumulation by promoting hepatic ß-oxidation.
Manuscript received January 13, 2004 and in revised form March 30, 2004.
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REFERENCES |
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and activation of a phospholipase C. Biochemistry. 29: 8735–8740.[CrossRef][Medline]
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