Glycerol-3-phosphate Acyltransferase (GPAT)-1, but Not GPAT4, Incorporates Newly Synthesized Fatty Acids into Triacylglycerol and Diminishes Fatty Acid Oxidation^*

Materials

Type I collagenase was from Worthington Biochemical Corp. [1-¹⁴C]Acetic acid and [1-¹⁴C]palmitic acid were purchased from PerkinElmer Life Sciences. Silica G gel plates were from Whatman. Tissue culture plates were from BD Biosciences, and media were obtained from Invitrogen. Sigma was the source of 4-methylene-2-octyl-5-oxotetrahydrofuran-3-carboxylic acid (C75) and all other chemicals, unless otherwise indicated.

Animal Care and Hepatocyte Isolation

Animal protocols were approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee. Mice were housed in a pathogen-free barrier facility on a 12-h light/dark cycle with free access to water and food (Prolab 5P76 Isopro 3000; 5.4% fat by weight). Gpat1^−/− (2) and Gpat4^−/− (3, 15) mice had been backcrossed at least eight times onto a C57BL6/J background. At 8 weeks of age, littermate controls and Gpat1^−/− and Gpat4^−/− mice were either fasted for 24 h or fasted for 24 h and then refed a high sucrose diet for 48 h before sacrifice. The high sucrose diet was modified from Research Diets D12450B (10% kcal of fat, 20% kcal of protein, and 70% kcal of carbohydrate (525 g/kg of sucrose, 175 g/kg of corn starch)).

To isolate hepatocytes, after mice were anesthetized with 250 mg/kg of Avertin, a 22-gauge catheter was inserted into the vena cava, and the liver was perfused with wash buffer (10 mm Hepes, 132 mm NaCl, 6.7 mm KCl, 20 mm glucose, 0.5 m adenosine, 100 mm EGTA, 140 nm insulin, pH 7.4) and then with digestion buffer (100 mm HEPES, 57 mm NaCl, 6.7 mm KCl, 5 mm CaCl₂, 20 mm glucose, 0.45 mg/ml type 1 collagenase, pH 7.4). The digested liver was removed and placed in a 100-mm dish on ice with Hanks'/BSA buffer (10 mm HEPES, 137 mm NaCl, 5.4 mm KCl, 1.26 mm CaCl₂, 0.8 mm MgSO₄, 4.2 mm NaHCO₃, 0.34 mm Na₂HPO₄, 0.44 mm KH₂PO₄, 5 mm glucose, pH 7.4). The hepatocytes were dissociated and filtered through a cell strainer, centrifuged at 4 °C at 50 × g for 3 min, and then washed once in Williams' medium E. The cells were resuspended in Williams' medium E:Percoll (16 ml:14 ml), centrifuged at 4 °C at 400 × g for 8 min, and washed with Williams' medium E. Cells were plated onto 60-mm collagen-coated dishes and incubated for 90 min in Williams' medium E + 10% FBS. After the cells attached, the medium was changed to Williams' medium E, 100 units of penicillin/streptomycin, 50 nm insulin, 0.1 μm dexamethasone, and 0.1% BSA.

GPAT Activity

Hepatocytes were cultured overnight in 60-mm plates and then scraped and homogenized in cold Medium I (250 mm sucrose, 10 mm Tris, pH 7.4, 1 mm EDTA, 1 mm dithiothreitol) with 10 up-and-down strokes with a Teflon glass motor-driven homogenizer. Liver was minced and then homogenized similarly. Total membranes were isolated by centrifuging the homogenate at 100,000 × g for 1 h. GPAT specific activity was assayed for 10 min at room temperature in a 200- μl reaction mixture containing 75 mm Tris-HCl, pH 7.5, 4 mm MgCl₂, 1 mg/ml BSA (essentially fatty acid-free), 1 mm dithiothreitol, 8 mm NaF, 800 μm [³H]glycerol 3-phosphate, and 80 μm palmitoyl-CoA (20). The reaction was initiated by adding 10–30 μg of membrane protein after incubating the membrane protein on ice for 15 min in the presence or absence of 2 mm N-ethylmaleimide (NEM), which inactivates GPAT isoforms 2, 3, and 4. The reaction products were extracted into CHCl₃, dried under N₂, resuspended in 4 ml of Cytoscint, and counted on a scintillation counter. NEM-resistant activity (GPAT1) was calculated by subtracting NEM-sensitive activity from total activity.

RNA Extraction and RT-PCR

Total RNA was extracted from overnight cultures of hepatocytes with TRIzol (Invitrogen). cDNA synthesis and real time RT-PCR were performed as described (3). Primer sequences for each GPAT isoform and data normalization used were described (3, 32).

Labeling, Oxidation, and Lipid Extraction

Sixteen to 20 h after plating, hepatocytes were labeled with 0.5 ml of labeling buffer (Krebs Ringer buffer, 10 mm HEPES, 5.5 mm glucose, 0.25% fatty acid-free bovine serum albumin (BSA), and 1 mm carnitine) containing 500 μm [1-¹⁴C]acetic acid or [1-¹⁴C]palmitic acid for 2 h. Media were removed, and CO₂ was liberated by adding 100 μl of 70% perchloric acid and trapped in 1 m NaOH. An aliquot of the NaOH was counted to measure radiolabeled CO₂. To quantify intracellular oxidation metabolites, fresh Krebs Ringer buffer and 100 μl of 70% perchloric acid were added to each plate, and acidified supernatants were collected. The acidified media and cell supernatants were incubated overnight with BSA and then centrifuged at 14,000 × g for 20 min. Aliquots of the supernatant were counted to measure radiolabeled acid-soluble metabolites (ASM), a measure of incomplete fatty acid oxidation. Cells were washed with 1% BSA and then with phosphate-buffered saline before lipids were extracted (16). Lipid extracts and standards were separated by thin layer chromatography on Partisil LK5D silica gel plates in a two-phase system: chloroform:methanol:ammonium hydroxide (65:25:4, v/v/v) run to 50% of the plate and then dried and followed by heptane:isopropyl ether:acetic acid (60:40:4, v/v/v) run to the top of the plate. Radiolabeled lipids were quantified with a Bioscan AR-2000 imaging scanner. Lipid standards were visualized by iodine staining.

Blood Chemistries and Lipids

Before blood collection, mice were either fasted for 24 h or fasted 24 h and then fed a high sucrose diet for 48 h. Plasma TAG (Stanbio, Boerne, TX), free fatty acid (Wako Diagnostics, Richmond, VA), glucose (glucose CII reagent, Wako Diagnostics), and β-hydroxybutyrate (Wako Diagnostics) were determined by enzymatic colorimetric methods.

Lipid Extraction and TAG Measurement in Liver

Liver lipids were extracted (12), dried under N₂ gas, resuspended in 200 μl of tert-butanol:methanol:Triton X-100 (3:1:1, v/v), and analyzed colorimetrically as described above.

Liver Acylcarnitine Measurement

Livers were snap-frozen in liquid nitrogen, pulverized, and weighed. The pulverized liver (50–100 mg) was homogenized in acetonitrile:water:formic acid (50:49.7:0.3; v/v) at a final concentration of 50 mg/ml. Acylcarnitines were analyzed by flow injection tandem mass spectrometry (17). Samples were stored at −80 °C before analysis.

Statistics

Data represent means ± S.E. of at least three independent experiments performed in triplicate unless indicated otherwise. In vitro models were analyzed by one-way analysis of variance comparing each genotype versus controls and post hoc comparisons of diet conditions within each genotype. Data were considered significant at p < 0.05.

Isolated and Cultured Primary Hepatocytes Retained GPAT Specific Activity

To compare the contributions of GPAT1 and GPAT4 to the total amount of GPAT activity, we isolated and cultured primary hepatocytes from control, Gpat1^−/−, and Gpat4^−/− mice and measured GPAT specific activity in the presence and absence of NEM, which inhibits GPAT2, 3, and 4 (Fig. 1A). When compared with wild type hepatocytes, total GPAT specific activity in Gpat1^−/− hepatocytes was 35% lower and minimal residual NEM-resistant GPAT activity remained, whereas NEM-sensitive GPAT activity was unchanged. Total GPAT specific activity in Gpat4^−/− hepatocytes was 42% lower than in control hepatocytes; NEM-resistant GPAT specific activity was unchanged, but NEM-sensitive GPAT activity was 60% lower. The residual NEM-sensitive GPAT activity is likely due to GPAT2 and -3. These results show that isolated and cultured primary hepatocytes retained GPAT activity and that the specific activity was similar in Gpat1^−/− and Gpat4^−/− hepatocytes.

To determine whether other GPAT isoforms were affected by deletion of either GPAT1 or GPAT4, we measured mRNA expression of each GPAT isoform (Fig. 1B). In Gpat1^−/− hepatocytes, Gpat3 mRNA abundance was 2-fold higher than in wild type hepatocytes, but NEM-sensitive GPAT activity did not change, indicating a lack of compensation. In the absence of GPAT4, the mRNA expression of Gpat2 and -3 increased 2.5- and 3.5-fold, respectively. GPAT2 activity is extremely low in liver (5), and even when the amount of Gpat2 mRNA doubles, it probably does not contribute substantially to total NEM-sensitive GPAT activity. However, because we cannot differentiate between GPAT3 and GPAT4 protein or activity, the 42% lower NEM-sensitive GPAT activity (Fig. 1B) could reflect partial compensation by GPAT3.

The Incorporation of de Novo Synthesized Fatty Acid into TAG Required GPAT1

Because GPAT1 is up-regulated by SREBP1c, we hypothesized that it might be functionally and specifically linked to de novo lipogenesis, such that newly synthesized fatty acids would be incorporated into TAG. Using [1-¹⁴C]acetate, we measured the ability of control, Gpat1^−/−, and Gpat4^−/− hepatocytes to incorporate de novo synthesized fatty acid into TAG and PL. The incorporation of acetate into TAG and PL was equal in hepatocytes from wild type and Gpat4^−/− mice; however, in Gpat1^−/− hepatocytes, the incorporation of [¹⁴C]acetate into PL was 78% lower than in controls, and the incorporation into TAG was virtually absent (Fig. 2, A and B). When wild type and Gpat4^−/− hepatocytes were cultured in the presence of the fatty acid synthase inhibitor C75, [¹⁴C]acetate incorporation into TAG was inhibited 80–85% (Fig. 2A). However, in Gpat1^−/− hepatocytes, C75 had no additional effect, indicating that the absence of GPAT1 in hepatocytes had abolished the incorporation of the de novo synthesized fatty acid. In wild type and Gpat4^−/− hepatocytes, C75 decreased acetate incorporation into PL by 60%, but only by 27% in Gpat1^−/− hepatocytes (Fig. 2B). In each group of hepatocytes, the addition of C75 decreased the incorporation of acetate into free fatty acids by more than 72% (data not shown). These results show that GPAT1, but not GPAT4, is required to incorporate de novo synthesized fatty acid into complex lipids.

Both GPAT1 and GPAT4 Can Incorporate Exogenous Fatty Acids into TAG and Phospholipid

In addition to de novo synthesis, liver fatty acids are also derived from exogenous sources. To distinguish between the functions of GPAT1 and GPAT4 in the use of exogenous fatty acids, we measured the incorporation of [1-¹⁴C]palmitate into glycerolipids in primary hepatocytes. In contrast to fatty acids derived from de novo synthesis, the incorporation of [1-¹⁴C]palmitate into TAG and PL was not altered by the absence of either GPAT1 or GPAT4 (Fig. 3, A and B), indicating that neither isoform was essential for the use of exogenous fatty acid. Thus, in both Gpat4^−/− and Gpat1^−/− hepatocytes, the remaining GPAT isoforms can fully incorporate exogenous fatty acid into glycerolipids.

In Hepatocytes, the Absence of GPAT1, but Not GPAT4, Increased Fatty Acid Oxidation

Previous studies have strongly suggested that GPAT1 competes with CPT1 for acyl-CoAs at the outer mitochondrial membrane (12). To confirm this competition for acyl-CoAs and to determine whether GPAT4 located on the endoplasmic reticulum competes similarly, we measured the incorporation of [1-¹⁴C]palmitate into ASM, a measure of incomplete fatty acid oxidation. Gpat1^−/− hepatocytes oxidized [¹⁴C]palmitate at a rate 200% higher than control hepatocytes, but the incorporation of [¹⁴C]palmitate into ASM by Gpat4^−/− hepatocytes was unchanged (Fig. 3C). Of the total ASM extracted from the media and cells, intracellular ASM comprises only 5% of the total ASM (Fig. 3C, inset); thus, media ASM reflect 95% of the total ASM produced. These data show that only GPAT1 competes with CPT1 for acyl-CoAs, thereby diminishing the flux of fatty acid into the mitochondria for oxidation.

Hepatic GPAT1 Diminished Fatty Acid Transport into Mitochondria in Vivo

To confirm the disparate effects of each GPAT isoform on the β-oxidation of exogenous versus de novo synthesized fatty acid in vivo, we measured hepatic acylcarnitine content in wild type, Gpat1^−/−, and Gpat4^−/− mice after food was removed for 24 h. Because fasting increases the flux of adipose-derived fatty acids into the liver, more fatty acids are transported to the mitochondria via CPT1, thereby elevating the content of hepatic long-chain acylcarnitines. These metabolites are subsequently reconverted to acyl-CoAs in the mitochondrial matrix, where they enter the β-oxidation pathway. In fasting mice of each genotype, the content of free carnitine (C0), acetylcarnitine (C2), and short-chain (C3-C6) and medium-chain (C8-C12) acylcarnitine species was similar (Fig. 4, A and C). Consistent with the hypothesized ability of GPAT1 to compete with CPT1 for acyl-CoAs, total long-chain (C14-C22) acylcarnitine content in fasted Gpat1^−/− liver was 3-fold higher than in control liver (Fig. 4C). The most abundant long-chain acylcarnitine species, 14:0, 16:1, 16:0, 18:2, 18:1, and 18:0, were 2–6-fold higher than in control liver (Fig. 4E). In contrast, and consistent with the in vitro data, the acylcarnitine content in livers of fasted Gpat4^−/− mice was similar to that of control mice.

Because GPAT1 was essential for the incorporation of de novo synthesized fatty acids into complex lipids in vitro, we predicted that the absence of hepatic GPAT1 in vivo would also increase the transport of de novo synthesized fatty acids into mitochondria. To test this hypothesis, we fasted mice for 24 h and then refed them with a high sucrose diet for 48 h to stimulate de novo lipogenesis. The total long-chain (C14-C22) acylcarnitine content of Gpat1^−/− liver remained 3.5-fold higher than that of controls, and the most abundant long-chain acylcarnitine species, 14:0, 16:1, 16:0, 18:2, 18:1 and 18:0, ranged from 2- to 5-fold higher (Fig. 4, D and F). In contrast, no differences were observed in the hepatic acylcarnitine content of Gpat4^−/− mice that had been fasted and then refed (Fig. 4, B, D, and F). Thus, GPAT1, but not GPAT4, was required in vivo to diminish CPT1-mediated transport of both de novo synthesized and exogenous fatty acid.

Hepatic GPAT1 Was Essential for the Incorporation of de Novo Synthesized Fatty Acid into TAG

Because the absence of GPAT1 increased hepatic β-oxidation of de novo synthesized fatty acids in vivo, we predicted that the liver TAG content would be low. After a 24-h fast, hepatic TAG content in control, Gpat4^−/−, and Gpat1^−/− liver was similar. After refeeding a high sucrose diet to enhance de novo lipogenesis, the TAG content in control and Gpat4^−/− liver increased 2–2.5-fold (Fig. 5A); however, the hepatic TAG content of Gpat1^−/− liver did not increase. These data support the conclusion that GPAT1 is essential for the incorporation of de novo synthesized fatty acid into TAG and for diverting those fatty acids away from mitochondrial oxidation.

The Absence of GPAT1, but Not GPAT4, Increased Ketogenesis during Fasting

When the concentration of plasma glucose decreases during an extended fast, adipocyte-derived fatty acids flood into the liver, their oxidation rate increases, and ketone bodies are synthesized and secreted into the blood. The accumulation of long-chain acylcarnitines in the liver together with an elevated plasma concentration of ketone bodies suggests an increased rate of hepatic fatty acid oxidation. Because GPAT1 diminishes the oxidation of exogenous fatty acid in vitro and the absence of GPAT1 in liver results in the accumulation acylcarnitines, we predicted that fasting would result in a higher rate of hepatic β-oxidation in Gpat1^−/− mice than in controls. To confirm this hypothesis, we measured plasma ketone bodies in control, Gpat1^−/−, and Gpat4^−/− mice. After a 24-h fast, the concentrations of plasma glucose and TAG in control and Gpat1^−/− mice were similar (Fig. 5, B and C), but the plasma fatty acid concentration in Gpat1^−/− mice was 1.5-fold higher than controls (Fig. 5D). Consistent with elevated fatty acid oxidation in the Gpat1^−/− mice, the plasma concentration of β-hydroxybutyrate was 2-fold higher than controls (Fig. 5E).

Ketogenesis Remained Elevated in Gpat1^−/− Mice during Refeeding

After a 24-h fast, refeeding with high sucrose drives hepatic de novo lipogenesis, promotes TAG storage, and blocks fatty acid oxidation (18). Because our data suggested that GPAT1 esterifies de novo synthesized fatty acids, we asked whether the rate of hepatic fatty acid oxidation would remain elevated during short-term high sucrose refeeding. Although a high carbohydrate diet diminishes PPARα gene targets related to β-oxidation and ketone body formation, in the absence of GPAT1, the plasma β-hydroxybutyrate concentration in Gpat1^−/− mice was 3-fold higher than in control or Gpat4^−/− mice (Fig. 5D), consistent with a continued higher rate of hepatic fatty acid oxidation. Consistent with diminished hepatic TAG synthesis in both Gpat1 and Gpat4 null mice, when compared with controls, the plasma TAG concentration was 48% lower in Gpat1^−/− mice and 25% lower in Gpat4^−/− mice (Fig. 5C). Plasma glucose and fatty acid concentrations in each genotype were similar (Fig. 5, B and E).