Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols

Abstract

Background: De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid.

Methods and results: Incubation of GPAT2-transfected CHO-K1 cells with [1-(14)C]arachidonate for 3 h increased incorporation of [(14)C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2's role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein.

Conclusions: These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

Figure 1. GPAT2 overexpression increased TAG storage in CHO-K1 cells.

CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs tagged with a FLAG epitope (Lanes 1–3 and 4–6 correspond to two different transient transfections). The expression of GPAT1 and GPAT2 was confirmed by western blot. Total particulate protein (50 µg) from GPAT1, GPAT2 and control cells was probed with anti-FLAG (A) and anti-GPAT2 (B) antibodies. The molecular mass of the expressed protein was 90 kDa (GPAT1) and 80 kDa (GPAT2). The membranes were probed with anti-β-actin antibody as a loading control. C) Lipid droplets were visualized in control, GPAT1, and GPAT2-overexpressing CHO-K1 cells by Oil-Red O staining. D) The average size of cellular lipid droplets and the average number of lipid droplets in each cell were quantified by Image Pro plus v5.1 software. Data represent mean ± SD of three independent experiments. (*p<0.05).

Figure 2. GPAT2 overexpression increased arachidonoyl-CoA esterification.

CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control), pcDNA3.1-GPAT1 (GPAT1) or pcDNA3.1-GPAT2 (GPAT2) constructs. A) NEM-sensitive GPAT activity was measured with [³H]glycerol-3-phosphate and the acyl-CoA esters substrates palmitoyl-CoA (16∶0-CoA), oleoyl-CoA (18∶1-CoA), linoleoyl-CoA (18∶2-CoA), arachidonoyl-CoA (20∶4-CoA), eicosapentaenoyl-CoA (20∶5-CoA) and docosahexanoyl-CoA (22∶6-CoA) in CHO-K1 cells. GPAT2 overexpression significantly increased GPAT activity only when arachidonoyl-CoA was used as a substrate. B) GPAT activity was measured in both control and GPAT2-overexpressing CHO-K1 and Vero cells with [³H]glycerol-3-phosphate and arachidonoyl-CoA in the absence (Total GPAT activity) and presence (NEM-resistant, NEM+) of 2 mM NEM. NEM-sensitive GPAT activity (NEM−) was calculated by difference of the other two activity values. C) NEM-resistant GPAT activity was measured in control and GPAT1-overexpressing CHO-K1 cells with the substrates palmitoyl-CoA (16∶0-CoA) and arachidonoyl-CoA (20∶4-CoA). D) The ratio between NEM-sensitive GPAT activity in control and GPAT2-overexpressing CHO-K1 cells with the same fatty acyl-CoA substrates as in A) and Vero cells with palmitoyl-CoA and arachidonoyl-CoA was calculated. Bars represent the mean ± SD of three independent experiments (**p<0.01).

Figure 3. GPAT2 overexpression increased phosphatidic acid synthesis.

A) The reaction products of GPAT reaction measured in control (C), GPAT1 (G1) and GPAT2 (G2)-overexpressing CHO-K1 cells with [¹⁴C]glycerol-3-phosphate and palmitoyl-CoA (16∶0-CoA) or arachidonoyl-CoA (20∶4-CoA) were visualized by a Storm radioactivity scanner. DAG, diacylglycerol, PA, phosphatidic acid, LPA, lysophosphatidic acid. B) AGPAT activity was measured with oleoyl-CoA or arachidonoyl-CoA and [¹⁴C]oleoyl-lysophosphatidic acid. GPAT2 overexpression significantly increased both GPAT and AGPAT activities only when arachidonoyl-CoA was used as a substrate (*p<0.05, **p<0.01).

Figure 4. GPAT2 overexpression stimulated AA incorporation in TAG.

CHO-K1 cells were transiently transfected with pcDNA3.1 empty vector (control) or pcDNA3.1-GPAT2 (GPAT2) constructs. A) Twenty-four h after transfection, cells growing in 6-well plates were incubated with 0.25 µCi of [¹⁴C]AA/well in standard medium plus 0.5% BSA for 3 h. Lipids were extracted and separated as described under “Materials and Methods”. Bars represent the mean ± SD of three independent experiments; PE, phosphatidylethanolamine, PI, phosphatidylinositol, PS, phosphatidylserine, PC, phosphatidylcholine, LPC, lysophosphatidylcholine, TAG, triacylglycerol. B) Twenty-four h after transfection, total lipids were extracted from control and GPAT2-transfected cells incubated with medium plus 10% FBS, and separated by TLC to isolate the TAG fraction and analyze the fatty acid composition. Values represent the mean ± SD of three independent experiments (*p<0.05, **p<0.01).

Figure 5. Gpat2 was expressed in mouse primary spermatocytes.

A) Adult mouse testis sections were hybridized in situ with a Gpat2 specific antisense probe (left panels) and the corresponding sense probe (right panels). Magnification: 100× (first row) and 600× (second row). A strong signal was detected in primary spermatocytes (black arrow). B) GPAT2 protein was detected in adult rat testis by immunofluorescence in the presence (left panel) or absence (right panel) of a specific GPAT2 antibody (green signal). The GPAT2 signal was detected in spermatocytes as well as in spermatides. Nuclei were stained with propidium iodide (red signal). Magnification: 400×. The highest GPAT2 expression was detected in the spermatocytes. Bar: 50 µm.

Figure 6. Gpat2 mRNA and protein expression were maximal in testes from 30-d old rats.

Testes from 19, 30, 40 and 60-d old rats were dissected, and either fixed and mounted on glass slides or total mRNA and mitochondria were isolated. A) Gpat2 mRNA was quantified by real time PCR and validated by Northern blot (insert). Values represent the mean ± SD of three independent experiments. B) Protein expression was measured by western blot with anti-GPAT2 antibody in mitochondria-enriched fraction obtained from 19-d (19), 30-d (30), 40-d (40) and 60-d (60) old rats, as well as in total particulate preparations of GPAT2-overexpressing CHO-K1 cells as a positive control (C+). Anti-voltage-dependent anion channel (VDAC) antibody was used as a loading control. The GPAT2 band corresponded to 80 kDa and VDAC to 30 kDa. Band intensities were quantified using the ImageJ program, and the number below each band represents the intensity ratio of GPAT2/VDAC relative to 19-d old (arbitrarily assigned a value of 1). C) Mitochondrial enrichment was monitored by assaying NADPH-cytochrome c reductase activity and D) by Western blot, probing the membrane with an antibody against the outer membrane protein VDAC. The enrichment of GPAT2 in the outer mitochondrial membrane was detected with anti-GPAT2 primary antibody. H, homogenate, C, crude mitochondria, ME, mitochondria enriched fraction, Mi, microsomes.

Figure 7. Expression of GPAT2 protein was greatest in 30 d and 40 d rat testis.

GPAT2 protein was detected in slides of testes from 19, 30, 40 and 60-d old rats by immunohistochemistry using an anti-GPAT2 antibody (brown signal). Nuclei were counterstained with haematoxylin (blue stain). Magnification: 400×. Bar = 80 µm. The results are representative of three independent experiments.

Figure 8. TAG mass and AA content were maximal in testes from 30-d-old rats.

Total lipids were isolated from testes from 19, 30, 40 and 60-d old rats. A) Lipids were separated by TLC and charred with 5% sulfuric acid in methanol, and the TAG spot was quantified by image processing. B) Fatty acid composition of the TAG fraction was determined by gas liquid chromatography at different stages of sexual development.