Glycerol-3-phosphate acyltransferase-1 regulates murine T-lymphocyte proliferation and cytokine production

Abstract

We have previously established a correlation between reduced mitochondrial glycerol-3-phosphate acyltransferase-1 (GPAT-1) activity and decreased proliferation in splenic T-lymphocytes from aged rats. To better understand the immunoregulatory role of GPAT-1, we examined T-lymphocyte function in young GPAT-1 knockout (KO) mice. We show that without GPAT-1, T-lymphocyte proliferation is inhibited and activation induced apoptosis is increased. Th-1 (IL-2 and IFN-gamma) cytokine secretion is reduced, and Th-2 (IL-4 and IL-10) cytokine secretion is increased. These changes may be due to alterations in membrane lipid composition since we found changes in the relative content of individual phospholipid species. Furthermore, we show increased arachidonate content and subsequent increased prostaglandin E(2) secretion, which may inhibit T-lymphocyte proliferation. Taken together, we show a novel link between GPAT-1 and changes in T-lymphocyte function. These data have broad health implications because GPAT-1 suppression has recently been implicated as a new target for preventing insulin sensitivity and hepatic steatosis and we show that immune function may also be affected. Interestingly, the changes in young GPAT-1 KO splenic T-lymphocytes are similar to defects commonly seen in T-lymphocytes from aged rodents, which further underscores the significance of GPAT-1 in T-lymphocyte function.

Fig. 1.

Splenic T-lymphocyte subsets are altered in glycerol-3-phosphate acyltransferase-1 (GPAT-1) knockout (KO) mice. A: spleens were removed from GPAT-1^+/+ (wild type; open bar), GPAT-1^+/− (heterozygous; shaded bar), and GPAT-1^−/− (homozygous; solid bar) KO mice and weighed. Splenic lymphocytes were then isolated as described in materials and methods. B: an aliquot of splenic lymphocytes was stained with the T-lymphocyte markers (anti-CD3) and anti-CD4 or anti-CD8 to distinguish the two major T-lymphocyte subsets, and 10,000 cells per sample were examined by flow cytometry. Each bar represents the mean ± SE of 4 individual mice. *Significantly different (P < 0.05) from GPAT-1^+/+ mice.

Fig. 2.

Effect of GPAT-1 KO on T-lymphocyte proliferation. A and B: splenic T-lymphocytes were isolated from GPAT-1^+/+, GPAT-1^+/−, and GPAT-1^−/− KO mice. T-lymphocytes, 1.5 × 10⁶ from each spleen, were left unstimulated (US) or were stimulated for 8, 16, 20, or 24 h with anti-CD3 and anti-CD28 (A) or phorbol myristate acetate (PMA) and ionomycin (B), and proliferation was measured using the MTT assay as described in materials and methods (8). Solid line, GPAT-1^+/+; dotted line, GPAT-1^+/−; dashed lined, GPAT-1^−/−. C and D: T-lymphocytes were stimulated for 20 h with anti-CD3 and anti-CD28 (C) or PMA and ionomycin (D), stained with annexin-V and propidium iodide, and analyzed by flow cytometry (13). Live cells are annexin-V and propidium iodide negative, early apoptotic cells stain annexin-V positive and propidium iodide negative, and late apoptotic/necrotic cells are both annexin-V and propidium iodide positive. Open bar, GPAT-1^+/+; shaded bar, GPAT-1^+/−; solid bar, GPAT-1^−/−. Values represent means ± SE of 4 individual mice. *Significantly different (P < 0.05) from GPAT-1^+/+ and GPAT-1^−/− mice. **Significantly different (P < 0.05) from GPAT-1^+/+ and GPAT-1^+/−.

Fig. 3.

Th-1 and Th-2 cytokine secretion is altered in GPAT-1 KO mice. Splenic T-lymphocytes, 2 × 10⁶ from each spleen, from GPAT-1^+/+ (open bar), GPAT-1^+/− (shaded bar), and GPAT-1^−/− (solid bar) mice were stimulated with anti-CD3 and anti-CD28 or PMA plus ionomycin for 20 h. Culture supernatants were collected and interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-4 (IL-4), and interleukin-10 (IL-10) were quantitated by ELISA kits as described in materials and methods. Each bar represents the mean ± SE of 4 individual mice. *Significantly different (P < 0.05) from GPAT-1^+/+ mice.

Fig. 4.

T-lymphocyte fatty acid composition is altered in GPAT-1 KO mice. Splenic T-lymphocytes, 5 × 10⁶ from each spleen, were isolated from GPAT-1^+/+ (open bar) and GPAT-1^−/− (solid bar) mice. Total lipids were extracted from unfractionated, whole T-lymphocytes without prior incubation or stimulation with a hexane/propanol solution and were converted to fatty acid methyl esters for fatty acid analysis by GC as described in materials and methods. Values represent means ± SE of 4 individual mice. *Significantly different (P < 0.05) from GPAT-1^+/+ mice.

Fig. 5.

GPAT-1 KO increases T-lymphocyte eicosanoid production. Splenic T-lymphocytes were isolated from GPAT-1^+/+ (open bar), GPAT-1^+/− (shaded bar), and GPAT-1^−/− (solid bar) mice. T-lymphocytes, 2 × 10⁶ from each spleen, were stimulated for 20 h with anti-CD3 or PMA plus ionomycin (Ion), and prostaglandin E₂ (PGE₂) (A) and leukotriene B₄ (LTB₄) (B) were measured in cell culture supernatants as described in materials and methods. Values represent means ± SE of 4 individual mice. *Significantly different (P < 0.05) from GPAT-1^+/+ mice.