GPAT3 and GPAT4 direct glycerolipid de novo synthesis in activated macrophages and their down-regulation alters macrophage functionality

QUIROGA IY; PELLON MAISON, MAGALI; COLEMAN, RA; GONZALEZ-BARO, MR

Waterville Valley

Congreso; Gordon Research Conference (GRC) on Molecular and Cellular Biology of Lipids; 2017

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first step in de novo glycerolipid synthesis. To study the role of GPAT isoforms during macrophage activation (a model of inflammation in which lipids accumulate), we analyzed the expression of the four mammalian GPAT isoforms by qRT-PCR in the murine RAW264.7 macrophage cell line and in bone-marrow derived macrophages (BMDM) from C57BL/6 mice after activation with Kdo2-Lipid A (KLA). In RAW264.7 cells, KLA treatment up-regulated the ER isoforms GPAT3 and GPAT4 by 6-fold. Although these GPAT isoforms were down-regulated in BMDM, in both RAW 264.7 and BMDM cells, GPAT3/4 enzymatic activity increased 20% and 3-fold, respectively, indicating a post-transcriptional/post-translational activation. KLA activation increased lipid droplet size and number, as well as the macrophage content of triacylglycerol (TAG) and phospholipid (PL) in both models. To understand the role of GPAT3 and GPAT4 in macrophage activation, we silenced GPAT3 in RAW 264.7 cells with shRNA (shGpat3 RAW) and obtained BMDM from Gpat3-/- and Gpat4-/- mice. Compared to scrambled and wt controls, activated shGpat3 RAW, GPAT3-/- and GPAT4-/- BMDM had lower TAG and PL content. To evaluate the effect of GPAT3 or GPAT4 down-regulation on de novo glycerolipid synthesis, we analyzed the incorporation of [14C]acetate and [14C]oleic acid into lipids. Compared to controls, after KLA activation, the incorporation of both radioactive substrates into total lipids, TAG, and phosphatidylcholine was lower in shGpat3 RAW, GPAT3-/- and GPAT4-/- BMDM. Lipid droplet size was also reduced when GPAT3 or GPAT4 were absent. To investigate the physiological effect of impaired lipid synthesis in activated shGpat3 RAW, GPAT3-/- and GPAT4-/- BMDM, we used E. coli conjugated with a pH-sensitive fluorescent dye to analyze phagocytic capacity. Compared to controls, phagocytosis capacity was 50, 19, and 25% lower, respectively. Furthermore, the lack of GPAT4 increased the release of pro-inflammatory cytokines and chemokines by activated macrophages. Taken together, these results suggest that GPAT3 and GPAT4 contribute to the increase in total glycerolipid content by inducing de novo synthesis during macrophage activation and that this process is important for macrophage phagocytosis and cytokine release.