Role of GPAT3 and GPAT4 in macrophage models

PELLON-MAISON, M.; COLEMAN, R.A.; QUIROGA IY; GONZALEZ BARO, M.R.

Vail, Colorado

Congreso; Kern Lipid Conference: Targets/Therapies for Diabetes, Atherosclerosis and NASH: A Dialogue between Academia and Industry on Innova􀆟on; 2016

Glycerol-3-phosphateacyltransferases (GPATs) catalyze the first step in the de novo glycerolipidsynthesis.  To study the role of GPATisoforms during the transition of macrophages to foam cells (a model of lipid accumulationduring atherosclerosis) and during macrophage activation (a model ofinflammation), we analyzed the expression of the four mammalian GPAT isoforms byqRT-PCR in the murine RAW264.7 macrophage line after differentiation wasstimulated by oxidized LDL (oxLDL) or activation was stimulated by Kdo2-LipidA (KLA).  These treatments down-regulatedthe mitochondrial isoform GPAT1 by 20 and 75%, respectively.  GPAT2 was not detected.  The endoplasmic reticulum isoform, GPAT3,increased 35-fold after 8h of exposure to oxLDL and 6-fold after 8h of treatmentwith KLA.  These data were consistentwith GPAT activity assays: N-ethylmaleimide(NEM) sensitive activity (GPAT2, 3 and 4) increased 10- and 2-fold after oxLDLand KLA treatment, respectively.  Bothtreatments increased lipid droplet size and number, as well as the macrophage contentof triacylglycerol (TAG) and phospholipid (PL).  To establish the role of GPAT3 and 4 inmacrophage differentiation models, we silenced GPAT3 in RAW 264.7 cells with shRNA. Compared to the scrambled controls afterKLA treatment, TAG and PL accumulation and [14C]acetateincorporation into total lipids decreased 39, 44 and 30%, respectively, inshGPAT3 cells.  To investigate thephysiological effect of impaired lipid synthesis, we analyzed the phagocyticcapacity of shGPAT3 cells; activated shGPAT3 cells had a 50% decrease in the phagocytosisof E. coli conjugated with a pH-sensitive fluorescent dye.  To validate these results, we obtainedbone-marrow derived macrophages (BMDM) from C57BL/6 mice.  Although GPAT mRNA expression was notup-regulated after KLA treatment, the NEM-sensitive GPAT activity increased 38%after 8h, suggesting post-translational activation of GPAT3/4 in BMDM.  Isolated BMDM from GPAT3-/- or GPAT4-/-mice incorporated 52 and 41% less [14C]acetate into total lipids,respectively, after KLA treatment, suggesting impaired lipid synthesis. Takentogether, these results suggest that GPAT3 and GPAT4 contribute to the increasein total glycerolipid content during both macrophage differentiation into foamcells and macrophage activation. Furthermore, lack of GPAT3 diminishes the phagocytic capacity ofactivated macrophages. Supported by ANPCyT PICT3214, CONICET PIP0310 (MRGB,Argentina) and NIH DK56598 (RAC).