GPAT3 AND GPAT4 DIRECT DE NOVO TRIACYLGLYCEROL SYNTHESIS DURING MACROPHAGE TO FOAM CELL TRANSITION AND THEIR DOWN-REGULATION ALTERS CYTOKINE RELEASE.

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

Steamboat Springs, Colorado

Congreso; FASEB Science Research Conference: Lipid Droplets on the Move from Health to Disease; 2018

FASEB

Atherosclerosis is achronic disease characterized by the deposition of excessive lipids in thearterial intima.  Macrophage to foam celltransition, characterized by the increase in lipid droplet formation, is amajor hallmark of early stage atherosclerotic lesions.  Cholesterol ester (CE) accumulates in these cellsand its role during foam-cell formation has been extensively studied, butlittle is known about the role of glycerolipids in this process.  Glycerol-3-phosphate acyltransferase (GPAT) catalyzesthe first step in de novoglycerolipid synthesis and is believed to be the rate-limiting enzyme of thispathway.  To study the role of GPATisoforms during the transition of macrophages to foam cells, we analyzed theexpression of the four mammalian GPAT isoforms by qRT-PCR in murine RAW264.7macrophages and in primary bone marrow derived macrophages (BMDM) after exposureto oxidized LDL (oxLDL).  In RAW 264.7cells, mRNA expression of the endoplasmic reticulum isoform GPAT3 increased 32-foldafter 8h of treatment while in BMDM we observed an 8-fold increase after 24h oftreatment.  These data were coincidedwith a ~2-fold increment in GPAT3/4 activity in both cellular models.  The lipid droplet area, as well astriacylglycerol (TAG) and phospholipid (PL) content also increased after oxLDLtreatment.  To establish the role ofGPAT3 and 4 during macrophage-to-foam celltransition, we silenced GPAT3 in RAW 264.7 cells (shGpat3 cell line) andobtained BMDM from Gpat3-/- andGpat4-/- mice.  GPAT3 and GPAT4 deficient foam cellsaccumulate ~30-40% fewer lipid droplets, ~25-40% less TAG and ~20% less PL comparedto wt or SCR (scrambled shRNA) controlfoam cells.  We also analyzed theincorporation of [14C]-acetate and [14C]-oleic acid intolipids in shGpat3 cells, and in Gpat3-/- and Gpat4-/- BMDM duringthe transition to foam cells.  Theincorporation of these substrates into total lipids, particularly TAG, waslower in GPAT3 and 4 deficient cells.  Toinvestigate the physiological effect of impaired lipid synthesis, we analyzed cytokinerelease in Gpat3-/- and Gpat4-/- BMDM.  No clear effects were observed in GPAT3-deficientcells, but when GPAT4 was absent, cytokine secretion after foam cell transitionincreased.  Taken together, these resultsshow that GPAT3 and 4 contribute to the increase in total glycerolipid content bycausing an increase in TAG de novo synthesis during macrophage to foam cell transition.  In addition, GPAT4 is required to regulatecytokine release during this process. Funding: ANPCyT-PICT3214, CONICET-PIP0310, UNLP-M202 (MRGB) and NIH-DK56598(RAC)