GPAT expression during macrophage differentiation

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

Waterville Valley, NH

Congreso; Gordon Research Conference "Molecular and Cellular Biology of Lipids"; 2015

GRC

Type-2 diabetes and obesity are characterized by an excessiveaccumulation of triacylglycerols (TAG) partially caused by a deregulation ofglycerol-3-phosphate acyltransferases (GPATs) which catalyze the first step in denovo glycerolipid synthesis, and carnitine palmitoyl transferase 1 (CPT1) whichregulates fatty acid oxidization.  Inorder to study the roles of these enzymes in monocyte-macrophage-foam-celltransition (a model of lipid accumulation during atherogenesis) and inmacrophage activation (a model of inflammation) we tested their expression byqRT-PCR in the murine macrophage RAW264.7 either differentiated into foam cellsby oxidized LDL (oxLDL) or activated by Kdo-Lipid A, and in the human THP-1monocyte cell line differentiated into macrophage by phorbol esters (PMA).  The treatment of macrophages with oxLDL andKdo increased lipid droplet size and number, as well as cellular TAG content.  We then analyzed the expression and activityof the four mammalian GPAT isoforms.  Ofthe mitochondrial isoforms, GPAT1 expression decreased in RAW cells after oxLDLand Kdo treatment and did not change in THP-1 cells, and GPAT2 expression wasnegligible in the three models.  We thenanalyzed the endoplasmic reticulum isoforms GPAT3 and 4.  Interestingly, GPAT3 expression increased 35-foldafter 8 h of oxLDL and 6-fold after of Kdo exposure of macrophages, and 6-fold after12 h of PMA activation of monocytes.  GPAT4expression significantly increased after 8 h in macrophage activation and after12 h in the monocyte to macrophage transition.  These results were consistent with GPATactivity assays, since N-ethylmaleimide (NEM) sensitive activity (GPAT2,3 and 4) but not NEM resistant activity (GPAT1) increased after oxLDL treatment,correlating with GPAT3 expression.  Wealso found that CPT1a was up-regulated during RAW264.7 cell differentiation tofoam cells and that a significant decrease was observed in the human macrophagesderived from THP1 monocytes and during macrophage activation, suggesting thatβ-oxidation may not be highly active in macrophages, consistent with theanaerobic metabolism hallmark of M1 pro-inflammatory macrophages.We have developed in our lab a RAW264.7 stable cell line knocked-downfor Gpat3 in 75% compared to the control. We expect to have data regarding the role of GPAT3 in macrophage functionby July and present them in the poster.