Figure 3.

EL preferentially hydrolyzes HDL to activate PPARα in a manner dependent on EL catalysis. Standard PPARα-LBD-GAL4 assays as described in Methods were performed in BAECs either with or without cotransfection of human EL. Results are expressed as fold activation over control after normalization. A, EL preferentially acts on phosphotidylcholine (PC) and HDL to activate the PPARα– LBD. EC transfected with either empty vector or the EL construct were exposed to PC, LDL, VLDL, and HDL at the concentrations shown and PPARα–LBD activity determined. In EL-transfected cells, the most potent activation was seen with HDL (*P<0.05); more modest activation was also evident with LDL and VLDL (all lipoproteins at 60 µg protein/mL). For comparison, the PPARα-LBD response to WY14643 (10 µmol/L) is shown. B, In untransfected BAEC, HDL increases PPARα-LBD activation in a concentration-dependent manner but not in the presence of tetrahydrolipstatin (10 µmol/L). C, Tetrahydrolipstatin preincubation (30 minutes, room temperature) decreases PPARα-LBD activation in a concentration-dependent manner as evident in control transfected (not shown) and EL-transfected BAEC exposed to HDL (60 µg/mL). D, PPARα activation by EL/HDL requires catalytically-active EL. PPARα–LBD-GAL4 assays were performed in BAEC after transfection of either wild-type or a catalytically-inactive EL point mutant in the presence of increasing concentrations of HDL. In cells expressing the catalytically-inactive EL mutant, no significant increase in PPARα–LBD activation is seen above basal levels across the HDL concentration tested. For comparison, concomitant PPARα-LBD activation in cells transfected with wild-type EL is shown; tetrahydrolipstatin (10 µmol/L) abrogated the EL/HDL/PPARα response.