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. 2004 Nov 23;384(Pt 2):295–305. doi: 10.1042/BJ20040955

Figure 6. Inhibition of PPARα by AICAR.

Figure 6

Cells were transfected with the reporter plasmid rAOX(PPRE)-CAT and co-transfected with the indicated expression plasmids for mPPARα and AMPKα mutants as described in the Experimental section. Fold induction represents CAT activity (means+S.E.M. for triplicate plates or means of duplicate plates differing by no more than 10%) normalized by CMV-β-galactosidase and further normalized by CAT activity of pSG5-transfected cells. (A) 293 cells were transfected with the reporter plasmid rAOX(PPRE)-CAT and the expression plasmids for mPPARα (0.1 μg) or mPPARα(S452A) (0.1 μg), and co-transfected with expression plasmids for rAMPKα1 (1.0 μg), rAMPKα1(T172A) (1.0 μg) and/or hAMPKβ1 (1.0 μg) plus rAMPKγ1 (1.0 μg) as indicated. CAT activity was determined in cells incubated overnight in the absence of added ligand (grey bars), as well as in cells incubated for an additional 7 h with 30 μM nafenopin in the absence (□) or presence (■) of added 250 μM AICAR. Note the nafenopin-dependent activity during the 7 h incubation period in the absence and presence of AICAR. (B) 293 cells were transfected with the reporter plasmid rAOX(PPRE)-CAT and the expression plasmid for mPPARα (0.02 μg), and co-transfected with expression plasmid for hAMPKα2(D157A) (2.0 μg). CAT activity was determined in cells incubated overnight in the absence of added ligand (grey bar), as well as in cells incubated for an additional 6 h with 10 μM nafenopin in the absence (□) or presence (■) of added 500 μM AICAR. Note the nafenopin-dependent activity during the 6 h incubation period in the absence and presence of AICAR. Significance: *P<0.05 compared with no added AICAR. (C) COS-7 cells were transfected with the reporter plasmid rAOX(PPRE)-CAT and the expression plasmid for mPPARα (0.05 μg). CAT activity was determined in cells incubated overnight in the absence of added ligands and incubated for an additional 7 h in the presence of DMSO vehicle, 50 nM iloprost, 10 μM Wy-14,643 or 10 μM nafenopin as indicated, in the absence (white bars) or presence of 200 (hatched bars), 250 (black bars) or 600 (cross-hatched bars) μM AICAR.