Activated AMPK inhibits PPAR-{alpha} and PPAR-{gamma} transcriptional activity in hepatoma cells

Abstract

AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor-α (PPAR-α) are critical regulators of short-term and long-term fatty acid oxidation, respectively. We examined whether the activities of these molecules were coordinately regulated. H4IIEC3 cells were transfected with PPAR-α and PPAR-γ expression plasmids and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid. The cells were treated with PPAR agonists (WY-14,643 and rosiglitazone), AMPK activators 5-aminoimidazole-4-carboxamide riboside (AICAR) and metformin, and the AMPK inhibitor compound C. Both AICAR and metformin decreased basal and WY-14,643-stimulated PPAR-α activity; compound C increased agonist-stimulated reporter activity and partially reversed the effect of the AMPK activators. Similar effects on PPAR-γ were seen, with both AICAR and metformin inhibiting PPRE reporter activity. Compound C increased basal PPAR-γ activity and rosiglitazone-stimulated activity. In contrast, retinoic acid receptor-α (RAR-α), another nuclear receptor that dimerizes with retinoid X receptor (RXR), was largely unaffected by the AMPK activators. Compound C modestly increased AM580 (an RAR agonist)-stimulated activity. The AMPK activators did not affect PPAR-α binding to DNA, and there was no consistent correlation between effects of the AMPK activators and inhibitor on PPAR and the nuclear localization of AMPK-α subunits. Expression of either a constitutively active or dominant negative AMPK-α inhibited basal and WY-14,643-stimulated PPAR-α activity and basal and rosiglitazone-stimulated PPAR-γ activity. We concluded that the AMPK activators AICAR and metformin inhibited transcriptional activities of PPAR-α and PPAR-γ, whereas inhibition of AMPK with compound C activated both PPARs. The effects of AMPK do not appear to be mediated through effects on RXR or on PPAR/RXR binding to DNA. These effects are independent of kinase activity and instead appear to rely on the activated conformation of AMPK. AMPK inhibition of PPAR-α and -γ may allow for short-term processes to increase energy generation before the cells devote resources to increasing their capacity for fatty acid oxidation.

Fig. 1.

A: treatment of H4IIEC3 cells with AMP-activated protein kinase (AMPK) activators results in increased phosphorylation of AMPK. H4IIEC3 cells were treated with metformin or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) for 24 h before harvesting and Western blotting for phospho-AMPK. Bars between columns signify noncontiguous lanes on the same gel. B: H4IIEC3 cells were transfected with a proliferator-activated receptor (PPAR)-α expression plasmid and a peroxisome-proliferator-response element (PPRE) luciferase reporter plasmid in addition to a tk-Renilla luciferase plasmid to control for transfection efficiency. Cells were treated for 24 h as described in the text before harvesting and measuring luciferase activity. Concentrations of the treatments were as follows: 1 μM WY-14,643 (WY), 100 μM oleic acid, 40 μM compound C (CC), 500 μM AICAR, 1 mM metformin (Met). N = 4. *P < 0.05 compared with control; #P < 0.05 compared with WY; $P < 0.05 compared with WY + AICAR; &P < 0.05 compared with metformin + WY; δP < 0.05 compared with WY + CC.

Fig. 2.

H4IIEC3 cells were transfected with PPAR-γ expression plasmids, along with PPRE reporter plasmid and tk-Renilla luciferase plasmid. Twenty-four hours before harvest, cells were exposed to the treatments listed above. Concentrations of the compounds were as described in Fig. 1; 30 μM rosiglitazone (Rosi) was used. N = 3. *P < 0.05 compared with control; #P < 0.05 compared with rosiglitazone alone, $P = 0.05 compared with AICAR + rosiglitazone; &P = 0.05 compared with metformin + rosiglitazone, δP < 0.05 compared with CC + rosiglitazone.

Fig. 3.

H4IIEC3 cells were transfected with a retinoic acid receptor (RAR) luciferase reporter plasmid and tk-Renilla-luciferase plasmid to control for transfection efficiency. Treatments were for 24 h, and concentrations of the compounds were the same as described in Fig. 1; 50 nM AM580 was used. N = 4. *P < 0.05 compared with control; #P < 0.05 compared with AM580.

Fig. 4.

A: H4IIEC3 cells were subjected to the treatments as described for 24 h before harvesting and cellular fractionation. Nuclear and cytoplasmic fractions (10 μg) were subjected to Western blotting for AMPK-α and -α2. B: cytoplasmic levels of total AMPK-α and -α2. Protein levels were normalized to GAPDH. *P < 0.05 compared with control. C: nuclear levels of total AMPK-α and α-2. Protein levels were normalized to the levels of lamin. *P < 0.05 compared with control. N = 3.

Fig. 5.

H4IIEC3 cells were transfected with PPAR-α and treated with the AMPK activators for 24 h as labeled. Lysates were then incubated with biotinylated double-stranded PPRE oligonucleotides overnight, before using streptavidin beads to pull down the biotinylated oligonucleotide and any protein bound to it. After being boiled with SDS-SB, samples were subjected to Western blotting for PPAR-α. The presence of nonbiotinylated PPRE oligonucleotide prevented the pull down of the lower, immunoreactive PPAR-α band, demonstrating that the upper band was attributable to a nonspecific interaction. NS, nonspecific band. Bars between columns signify noncontiguous lanes on the same gel. There was no consistent effect of AICAR, WY, or metformin on binding of PPAR-α to its consensus binding site in cellular extracts from 4 independent experiments.

Fig. 6.

H4IIEC3 cells were transfected with the reporter plasmids and PPAR-α (A) or -γ (B), and the AMPK-α1³¹² expression plasmid as noted by CA (constitutively active). The cells were treated with WY, rosiglitazone, and AM580 at the same concentrations noted in Figs. 1–3 for 24 h. A: PPAR-α, *P < 0.05 compared with control; $P < 0.05 compared with WY-stimulated cells. B: PPAR-γ, *P < 0.05 compared with control. C: RAR. *P < 0.05 compared with control.

Fig. 7.

H4IIEC3 cells were transfected with the reporter plasmid and PPAR-α or -γ as labeled. Dominant negative (α1-DN) AMPK was transfected at the same time as the reporter and expression plasmids. Concentrations of WY, rosiglitazone, and AM580 were as described in Figs. 1–3, and treatments were for 24 h. A: PPAR-α, *P < 0.05 compared with control; $P < 0.05 compared with WY-stimulated cells. B: PPAR-γ, *P < 0.05 compared with control; $P < 0.05 compared with rosiglitazone-stimulated cells. C: RAR, *P < 0.05 compared with control.

Fig. 8.

H4IIEC3 cells were transfected with the expression plasmids for PPAR-α or -γ (A and B) and with the constitutively active (CA AMPK) or dominant negative (DN AMPK) expression plasmids. Where noted, the cells were treated with WY, rosiglitazone, or AM580 as described in Figs. 1–3. The cells were harvested after 24 h and the cellular extracts blotted for PPAR-α, -γ, or RAR-α. GAPDH was used as the loading control. There was no effect of the transfection of the AMPK variants or treatment with receptor ligands on the expression of the nuclear receptors. C, control.