5-Aminoimidazole-4-carboxyamide-ribonucleoside (AICAR)-stimulated hepatic expression of Cyp4a10, Cyp4a14, Cyp4a31, and other peroxisome proliferator-activated receptor α-responsive mouse genes is AICAR 5'-monophosphate-dependent and AMP-activated protein kinase-independent

Abstract

5-Aminoimidazole-4-carboxyamide-ribonucleoside (AICAR), a prodrug activator of AMP-activated protein kinase (AMPK), increased hepatic expression of cytochrome P450 4a10, 4a14, and 4a31 mRNAs 2-, 3-, and 4-fold, respectively, and liver microsomal lauric acid ω-hydroxylation increased 2.8-fold. Likewise, mRNA levels of the peroxisome proliferator-activated receptor α (PPARα)-responsive genes, Acox1, Acadm, Cpt1a, and Fabp1, were also increased by AICAR treatment. AICAR did not elicit these changes in PPARα null mice. In isolated murine hepatocytes, AICAR and adenosine produced similar effects, and these responses were blocked by the PPARα antagonist [(2S)-2-[[(1Z)-1-methyl-3-oxo-3-[4-(trifluoromethyl)phenyl]-1-propenyl]amino]-3-[4-[2-(5-methyl-2-phenyl-4-oxazolyl)ethoxy]phenyl]propyl]-carbamic acid ethyl ester (GW6471). Inhibition of AMPK using compound C (dorsomorphin or 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-a]pyrimidine) did not block the induction of the PPARα-responsive genes by AICAR or adenosine, and 6,7-dihydro-4-hydroxy-3-(2'-hydroxy[1,1'-biphenyl]-4-yl)-6-oxo-thieno[2,3-b]pyridine-5-carbonitrile (A-769662), a non-nucleoside, direct activator of AMPK, did not increase expression of PPARα-responsive genes. An inhibitor of adenosine kinase, 5-iodotubercidin, blocked these responses, suggesting that the phosphorylation of AICAR and adenosine to AICAR 5'-monophosphate (ZMP) or AMP, respectively, was required. Concentrations of ZMP and AMP were elevated and ATP levels diminished at 24 h. The PPARα-dependent responses were associated with increased concentrations of oleic acid, a potent PPARα agonist, and diminished levels of oleoyl-CoA. Oleoyl-CoA synthase activity was inhibited by ZMP and AMP with IC(50) values of 0.28 and 0.41 mM, respectively. These results suggest that PPARα is activated by increased concentrations of free fatty acids that may arise from impaired fatty acid metabolism caused by altered levels of ATP, AMP, and ZMP after AICAR or adenosine treatment.

Fig. 1.

Time course for induction of hepatic Cyp4 mRNA after treatment with AICAR. Eight-week-old male C57BL/6 mice were injected intraperitoneally with saline (control) or AICAR (0.7 mg/g) then sacrificed at the indicated times after injection, and mRNA was analyzed using real-time PCR. Data are expressed relative to control (n = 4 mice per time point). Student's t tests were performed to determine significance. *, p < 0.05; **, p < 0.01. Results were similar when the same studies were performed with female mice.

Fig. 2.

Repeated doses of AICAR increase the metabolism of lauric acid (a Cyp4a substrate). Liver microsomes were isolated from 8-week-old male C57BL/6 mice that were intraperitoneally injected with saline (control) or AICAR daily for 2 days. Mice were euthanized 24 h after the final injection. Liver microsomes and total mRNA were prepared as described under Materials and Methods. Left, relative mRNA expression was determined as described in the legend to Fig. 1. Right, rates of liver microsomal metabolism of [¹⁴C]lauric acid were determined as described under Materials and Methods (n = 4 mice). Student's t tests were performed to determine significance. **, p < 0.01.

Fig. 3.

AICAR-mediated induction of Cyp4a mRNA is PPARα-dependent. Eight-week-old wild-type 129/Sv mice [PPARα(+/+)] (left) or PPARα-null(−/−) (right) mice (129/Sv background) were injected intraperitoneally with AICAR or saline. Livers were harvested 24 h after injection, and mRNA expression was analyzed using qPCR (n = 4). Shp was used as an AMPK-responsive positive control. Student's t tests were performed to determine significance. *, p < 0.05; **, p < 0.01.

Fig. 4.

AICAR-treatment increases the expression of the PPARα-responsive genes Acox1, Acsmd, Cpt1a, and Fabp1 in a PPARα-dependent manner. Eight-week-old wild-type 129/Sv (left) or PPARα-null mice (129Sv background) (right) were injected intraperitoneally with AICAR or saline. Livers were harvested 24 h after injection, and mRNA expression was analyzed using qPCR (n = 4). Student's t tests were performed to determine significance. **, p < 0.01.

Fig. 5.

Increased Cyp4a10, Cyp4a14, Cyp4a31, Acox1, Acsmd, Cpt1a, and Fabp1 mRNA expression in response to AICAR is not affected by AMPK inhibition. Hepatocytes were pretreated with compound C or DMSO (vehicle control). After 30 min, AICAR was added to the culture medium, and the cells were harvested after 24 h by the addition of TRIzol. Expression was then analyzed using qPCR and normalized to L27 mRNA for triplicate samples. The data are representative of hepatocyte preparations from four individual mice. Student's t test was performed to determine significance. **, p < 0.01.

Fig. 6.

The AMPK activator A769662 does not increase Cyp4a10, Cyp4a14, Cyp4a31, Acox1, Acsmd, Cpt1a, and Fabp1 mRNA expression. Hepatocytes were treated with A-769662 or DMSO (vehicle control). Twenty-four hours after treatment, the cells were harvested by the addition of TRIzol. Expression was then analyzed using qPCR and normalized to L27 mRNA for triplicate samples. The data are representative of hepatocyte preparations from four individual mice. Student's t tests were performed to determine significance. **, p < 0.01.

Fig. 7.

Adenosine increases Cyp4a10, Cyp4a14, Cyp4a31, Acox1, Acsmd, Cpt1a, and Fabp1 mRNA expression in a AMP-dependent and AMPK-independent manner. Hepatocytes were isolated from 8-week-old male C57BL/6 mice by EGTA/collagenase perfusion. Hepatocytes were pretreated with 5-iodotubercidin (adenosine kinase inhibitor) or DMSO (vehicle control). After 30 min, adenosine (10 μM) was added to the culture medium, and the cells were harvested after 24 h by the addition of TRIzol. Expression was then analyzed using qPCR and normalized to L27 mRNA for triplicate samples. The data are representative of hepatocyte preparations from four individual mice. Student's t tests were performed to determine significance. **, p < 0.01.

Fig. 8.

Conversion of AICAR to ZMP is necessary for the activation of Cyp4a, Acox1, Acsmd, Cpt1a, and Fabp1 mRNA expression. Hepatocytes were isolated from 8-week-old male C57BL/6 mice by EGTA/collagenase perfusion. Hepatocytes were pretreated with 5-iodotubercidin (adenosine kinase inhibitor) or DMSO (vehicle control). After 30 min, AICAR was added to the culture medium, and the cells were harvested after 24 h by the addition of TRIzol. Expression was then analyzed using qPCR and normalized to L27 mRNA for triplicate samples. The data are representative of hepatocyte preparations from four individual mice. Student's t tests were performed to determine significance. **, p < 0.01.

Fig. 9.

Ligand activation of PPARα is required for AICAR- and adenosine-stimulated increased mRNA expression of PPARα-responsive genes. A to D, hepatocytes were treated with AICAR (A and C) or adenosine (B and D) in the presence and absence of compound C or 5-iodotubercin, and free fatty acids and fatty acyl-CoAs were isolated from hepatocytes followed by quantitation using ultra performance liquid chromatography- tandem mass spectrometry as described under Materials and Methods. Free fatty acid and fatty acyl-CoA concentrations were normalized to total protein concentration of cells that were lysed for analysis. E and F, for the experiments using the PPARα antagonist GW6471 hepatocytes were preincubated with this compound or DMSO (vehicle control) followed by treatment with AICAR (E) or adenosine (F) 30 min later. The hepatocytes were harvested after 24 h by the addition of TRIzol, and the mRNA levels were measured using qPCR and normalized to L27 mRNA for triplicate samples. Hepatocytes were isolated from four individual mice, and Student's t tests were performed to determine significance. **, p < 0.01.

Fig. 10.

ZMP inhibits oleoyl-CoA formation by hepatocyte lysates. Lysates (200 μg of total protein) from primary mouse hepatocytes were incubated in the presence of [³H]oleic acid, ATP, CoA, and ZMP (A), AMP (B), or triascin C (C) for 20 min at 37°C. The resulting [³H]oleoyl-CoA was isolated, and radioactivity was measured by using a scintillation counter. The results represent experiments that were performed using hepatocytes from four separate isolations, and the incubations were carried out in duplicate.