CAR was originally characterized as a “xenobiotic sensor” that, together with its closest relative PXR, provides a functionally redundant “metabolic safety net” that is activated in response to xenobiotic stress. Recent biochemical and structural data suggest that, as opposed to merely sensing xenobiotic stress, CAR is activated in response to metabolic or nutritional stress in a ligand-independent manner. Our data reveal a novel molecular mechanism of ligand-independent CAR activation that involves a tightly controlled network of interactions between cAMP, PGC-1α, HNF4α, and CAR. This mode of activation positions CAR as the key regulator of both drug and hormone disposition in liver during β-adrenergic/glucagon-mediated stimulation of liver such as occurs during fasting and caloric restriction. The data presented in this study further distinguish CAR from PXR and suggest that CAR functions as a sensor of energy stores and is activated by increased intracellular cAMP and increases expression of genes involved in transport and metabolism of thyroid and steroid hormones.
A model illustrating this mechanism is shown in . In response to fasting or caloric restriction, the level of cAMP rises due to increased action of epinephrine and glucagon in liver. Increased intracellular concentrations of cAMP then activate the cAMP-dependent protein kinase (PKA), which then phosphorylates and activates the cAMP-response element binding protein (CREB). Activated CREB then turns on the expression of PGC-1α, which induces the expression of CAR through coactivation of HNF4α. Subsequently, the interaction between CAR and PGC-1α increases the expression of selected CAR target genes involved in transport and metabolism in liver.
Model for the induction of CAR and CAR target genes by cAMP in mouse liver during caloric restriction or fasting
It is worth noting here that one hallmark of PGC-1α
-mediated activation of nuclear receptors is that it does so in a ligand-independent manner. It is also worth noting that, with the exception of TCPOBOP (1,4-bis[2-(3,5-dichloropyridyloxy)]) benzene and CITCO (6-(4-chlorophenyl)imidazo[2,1-b](1
-(3,4-dichlorobenzyl)oxime), the vast majority of CAR activators identified to date activate CAR in an indirect- and phosphorylation-dependent manner. Although the extent to which the mode of CAR activation described here has an impact upon the metabolic disposition of thyroid hormones and other steroidal endobiotic compounds involved in regulating weight, it is clear that ablation of CAR function in vivo
in mice produces a profound decrease in the resistance to weight loss observed during extended periods of caloric restriction (9
). Future experiments utilizing in vivo
gain-of-function and knockdown approaches that target this newly identified novel signaling pathway in rodent models will directly address this question.
The data we present here cannot rule out the possibility that other PGC-1α
-independent mechanisms are also involved in the activation of CAR during fasting. For example, a recent study shows that the coactivator PPAR-binding protein is critical for the induction of CAR target genes by xenobiotics (51
), and our data do not rule out a role for PPAR-binding protein in the activation of CAR during fasting. It is possible that the xenobiotic response of CAR is differentiated from its metabolic response and is produced through interaction with distinct cofactors, such as PGC-1α
, during fasting. The respective CAR-cofactor complexes might then function to regulate the expression of different groups of genes in response to metabolic versus
xenobiotic stress. In addition, previous work indicates that protein kinase signaling pathways regulate the activity of PXR, a close relative of CAR, by modulating the interactions between PXR and cofactor proteins (52
). Moreover, a study shows that phosphorylation/dephosphorylation of serine 202 regulates the subcellular location of CAR (53
). Therefore, fasting may modulate CAR-cofactor interactions through alterations in the phosphorylation status of cofactor proteins, CAR, or both. Fasting may also induce the expression of HNF4α
in liver (38
), and this might further contribute to induction of CAR expression and activity in response to metabolic stress. It is noteworthy that our kinetic analysis shows that treatment with 8-Br-cAMP produced relatively early increases in the expression of the Oatp2 and Sult2a1 genes when compared with CAR, Cyp2b10, and Ugt1a1. This indicates that cAMP signaling may play a role in regulating the expression of these genes that is independent of CAR, and this represents an interesting topic for future investigations. Finally, further dissection of the mouse 4.6-kb CAR promoter fragment identified here should yield additional information regarding the molecular mechanisms that regulate the expression of the car
gene in mice.
It should be pointed out that our current studies have been focused on mRNA levels. Although work from other groups has revealed that the mRNA levels of UGTs and SULTs are correlated well with TH metabolism (9
), it still remains to be determined in the future whether the changes in gene expression observed in our studies are associated with corresponding changes in protein levels and enzymatic activities for UGTs and SULTs.
Although activation of TH receptor by T3 increases metabolic rate, the lack of tissue-selectivity of THs and the associated cardiac side-effects has prevented the use of these hormones as anti-obesity agents (55
). A number of recent studies indicate that selective activation of TH receptor in liver can raise the metabolic rate with only minimal cardiac side-effects (57
). Therefore, drugs that selectively decrease hepatic TH metabolism could in principle have important anti-obesity properties that have diminished cardiac-related side-effects. Our findings suggest that the potential exists to target a number of hepatic factors to decrease the metabolism of THs in liver, thereby generating a gradient of TH concentrations with the local TH concentrations in liver being relatively higher. First, selective antagonism of hepatic β
-adrenergic receptors would be expected to prevent the rise of cAMP in liver during fasting or caloric restriction. Second, liver-specific PKA or CREB inhibitors should prevent the induction of PGC-1α
and CAR in liver during fasting or caloric restriction. Third, an HNF4α
inverse agonist might prevent the induction of CAR gene expression during fasting or caloric restriction. Finally, small molecules that disrupt the interaction between PGC-1α
and CAR could in principle repress the induction of CAR target genes involved in the uptake, metabolism, and excretion of THs or other important endobiotics during fasting and caloric restriction. It should be pointed out that targeting any of these factors for therapeutic purposes requires achieving tissue specificity to reduce any potential side-effects, and this remains challenging work for the future.
Since its identification in 1998, PGC-1α
has gained attention as a promising drug target for diabetes and obesity due to its key role in both glucose and energy homeostasis (61
). However, our findings suggest that selective targeting PGC-1α
function in liver may have profound effects on the activity of CAR, including alterations in the drug and endobiotic-metabolizing pathways regulated by this nuclear receptor. Although drugs that selectively target the activity of hepatic PGC-1α
may favorably modulate glucose and energy homeostasis, they may also disturb xenobiotic and endobiotic homeostasis and hence cause unwanted and potentially lethal side-effects. A recent report indicates that xenobiotic-inducible Cyp2b10 expression is intact in mice lacking PGC-1α
in liver (65
). It will therefore be important to determine the extent to which the CAR-mediated metabolic response we describe is altered in this animal model. We also note that the basal expression level of Cyp2b10 in the liver of CAR-KO mice is drastically reduced when compared with their wild-type littermates, indicating that CAR plays a key role in maintaining the target gene basal expression levels (9
). However, it should be pointed out that the increase in CAR expression and activity during fasting and 8-Br-cAMP treatment represent acute effects, whereas the gene-knock-out studies reflect a chronic effect.
Our experiments with liver-specific HNF4α-KO mice clearly show fasting-inducible CAR activity is abolished and therefore support a central role for HNF4α in the fasting response. Future in vivo genetic animal studies will determine the extent to which acute stimulation of this pathway in liver, such as occurs during fasting, produces alteration in the circulating levels of serum THs and other steroids, including cortisol. Our future studies will also be designed to investigate the extent to which chronic overexpression of PGC-1α in liver such as occurs during caloric restriction produces increased sensitivity to diet-induced obesity or increased resistance to weight loss.
THs and steroid hormones are transported into hepatocytes, which can be mediated by the sinusoidal transporter Oatp2 (66
). Subsequent biotransformation reactions are catalyzed by UGTs and SULTs. Conjugated and unconjugated molecules are then excreted into bile via transporters localized on the canalicular membranes such as multidrug resistance-associated proteins (44
). It will therefore be of interest to determine the extent to which canalicular transport of THs and other hormones is regulated by fasting and cAMP. Finally, although Cyp2b10 is often used as a marker for CAR activity, the physiological significance of the induction of this cytochrome P-450 enzyme during fasting remains totally unknown.