An increase in liver microsomal drug-metabolizing activity in alloxan-treated diabetic rats was first reported in the early 1960s when drugs such as barbiturates were also found to induce the same activity (3
). It was later confirmed that the increased drug metabolism activity was due to the induction of enzymes, such as CYP2B and CYP3A. For example, P450b (presently called CYP2B1) was increased 25- to 30-fold in the liver microsomes of alloxan-treated rats, and insulin treatment restored P450b to normal levels (56
). In the present study, we have demonstrated that the regulatory mechanisms of the insulin response and of the drug-induced transcription cross paths through direct interaction between FOXO1 and the nuclear receptors CAR and PXR. Accordingly, drug metabolism and gluconeogenesis could be reciprocally coregulated by the transcription factors in response to insulin and/or drugs (Fig. ).
Schematic representation of cross-talk. Arrows indicate activation and coactivation, while stop bars indicate repression and corepression.
Our study first showed that FOXO1 binds directly to CAR. While both FOXO1- and insulin-nonresponsive FOXO13A
coactivated CAR activity, insulin effectively repressed FOXO1 coactivation but not FOXO13A
, but not AktKN
, inhibited FOXO1-CAR activity. Finally, insulin represses the induction of CYP2B10 mRNA in TCPOBOP-treated mouse primary hepatocytes. CAR accumulates in the nucleus after treatment with its activators, such as PB, TCPOBOP, and estradiol (15
). FOXO1 is in a position to coactivate CAR when the receptor accumulates in the nucleus. By virtue of the PI3K-Akt pathway, insulin inactivates FOXO1 by exporting it from the nucleus to the cytoplasm (2
). When in the nucleus, FOXO1 upregulates CAR activity, augmenting the expression of CAR target genes, such as CYP2B
genes. Insulin, on the other hand, downregulates the receptor by exporting its coactivator FOXO1 from the nucleus, which could result in the repression of the CAR-regulated genes. Thus, coregulation of CAR by FOXO1 provides insulin with a regulatory mechanism to repress hepatic drug metabolism. In addition to CAR, FOXO1 also activates PXR, suggesting that insulin repression of drug metabolism via FOXO1 may be a general pathway. A gel shift assay with the CAR-binding sequence NR1 as a probe showed that binding of CAR-RXR to NR1 was decreased in the presence of FOXO1, suggesting that CAR interacted with FOXO1 (data not shown). However, a supershift band that represented a FOXO1-CAR-RXR complex was not detected under the experimental conditions used. While one can argue that the ternary complex is simply too large in size to enter into a gel or is not stable enough to be detected, failing to detect it would also pose the question of whether FOXO1 forms a stable complex with CAR-RXR-NR1 to activate it. If so, how could FOXO1 activate CAR or RXR? These molecular mechanisms remain elusive and will be an interesting research objective for future investigations.
Our studies with CAR-null mice suggested that the receptor regulated the PB-mediated decrease of PEPCK1 mRNA. Our present investigations revealed the possible regulatory mechanism for PB-induced repression of gluconeogenic genes (Fig. ). Following activation by PB, CAR binds to FOXO1 and prevents its binding to the IRS, which could result in transcriptional repression of the genes that are regulated by IRS, such as PEPCK1
. In addition, future investigations may find the other gluconeogenic enzymes, including G6P and also key enzymes such as pyruvate dehydrogenase kinase 4 in glycolysis, to be regulated by CAR-FOXO1. This repression mechanism of FOXO1 by CAR is consistent with previous findings that nuclear hormone receptors (e.g., estrogen and androgen receptors, retinoid A receptor, and HNF4) bind to the DNA-binding domain of FOXO1 (21
) and that FOXO1 binding to IRS is decreased in the presence of the androgen receptor in a gel shift assay (21
). CAR and PXR have now extended the list of nuclear receptors (e.g., ER, AR, GR, TR, RAR, HNF4, and PPARγ) that interact with and coregulate FOXO1. In addition to cell proliferation (22
), glucose metabolism (29
), and adipogenesis (27
), drug metabolism becomes a novel target of coregulation by FOXO1 cross-talking with these nuclear receptors. Because the constitutively active AktCA
inhibited CAR activity in HepG2 cells, endogenous FOXO1 could be functional in coactivating the receptor. This functionality was further substantiated by the concomitant reduction of endogenous FOXO1 by RNAi and the repression of CAR activity. FOXO1 is the first factor to coregulate the nuclear receptors in response to an endogenous signal among various coregulators, such as SRC-1, NCoR, SMRT, and SHP, that are already known to regulate CAR and PXR (8
). PGC1 is another signal-responsive coregulator that can activate both CAR and PXR (41
). Whether it regulates the receptor in a signal-dependent manner has not been demonstrated.
While our present study concerned the mouse nuclear receptors and FOXO1, we also examined and confirmed that FOXO1 coactivated human CAR while the human receptor repressed FOXO1 (data not shown). It has been known for many years that diabetes elevates the activity of hepatic drug metabolism in humans. Moreover, PB was clinically used in diabetic patients to decrease their plasma glucose (20
). The CAR and PXR response elements, such as PBREM and XREM, are conserved in many human genes, such as CYP2B6
), and UGT1A1
). Although a physiological implication of this cross-talk regulation remains only speculative at the moment, one possible scenario may be to balance NADPH and NADH consumption. Insulin acts to decrease NADPH consumption by drug metabolism, while drugs act to repress gluconeogenesis to increase NADPH supply for drug metabolism. NADPH is an essential electron donor for cytochrome P450-dependent monooxygenase activity. The concept of reciprocal coregulation by CAR or PXR and FOXO1 may have clinical implications in the understanding of disease prevention, drug-drug interactions, and the development of better drug treatments.