Metformin Represses Drug-Induced Expression of CYP2B6 by Modulating the Constitutive Androstane Receptor Signaling

Abstract

Metformin is currently the most widely used drug for the treatment of type 2 diabetes. Mechanistically, metformin interacts with many protein kinases and transcription factors that alter the expression of numerous downstream target genes governing lipid metabolism, cell proliferation, and drug metabolism. The constitutive androstane receptor (CAR, NR1i3), a known xenobiotic sensor, has recently been recognized as a novel signaling molecule, in that its activation could be regulated by protein kinases in addition to the traditional ligand binding. We show that metformin could suppress drug-induced expression of CYP2B6 (a typical target gene of CAR) by modulating the phosphorylation status of CAR. In human hepatocytes, metformin robustly suppressed the expression of CYP2B6 induced by both indirect (phenobarbital) and direct CITCO [6-(4-chlorophenyl)imidazo[2,1-b]1,3thiazole-5-carbaldehyde O-(3,4-dichlorobenzyl)oxime] activators of human CAR. Mechanistic investigation revealed that metformin specifically enhanced the phosphorylation of threonine-38 of CAR, which blocks CAR nuclear translocation and activation. Moreover, we showed that phosphorylation of CAR by metformin was primarily an AMP-activated protein kinase-and extracellular signal-regulated kinase 1/2-dependent event. Additional two-hybrid and coimmunoprecipitation assays demonstrated that metformin could also disrupt CITCO-mediated interaction between CAR and the steroid receptor coactivator 1 or the glucocorticoid receptorinteracting protein 1. Our results suggest that metformin is a potent repressor of drug-induced CYP2B6 expression through specific inhibition of human CAR activation. Thus, metformin may affect the metabolism and clearance of drugs that are CYP2B6 substrates.

Phenobarbital Meets Phosphorylation of Nuclear Receptors

“…2B). Subsequently, phosphorylation and dephosphorylation were shown to occur in human primary hepatocytes (Yang et al, 2014), confirming that PB-induced dephosphorylation at threonine 38 is conserved in both mice and humans. In fact, the phosphomimetic CAR T38D mutant was unable to bind NR1 in gel-shift assays and was retained in the cytoplasm of human hepatoma-derived HepG2 cells.…”

Investigational Small-Molecule Drug Selectively Suppresses Constitutive CYP2B6 Activity at the Gene Transcription Level: Physiologically Based Pharmacokinetic Model Assessment of Clinical Drug Interaction Risk

“…of triplicate measurements per donor per concentration; *P , 0.05 versus vehicle control. and nuclear translocation (Yang et al, 2014). LY2090314 potency toward inhibition of GSK-3 (IC 50 ;1 nM) is approximately 50-fold more potent than CYP2B6 suppression in hepatocytes (IC 50 ;50 nM).…”

“…The lack of CYP2B6 functional suppression by known CAR inverse agonists, such as clotrimazole and 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinoline-carboxamide (PK11195), is attributed to their PXR activation, which overcompensates for decreased CAR activity yielding net CYP2B6 induction (Faucette et al, 2004;Li et al, 2008). Likewise, metformin was recently shown to indirectly inhibit CAR activation by enhancing phosphorylation and limiting nuclear translocation; however, although metformin suppressed CARmediated CYP2B6 induction, it did not decrease constitutive CYP2B6 levels (Yang et al, 2014).…”

“…Although CAR inhibition can occur by direct (inverse agonism) or indirect mechanisms (e.g. altered phosphorylation and diminished nuclear translocation), it is not known to elicit decreased constitutive CYP2B6 enzyme activity (Moore et al, 2000;Faucette et al, 2004;Li et al, 2008;Tolson and Wang, 2010;Yang et al, 2014). The lack of CYP2B6 functional suppression by known CAR inverse agonists, such as clotrimazole and 1-(2-chlorophenyl-N-methylpropyl)-3-isoquinoline-carboxamide (PK11195), is attributed to their PXR activation, which overcompensates for decreased CAR activity yielding net CYP2B6 induction (Faucette et al, 2004;Li et al, 2008).…”

Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats

“…SDs contain caffeine as a main ingredient and caffeine was shown to stimulate 5'-AMP-activated protein kinase (AMPK) in the extensor digitorum longus muscle (36). Furthermore, metformin was recently demonstrated to enhance constitutive active/androstane receptor (CAR) phosphorylation in human hepatocytes in part via an AMPK-dependent signaling pathway and suppression of CYP2B6 (37). Thus, the suppressive effect of SDs on CYP2B1 mRNA expression, which was revealed in the current study, may be mediated through caffeine stimulation of AMPK-dependent enhancement of CAR phosphorylation (38).…”