Mechanistic studies of the phenobarbital-type induction of cytochromes P450 : role of AMP-activated protein kinase

Inside the liver cells there are sophisticated mechanisms that have evolved over millions of years to metabolize toxic substances, many of which are fat-soluble compounds making them difficult for the body to excrete. Cytochromes P450 (CYPs) are drug-metabolizing enzymes predominantly expressed in the liver that catalyze the first step in the metabolism of lipophilic compounds into water-soluble and thus excretable compounds. Transcriptional activation of CYPs and other drug-metabolizing enzymes by drugs and xenobiotics in the liver is mediated by the mammalian constitutive androstane receptor (CAR) and pregnanes X receptor (PXR), whereas the chicken xenobiotic receptor (CXR) mediates drug responses in chicken hepatocytes. This phenomenon, which is called drug induction, is fully reversible and dose-dependent. By increasing the capability for metabolic detoxification and elimination, induction of CYPs is an integral part of the defense mechanism against xenochemical insult. However, it also has important negative clinical consequences, such as altered pharmacokinetics of drugs and carcinogens, drug-drug interactions, and changes in the metabolism of steroids, vitamin D, and other endogenous compounds. For these reasons, it is of great importance to understand the molecular mechanisms leading to drug induced gene expression of hepatic drugmetabolizing enzymes. In this thesis the role of the AMP-activated protein kinase (AMPK) in the phenobarbital-mediated transcriptional regulation of CYPs was studied in hepatoma cells (chicken and human), and in hepatocytes (human and mouse). We show that AMPK activation by phenobarbital occurs via increased reactive oxygen species (ROS) formation in mitochondria and that increased AMPK activity is necessary for drug induction. Furthermore, we demonstrate that phenobarbital-type drugs affect the AMPK upstream kinase LKB1 and the protein kinase C zeta (PKCζ) activities. Importantly, these studies reveal that the signaling cascade involved in drug responses is conserved in evolution from birds to mice and humans. In addition, CXR interacting proteins were identified, which may also bind CAR in mammalian liver. In conclusion, these findings provide new insights into the so far poorly characterized molecular mechanism by which drugs lead to transcriptional regulation of CYP enzymes.