| dc.description.abstract | "We may be born 100% human but will die 90% bacterial--a truly complex organism!" (Goodacre, 2007). This statement reflects the fact that there are 10 times more bacterial cells in the human body compared to the number of human cells, and there are 100 times more genes in the human microbiome compared to the number of genes in the human genome. Gut bacteria and host communicate with each other and collectively determine many aspects of host physiology such as bile acid (BA) and drug metabolism. Gut bacteria varies significantly between individuals and therefore, may be responsible for the inter-individual differences in BA concentrations and drug responses. There are a number of diseases such as obesity, inflammatory bowel disorder, and autism, that have been associated with an abnormal bloom in certain gut bacteria or a decrease in the diversity of gut bacteria. Therefore, modulating gut bacteria by probiotics, prebiotics, and by fecal transplantation have become viable therapeutic strategies. Alterations of gut bacteria in diseases or the therapeutic modulation of gut bacteria has the potential to alter host BA signaling and drug responses. Germ-free (GF) mice provide an excellent model system for understanding the functions of gut bacteria. The overall goal of this dissertation is to expand the understanding of the role of gut bacteria in regulating host BA homeostasis and hepatic drug metabolism. In Specific Aim 1, I determined the changes in BA homeostasis and BA signaling in GF mice. BAs are amphipathic cholesterol metabolites that are synthesized in liver and secreted into bile. Gut bacteria metabolize primary BAs to secondary BAs. The majority of BAs are reabsorbed from the intestine, effluxed into the portal vein and return to the liver. Therefore, the BA profile in the host is the result of the host hepatic enzyme activity and the gut bacterial enzyme activity. The BA profile is important because, BAs act like hormones and regulate host physiology by activating the BA receptors, namely the farnesoid X receptor (FXR) and transmembrane G-protein-coupled receptor (TGR5). The BA profiles of both male and female GF mice are markedly altered compared to conventional (CV) mice. GF mice have an increase in total BAs in all the tissue compartments analyzed and decreased total fecal excretion of BAs compared to CV mice. This could be due to slower intestinal propulsion rates and increased BA reabsorption from the intestines. The dominant BAs in GF mice are taurine conjugated α and β muricholic acids (Tα+β MCA). There is an increase in both ursodeoxycholic acid (UDCA) and MCAs and in the proportion of taurine conjugated BAs and these BAs result in a more hydrophilic BA pool in GF mice. UDCA which was previously considered to be a secondary BA that is synthesized by gut bacteria increases in GF mice. Biotransformation experiments in vitro demonstrated that UDCA can be synthesized from CDCA by enzymes present in hepatic microsomes isolated from both GF and CV mice. This explains why UDCA is increased in GF mice, and is evidence that UDCA is a primary BA synthesized by hepatic enzymes in mice. The altered BA profile in GF mice results in the activation of TGR5 signaling. Therefore, GF mice display all the characteristics of TGR5 activation, such as increased gallbladder size, increased serum GLP-1 levels, and increased mRNA of type 2 iodothyronine deiodinase (D2), the enzyme which increases energy expenditure as heat in brown adipose tissue. In Specific aim 2, I determined alterations in the mRNA of drug metabolizing enzymes in livers of GF mice by RNA-Seq. Gene expression of a many hepatic Phase-1 and Phase-2 enzymes was altered in the absence of gut bacteria. Based on this study, I was able to generate of a list of genes in the liver that are altered by gut bacteria. Among these genes are Cyp2b10 and Cyp3a11 as their mRNAs were decreased in GF mice. In order to test the functional consequences, GF and CV mice were treated with the anesthetic pentobarbital and I observed that GF mice sleep longer than CV mice suggesting that pentobarbital metabolism is slower in GF mice. These observations strongly suggest that gut bacteria play an important role in regulating drug metabolizing enzymes in the liver. Drug metabolizing enzymes with decreased mRNA levels in GF mice are probably increased in CV mice to help biotransform chemicals formed by gut bacteria. In contrast, drug metabolizing enzymes with increased mRNA levels in GF mice are likely enzymes whose function normally can be performed by the gut bacterial enzymes, and therefore in the absence of gut bacteria, are induced in the liver. In conclusion, this dissertation work has provided a detailed roadmap of alterations in BA composition, BA signaling, and expression of hepatic drug metabolizing enzymes in the absence of gut bacteria and will help to understand how to alter gut bacteria in a beneficial way. | |
