Date of Award

2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Pharmaceutical Sciences

Department

Biomedical and Pharmaceutical Sciences

First Advisor

Ruitang Deng

Abstract

Bile acids are physiological detergents and surfactants with recently identified roles as signaling hormones. Maintenance of physiologically normal bile acids levels is fundamentally important as a deregulation within the bile acid synthesis pathway has the potential to result in an unbalanced bile acid pool. This may result in any number of pathological down stream effects and can exacerbate various diseases and disorders. Chenodeoxycholic acid (CDCA) and cholic acid (CA) are two major primary bile acids in humans with CDCA being more hydrophobic and toxic than CA. Aldo-keto reductase 1D1 (AKR1D1) and 12-alpha-hydroxylase (CYP8B1) are the two key enzymes responsible for the synthesis of CDCA and CA, respectively.

It remains largely unknown how AKR1D1 and CYP8B1 are regulated to maintain homeostatic CDCA and CA concentrations under physiological conditions. Likewise, little is known regarding the regulation of their synthesis under pathological conditions, or the mechanisms by which this regulation occurs. To date, much focus has been on CYP8B1 expression as the key regulator of bile acid synthesis, and also as the determining factor for the CDCA to CA ratio within the bile acid pool. We hypothesize that due to the increased toxicity associated with elevated CDCA concentrations, combined the knowledge that CDCA is a potent ligand for various signaling pathways, the liver is consistently altering AKR1D1 expression, and not CYP8B1, in an effort to maintain physiologically normal liver function.

Further knowledge pertaining to the regulation of individual primary bile acids may shed light on novel mechanisms by which various disorders and diseases, which are attributed to bile acid dysregulation, can be treated or prevented. For example, it has been established that the CDCA composition of the bile acid pool is lower in patients with diabetes; however, the mechanism by which this occurs is unclear. Understanding the role of bile acid synthesis in diabetic models may identify a mechanism by which CDCA production is being regulated. Hepatocellular carcinoma (HCC) is the most prevalent type of liver cancer. It has been identified that high concentrations of CDCA are toxic to the hepatocytes and can have carcinogenic effects; therefore a better elucidation of the regulation of CDCA production may prove important for prevention or progression of HCC. Intrahepatic cholestasis of pregnancy (ICP) is a disorder that occurs within the third trimester of pregnancy as a result of elevated bile acid levels. Although AKR1D1 expression is known to increase during pregnancy, the role of AKR1D1 in pregnancy has not yet been established.

Experiments were carried out in-vitro in a human liver carcinoma cell line and in-vivo in mice as well as on human liver tissue and tissue sections. Our data revealed that endogenous AKR1D1, and not CYP8B1, is transcriptionally and translationally regulated by primary bile acids, CDCA and CA, by a negative feedback or positive feed-forward mechanism, respectively. Likewise, with the use of a human AKR1D1 promoter reporter, the transcriptional regulation of AKR1D1 expression was investigated, and confirmed endogenous results. Additionally, results indicated that in conditions of metabolic disorder, (i.e., diabetes) or in liver cancer, human hepatic AKR1D1 expression is significantly decreased as compared to normal human tissue. Furthermore, a substantial link was established between AKR1D1 expression, estrogen and conditions of ICP in in-vitro and in-vivo experiments.

With knowledge of the cooperative capabilities of primary bile acids on the expression of AKR1D1, we next investigated potential signaling pathways involved in AKR1D1 regulation. In-vitro and in-vivo activation of critical bile acid (Farnesoid X Receptior, FXR), lipid (peroxisome proliferator activated-receptor, PPAR) and cholesterol (Liver X Receptor, LXR) signaling pathways were examined for implications on AKR1D1 expression. Our results demonstrated that while FXR was not involved in the regulation of AKR1D1, PPAR activation decreased AKR1D1 expression and LXR activation resulted in an increase in AKR1D1 expression. Furthermore, a substantial link was established between estrogen signaling and AKR1D1 expression in-vitro as well as in pregnant mice.

In conclusion, CDCA and CA are key regulators of AKR1D1, but not CYP8B1, expression. Such coordinated down-regulation and up-regulation of AKR1D1 by primary bile acids represents a mechanism by which the liver maintains homeostatic CDCA and CA levels under physiological condition. The data also indicates that LXR and PPAR signaling pathways are involved in regulating AKR1D1 with the possible identification of response elements in the promoter region. Moreover, the data demonstrates that changes to the bile acid composition of diabetic patients are due to a down regulation of AKR1D1 resulting in a decreased production of CDCA. Our data also demonstrates that in circumstances of HCC, the liver may alter bile acid synthesis through the down regulation of AKR1D1 expression, in a negative feedback manner to decrease production of CDCA. Finally, the role of AKR1D1 with respect to pregnancy was identified, substantiating a link between estrogen and AKR1D1 expression. In summary, our results confirm our hypothesis that the liver alters AKR1D1 expression in an effort to minimize toxicity associated with CDCA production and maintain homeostatic CDCA to CA levels.

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