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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Physiology and Pharmacology

Supervisor

Kim, Richard B.

Abstract

Sodium-taurocholate co-transporting polypeptide (NTCP, SLC10A1) is the central bile acid uptake transporter on the basolateral membrane of hepatocytes. Pharmacological inhibition of NTCP is also being used to treat Hepatitis B and D, and NTCP transports a variety of drugs including cholesterol-lowering statins. Despite these crucial roles, limited knowledge exists regarding the effects of genetic variation in SLC10A1 on bile acid and rosuvastatin transport.

To address this, we characterized activity and protein expression of genetically variant SLC10A1 in vitro. Seven SLC10A1 genetic variants displayed robust reductions in NTCP-mediated transport of taurocholic acid and rosuvastatin and virtually absent NTCP protein expression at the plasma membrane. In silico tools were employed to assess their performance to predict deleterious function, however these did not generate robust enough predictions to replace in vitro studies.

To elucidate the in vivo effects of targeted Slc10a1 disruption, serum bile acid composition and hepatic, renal, and ileal gene expression were assessed in male Slc10a1-/- mice. Conjugated serum hypercholanemia and absence of Oatp1a1 (Slco1a1) mRNA were observed in a subset of Slc10a1-/- mice. Additional changes in gene mRNA expression and mouse necropsy studies suggest these mice were unable to thrive as a result of nutrient malabsorption and disrupted nuclear receptor signaling.

Sex-related differences were evaluated in serum bile acid composition and hepatic gene expression in Slc10a1-/-mice. No important sex-related differences were observed in serum bile acid composition. Oatp1a1 mRNA was nearly undetectable in both male and female hypercholanemic mice. Sex associated differences in hepatic gene expression in control and normocholanemic Slc10a1-/-mice were consistent with literature, however these sex-specific differences were reversed for certain bile acid genes in hypercholanemic mice.

These findings identify novel loss of function genetic variants in the SLC10A1 gene in vitro. Additionally, our studies in Slc10a1-/-mice provide evidence of altered nuclear receptor signaling that may have important implications on bile acid physiology and drug response.

Summary for Lay Audience

Bile acids are important in digesting food but also play important roles in liver and intestinal health. Genetic changes that affect bile acid movement throughout the gastrointestinal tract can therefore have important implications on health and disease. Specifically, proteins located on the outer membranes of cells in the liver and intestine act as entry and exit points for the movement of bile acids through these tissues. These proteins are called transporters and are also involved in cellular entry and exit of drugs in clinical use. Of importance to this thesis is the commonly prescribed drug rosuvastatin, known by its brand name Crestor™. The ability of rosuvastatin to enter into liver cells is important for its cholesterol-lower capabilities. Therefore, in addition to transporter proteins being important in gastrointestinal health, they are important in drug response. Genetic changes that affect transporters can decrease entry of rosuvastatin into the liver, thereby reducing its cholesterol-lowering benefit. Moreover, reduced entry of rosuvastatin into the liver increases rosuvastatin concentrations in the blood, which has been associated with the adverse drug event of muscle pain and weakness.

Limited information is available regarding the effects of genetic variation in a specific transporter protein that is expressed on the outer cellular membrane of liver cells. This thesis aimed to gather information related to bile acid and drug transport into the liver by using cellular and mouse models to study the effects of genetic changes in our transporter protein of interest. Results from these studies indicate that previously untested genetic changes in our transporter protein result in reduced protein function and expression, reflected by reduced transport of a bile acid and of the drug rosuvastatin. Furthermore, we genetically disrupted our transporter of interest in mice and identified important changes in bile acid physiology and signaling. We did not observe any differences in rosuvastatin concentrations in the blood or livers of these genetically modified mice, as additional transport proteins are sufficient to maintain rosuvastatin uptake into the liver.

Taken together, these studies used cellular and animal models to identify important effects of genetic changes in our transporter protein. Future studies should assess these effects in humans to determine the overall clinical relevance of our findings. These studies will help contribute to better understanding bile acid transport, signaling, and drug response.

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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