Date of Award
Doctor of Philosophy (PhD)
In MODY3 patients, as well in the experimental mouse model, the Tcf1 -/- mouse, there is a defect in arginine-induced insulin secretion. In this thesis, we examined the role of arginine transporters in the insulin secreting cells in arginine-induced insulin secretion. We first characterized arginine uptake by MIN6 cells as having a Km of 102.6 μM and being partially Na-dependent and entirely Cl-dependent. We then examined Tcf1 -/- pancreatic islets, which are defective for arginine-induced insulin secretion. Using gene expression array analysis and semi-quantitative RT-PCR analysis on pancreatic islets from Tcf1 +/+ and Tcf1 -/- mice, we found eight arginine transporters expressed in the pancreatic islets with only two transporters, mNAT3 and CAT3, regulated by Tcf1 in the islets. Using siRNA-mediated knockdown of both transporters, we found that these transporters are not required for arginine-induced insulin secretion in MIN6 cells. We conclude that there is a high level of redundancy for arginine transport into insulin-secreting cells. We also examined the regulation of mNAT3 in the liver. The liver is the main site for gluconeogenesis during fasting, and has been shown to increase amino acid uptake during periods of low nutrient intake to increase its substrate iv pool for glucose production. We found that mNAT3 is upregulated during fasting, and that this response is abolished with insulin. Using mice injected with adenovirus expressing either Foxa2 or GFP as a control, we found that Foxa2 mediates this insulin-sensitive increase in hepatic mNAT3 expression. Through electrophoretic mobility shift assays and chromatin immunoprecipitation experiments, we found that Foxa2 binds to promoter elements of the mNAT3 gene, Slc38a4. We also showed these promoter elements to be important for Foxa2 transactivation using luciferase reporter gene assays. In addition, with liver perfusion experiments using mice infected with adenovirus, we found increased arginine uptake by livers overexpressing Foxa2. Thus, we have identified Foxa2 as a mediator for increased hepatic amino acid uptake during fasting.
Lee, Vivian M., "Energy Homeostasis During Fasting and Fed States: Foxa2 Regulates Hepatic Amino Acid Uptake" (2007). Student Theses and Dissertations. 189.