Abstract 461: Hepatic Nuclear Factor 4 Alpha as a Regulator of Alanine: Glyoxylate Aminotransferase 2 Expression and Systemic Levels of Endogenous Methylarginines

Abstract

Introduction: Endogenous methylarginines have been proposed as markers and potentially mediators of cardiovascular diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) is the only enzyme capable of regulation of plasma levels of all three endogenous methylarginines. It has also been demonstrated that AGXT2 and its alternative substrate beta-aminoisobutyric acid (BAIB) can play an important modulatory role in lipid metabolism. Using bioinformatic analysis we identified a highly conserved putative binding site for the diabetes-associated transcription factor hepatic nuclear factor 4 alpha (HNF4A) in the mammalian AGXT2 promoter region. The aim of this study was to test the hypothesis that HNF4a is the major regulator of AGXT2 expression and activity. Methods and results: We introduced several point mutations in the putative HNF4A binding site and investigated their influence on activity of the murine Agxt2 promoter using luciferase reporter assay. The mutated constructs decreased the activity of the reporter gene by 75% as compared to the native promoter sequence. We showed direct binding of HNF4a to Agxt2 promoter using chromatin immunoprecipitation. We were able to demonstrate that siRNA-mediated knockdown of HNF4a leads to 50% reduction of Agxt2 expression in the murine hepatic cell line Hepa 1-6. In the in-vivo part of the project we showed that liver-specific Hnf4a knockout mice have a 90% reduction in liver Agxt2 mRNA levels, a 85% decrease in liver AGXT2 activity and significantly increased plasma levels of endogenous methylarginines and BAIB. Conclusions: In the current study we showed direct binding of HNF4a to the mammalian AGXT2 promoter region. We also demonstrated using in-vitro and in-vivo approaches that HNF4A is the major regulator of Agxt2 expression and has direct influence on systemic levels of endogenous methylarginines and BAIB. These findings suggest a novel link between NO-mediated impairment of vascular and renal function and lipid metabolism.