Abstract 551: Deficiency of Hepatic Nuclear Factor 4 Alpha Results in Impaired Metabolism of Endogenous Methylarginines and Beta-aminoisobutiric Acid - A Novel Mechanism of Cardiovascular Complications in Patients With Type 2 Diabetes?

Abstract

Introduction: Alanine-glyoxylate aminotransferase 2 (AGXT2) is the only known enzyme capable of degradation of all three endogenous methylarginines, which serve as markers and potentially mediators of cardiovascular disease. Recent studies also suggest that AGXT2 and its alternative substrate beta-aminoisobutyric acid (BAIB) play important role in lipid metabolism. The predicted core promoter region of mammalian AGXT2 promoter contains a highly conserved putative binding site for hepatic nuclear factor 4 alpha (HNF4A). Patients with severe deficiency in HNF4a develop maturity onset diabetes of young 1. Furthermore, polymorphisms of HNF4A are associated with increased risk of diabetes type 2. The aim of this study was to test the hypothesis that HNF4A is a major regulator of AGXT2 expression and activity. Methods and results: We demonstrated direct binding of HNF4A to the Agxt2 promoter region in hepatic cell line Hepa 1-6 using chromatin immunoprecipitation assays. Then we showed that mutations of the predicted HNF4A binding site in the Agxt2 core promoter result in up to 80% decrease in the promoter activity as assessed by luciferase reporter assays (p<0.001). We used siRNA-mediated knockdown of HNF4A to determine whether this factor is required for basal Agxt2 expression in Hepa 1-6 cells. Knockdown of HNF4A led to almost 50% reduction in Agxt2 mRNA levels compared to controls (p<0.01). We took advantage of the previously characterized inducible liver-specific Hnf4a knockout (KO) mice to determine whether HNF4A regulates Agxt2 expression in vivo and showed a 90% (p<0.001) decrease in liver Agxt2 expression and a 85% (p<0.01) decrease in liver AGXT2 activity towards methylarginines in Hnf4a KO mice compared with the wild-type littermates. Finaly, on a functional level, Hnf4a KO mice had significant amounts of BAIB present in plasma, whereas BAIB was not detectable in the plasma of the wild-type littermates. Conclusions: In our study we identified HNF4A as the major regulator of Agxt2 gene expression. This finding suggests that diabetic patients with HNF4A deficiency might have a unique mechanism for development of cardiovascular complication via AGXT2-dependent impairment of lipid metabolism and methylarginines-mediated vascular dysfunction.