Low plasma concentrations of L-homoarginine are associated with an increased risk of cardiovascular events, while homoarginine supplementation is protective in animal models of metabolic syndrome and stroke. Catabolism of homoarginine is still poorly understood. Based on the recent findings from a Genome Wide Association Study we hypothesized that homoarginine can be metabolized by alanine:glyoxylate aminotransferase 2 (AGXT2). We purified human AGXT2 from tissues of AGXT2 transgenic mice and demonstrated its ability to metabolize homoarginine to 6-guanidino-2-oxocaproic acid (GOCA). After incubation of HepG2 cells overexpressing AGXT2 with isotope-labeled homoarginine-d4 we were able to detect labeled GOCA in the medium. We injected wild type mice with labeled homoarginine and detected labeled GOCA in the plasma. We found that AGXT2 knockout (KO) mice have higher homoarginine and lower GOCA plasma levels as compared to wild type mice, while the reverse was true for AGXT2 transgenic (Tg) mice. In summary, we experimentally proved the presence of a new pathway of homoarginine catabolism – its transamination by AGXT2 with formation of GOCA and demonstrated that endogenous AGXT2 is required for maintenance of homoarginine levels in mice. Our findings may lead to development of novel therapeutic approaches for cardiovascular pathologies associated with homoarginine deficiency.

Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and increase in blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency. We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling. Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Objective: Elevated plasma concentrations of asymmetric dimethylarginine (ADMA) are associated with an increased risk of mortality and adverse cardiovascular outcomes. ADMA can be metabolized by dimethylarginine dimethylaminohydrolases (DDAHs) and by alanine-glyoxylate aminotransferase 2 (AGXT2). Deletion of DDAH1 in mice leads to elevation of ADMA in plasma and blood pressure, while overexpression of human DDAH1 is associated with a lower plasma ADMA concentration and protective cardiovascular effects. The possible role of alternative metabolism of ADMA by AGXT2 remains to be elucidated. The goal of the current study was to test the hypothesis that transgenic overexpression of AGXT2 leads to lowering of plasma levels of ADMA and protection from vascular damage in the setting of DDAH1 deficiency.Approach and Results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2. qPCR and Western Blot confirmed the expression of the transgene. Systemic ADMA levels were decreased by 15% in TG mice. In comparison with wild type animals plasma levels of asymmetric dimethylguanidino valeric acid (ADGV), the AGXT2 associated metabolite of ADMA, were six times higher. We crossed AGXT2 TG mice with DDAH1 knockout mice and observed that upregulation of AGXT2 lowers plasma ADMA and pulse pressure and protects the mice from endothelial dysfunction and adverse aortic remodeling.Conclusions: Upregulation of AGXT2 led to lowering of ADMA levels and protection from ADMA-induced vascular damage in the setting of DDAH1 deficiency. This is especially important, because all the efforts to develop pharmacological ADMA-lowering interventions by means of upregulation of DDAHs have been unsuccessful.

Introduction: Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthase, which has been proposed to play a direct role in the pathogenesis of cardiovascular disease. There are two enzymatic pathways for degradation of ADMA: hydrolysis to citrulline by dimethylarginine dimethylaminohydrolase (DDAH) and transamination by alanine-glyoxylate aminotransferase 2 (AGXT2) with formation of asymmetric dimethylguanidino valeric acid (ADGV). The first pathway is well characterized, whereas the physiological role of AGXT2 is still poorly understood. The goal of our study was to test the hypothesis that transgenic overexpression of AGXT2 would lead to lowering of systemic levels of ADMA and improved vasomotor function. Methods and results: We generated transgenic mice (TG) with ubiquitous overexpression of AGXT2 under control of the chicken beta actin (CAG) promoter. Ubiquitous overexpression of the transgene was confirmed by qPCR and Western Blot. TG animals had normal weight and no observed developmental abnormalities. Biochemical data were generated using HPLC-MS/MS. ADMA plasma levels in AGXT2 TG animals were decreased by 15% (p<0.05), whereas ADGV levels were 6 times higher in TG animals in comparison with wild-types (p<0.001). Lung and heart of TG animals exhibited 2 times lower tissue ADMA content in comparison with controls (p<0.05). Isolated aortic rings were used to estimate endothelium-dependent and -independent relaxation in response to acetylcholine (Ach) and sodium nitroprusside (SNP), respectively. Aortas from AGXT2 TG mice demonstrated an increase in maximal response to ACh (p<0.05). There was a similar relaxation in response to SNP in both groups. Conclusions: Our findings show that upregulation of AGXT2 results in lower ADMA levels and improved endothelial-dependent relaxation in vivo. AGXT2 thereby may be a therapeutic target for long-term reduction of systemic ADMA levels and improvement of vascular function in vivo. This is especially important, because all the efforts to develop ADMA-lowering interventions by means of upregulation of DDAH have not been successful so far. Our data suggest that AGXT2 might be a promising drug target for cardiovascular pathologies associated with elevated ADMA levels.

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.