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Roman N. Rodionov - One of the best experts on this subject based on the ideXlab platform.
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kidney and liver are the main organs of expression of a key metabolic enzyme alanine glyoxylate aminotransferase 2 in humans
Atherosclerosis Supplements, 2019Co-Authors: Natalia Jarzebska, Renke Maas, Roman N. Rodionov, Silke Brilloff, Bernd Hohenstein, Normund Jabs, Sophia Georgi, Christian Zietz, Sabrina Montresor, Norbert WeissAbstract:Abstract Background The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that AGXT2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of AGXT2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. Material and methods We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. Results We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. Conclusions Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.
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a novel pathway for metabolism of the cardiovascular risk factor homoarginine by alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Anton V Demyanov, Silke Brilloff, Natalia Jarzebska, James Leiper, Jens Martenslobenhoffer, Elisa Oppici, Anne Kolouschek, Renke MaasAbstract: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.
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Abstract 461: Hepatic Nuclear Factor 4 Alpha as a Regulator of Alanine: Glyoxylate Aminotransferase 2 Expression and Systemic Levels of Endogenous Methylarginines
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Alexey A Kolobov, Anton V Demyanov, Alexey A. Soshnev, Nikolay Samusik, Silke Brilloff, Natalia Jarzebska, Chad Brocker, Jens Martens-lobenhofferAbstract: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.
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role of alanine glyoxylate aminotransferase 2 in metabolism of asymmetric dimethylarginine in the settings of asymmetric dimethylarginine overload and bilateral nephrectomy
Nephrology Dialysis Transplantation, 2014Co-Authors: Roman N. Rodionov, Anja Kittel, Renke Maas, Silke Brilloff, Natalia Jarzebska, Jens Martenslobenhoffer, Bernd Hohenstein, Normund Jabs, Norbert Weiss, Stefanie M BodebogerAbstract:BACKGROUND Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. METHODS ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. RESULTS Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. CONCLUSIONS Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.
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human alanine glyoxylate aminotransferase 2 lowers asymmetric dimethylarginine and protects from inhibition of nitric oxide production
Journal of Biological Chemistry, 2010Co-Authors: Roman N. Rodionov, Daryl J Murry, Sarah F Vaulman, Jeff W Stevens, Steven R LentzAbstract:Elevated blood concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric-oxide (NO) synthase, are found in association with diabetes, hypertension, congestive heart failure, and atherosclerosis. ADMA levels are controlled by dimethylarginine dimethylaminohydrolases (DDAHs), cytosolic enzymes that hydrolyze ADMA to citrulline and dimethylamine. ADMA also has been proposed to be regulated through an alternative pathway by alanine-glyoxylate aminotransferase 2 (AGXT2), a mitochondrial aminotransferase expressed primarily in the kidney. The goal of this study was to define the subcellular localization of human AGXT2 and test the hypothesis that overexpression of human AGXT2 protects from ADMA-induced inhibition in nitric oxide (NO) production. AGXT2 was cloned from human kidney cDNA and overexpressed in COS-7 cells and human umbilical vein endothelial cells with a C-terminal FLAG epitope tag. Mitochondrial localization of human AGXT2 was demonstrated by confocal microscopy and a 41-amino acid N-terminal mitochondrial cleavage sequence was delineated by N-terminal sequencing of the mature protein. Overexpression of human AGXT2 in the liver of C57BL/6 mice using an adenoviral expression vector produced significant decreases in ADMA levels in plasma and liver. Overexpression of human AGXT2 also protected endothelial cells from ADMA-mediated inhibition of NO production. We conclude that mitochondrially localized human AGXT2 is able to effectively metabolize ADMA in vivo resulting in decreased ADMA levels and improved endothelial NO production.
Renke Maas - One of the best experts on this subject based on the ideXlab platform.
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kidney and liver are the main organs of expression of a key metabolic enzyme alanine glyoxylate aminotransferase 2 in humans
Atherosclerosis Supplements, 2019Co-Authors: Natalia Jarzebska, Renke Maas, Roman N. Rodionov, Silke Brilloff, Bernd Hohenstein, Normund Jabs, Sophia Georgi, Christian Zietz, Sabrina Montresor, Norbert WeissAbstract:Abstract Background The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that AGXT2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of AGXT2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. Material and methods We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. Results We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. Conclusions Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.
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a novel pathway for metabolism of the cardiovascular risk factor homoarginine by alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Anton V Demyanov, Silke Brilloff, Natalia Jarzebska, James Leiper, Jens Martenslobenhoffer, Elisa Oppici, Anne Kolouschek, Renke MaasAbstract: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.
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role of alanine glyoxylate aminotransferase 2 in metabolism of asymmetric dimethylarginine in the settings of asymmetric dimethylarginine overload and bilateral nephrectomy
Nephrology Dialysis Transplantation, 2014Co-Authors: Roman N. Rodionov, Anja Kittel, Renke Maas, Silke Brilloff, Natalia Jarzebska, Jens Martenslobenhoffer, Bernd Hohenstein, Normund Jabs, Norbert Weiss, Stefanie M BodebogerAbstract:BACKGROUND Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. METHODS ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. RESULTS Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. CONCLUSIONS Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.
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alanine glyoxylate aminotransferase 2 AGXT2 polymorphisms have considerable impact on methylarginine and β aminoisobutyrate metabolism in healthy volunteers
PLOS ONE, 2014Co-Authors: Anja Kittel, Maren Mieth, Martin F. Fromm, Fabian Müller, Ana Kralj, Oliver Zolk, Heinrich Sticht, Markus R. Heinrich, Jörg König, Renke MaasAbstract:Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p<0.001). ADMA concentrations were not affected by both SNPs. A haplotype analysis revealed that the second investigated AGXT2 SNP rs16899974, which was not significantly linked to the other AGXT2 SNP, further aggravates the effect of rs37369 with respect to BAIB concentrations in plasma and urine. To investigate the impact of the amino acid exchange p.Val140Ile, we established human embryonic kidney cell lines stably overexpressing wild-type or mutant (p.Val140Ile) AGXT2 protein and assessed enzyme activity using BAIB and stable-isotope labeled [2H6]-SDMA as substrate. In vitro, the amino acid exchange of the mutant protein resulted in a significantly lower enzyme activity compared to wild-type AGXT2 (p<0.05). In silico modeling of the SNPs indicated reduced enzyme stability and substrate binding. In conclusion, SNPs of AGXT2 affect plasma as well as urinary BAIB and SDMA concentrations linking methylarginine metabolism to the common genetic trait of hyper-β-aminoisobutyric aciduria.
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Alanine-glyoxylate aminotransferase 2 (AGXT2) polymorphisms have considerable impact on methylarginine and β-aminoisobutyrate metabolism in healthy volunteers.
PLOS ONE, 2014Co-Authors: Anja Kittel, Maren Mieth, Martin F. Fromm, Fabian Müller, Ana Kralj, Oliver Zolk, Heinrich Sticht, Markus R. Heinrich, Jörg König, Renke MaasAbstract:Elevated plasma concentrations of asymmetric (ADMA) and symmetric (SDMA) dimethylarginine have repeatedly been linked to adverse clinical outcomes. Both methylarginines are substrates of alanine-glyoxylate aminotransferase 2 (AGXT2). It was the aim of the present study to simultaneously investigate the functional relevance and relative contributions of common AGXT2 single nucleotide polymorphisms (SNPs) to plasma and urinary concentrations of methylarginines as well as β-aminoisobutyrate (BAIB), a prototypic substrate of AGXT2. In a cohort of 400 healthy volunteers ADMA, SDMA and BAIB concentrations were determined in plasma and urine using HPLC-MS/MS and were related to the coding AGXT2 SNPs rs37369 (p.Val140Ile) and rs16899974 (p.Val498Leu). Volunteers heterozygous or homozygous for the AGXT2 SNP rs37369 had higher SDMA plasma concentrations by 5% and 20% (p = 0.002) as well as higher BAIB concentrations by 54% and 146%, respectively, in plasma and 237% and 1661%, respectively, in urine (both p
Natalia Jarzebska - One of the best experts on this subject based on the ideXlab platform.
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the role of alanine glyoxylate transaminase 2 AGXT2 in β alanine and carnosine metabolism of healthy mice and humans
European Journal of Applied Physiology, 2020Co-Authors: Jan Stautemas, Natalia Jarzebska, Zhou Xiang Shan, Laura Blancquaert, Inge Everaert, Sarah De Jager, Siegrid De Baere, Arne Hautekiet, Anneke Volkaert, Filip LefevereAbstract:Chronic β-alanine supplementation leads to increased levels of muscle histidine-containing dipeptides. However, the majority of ingested β-alanine is, most likely, degraded by two transaminases: GABA-T and AGXT2. In contrast to GABA-T, the in vivo role of AGXT2 with respect to β-alanine metabolism is unknown. The purpose of the present work is to investigate if AGXT2 is functionally involved in β-alanine homeostasis. Muscle histidine-containing dipeptides levels were determined in AGXT2 overexpressing or knock-out mice and in human subjects with different rs37369 genotypes which is known to affect AGXT2 activity. Further, plasma β-alanine kinetic was measured and urine was obtained from subjects with different rs37369 genotypes following ingestion of 1400 mg β-alanine. Overexpression of AGXT2 decreased circulating and muscle histidine-containing dipeptides (> 70% decrease; p 0.05). In humans, the results support the evidence that decreased AGXT2 activity is not associated with altered histidine-containing dipeptides levels (p > 0.05). Additionally, following an acute dose of β-alanine, no differences in pharmacokinetic response were measured between subjects with different rs37369 genotypes (p > 0.05). Interestingly, urinary β-alanine excretion was 103% higher in subjects associated with lower AGXT2 activity, compared to subjects associated with normal AGXT2 activity (p < 0.05). The data suggest that in vivo, β-alanine is a substrate of AGXT2; however, its importance in the metabolism of β-alanine and histidine-containing dipeptides seems small.
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kidney and liver are the main organs of expression of a key metabolic enzyme alanine glyoxylate aminotransferase 2 in humans
Atherosclerosis Supplements, 2019Co-Authors: Natalia Jarzebska, Renke Maas, Roman N. Rodionov, Silke Brilloff, Bernd Hohenstein, Normund Jabs, Sophia Georgi, Christian Zietz, Sabrina Montresor, Norbert WeissAbstract:Abstract Background The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that AGXT2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of AGXT2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. Material and methods We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. Results We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. Conclusions Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.
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diabetes linked transcription factor hnf4α regulates metabolism of endogenous methylarginines and β aminoisobutyric acid by controlling expression of alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Dmitri Burdin, Alexey A Kolobov, Anton V Demyanov, Alexey A. Soshnev, Nikolay Samusik, Silke Brilloff, Natalia Jarzebska, Chad Brocker, Jens Martenslobenhoffer, Maren MiethAbstract:Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of AGXT2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to AGXT2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in AGXT2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver AGXT2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of AGXT2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.
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a novel pathway for metabolism of the cardiovascular risk factor homoarginine by alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Anton V Demyanov, Silke Brilloff, Natalia Jarzebska, James Leiper, Jens Martenslobenhoffer, Elisa Oppici, Anne Kolouschek, Renke MaasAbstract: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.
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Abstract 461: Hepatic Nuclear Factor 4 Alpha as a Regulator of Alanine: Glyoxylate Aminotransferase 2 Expression and Systemic Levels of Endogenous Methylarginines
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Alexey A Kolobov, Anton V Demyanov, Alexey A. Soshnev, Nikolay Samusik, Silke Brilloff, Natalia Jarzebska, Chad Brocker, Jens Martens-lobenhofferAbstract: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.
James Leiper - One of the best experts on this subject based on the ideXlab platform.
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a novel pathway for metabolism of the cardiovascular risk factor homoarginine by alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Anton V Demyanov, Silke Brilloff, Natalia Jarzebska, James Leiper, Jens Martenslobenhoffer, Elisa Oppici, Anne Kolouschek, Renke MaasAbstract: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.
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alanine glyoxylate aminotransferase 2 metabolizes endogenous methylarginines regulates no and controls blood pressure
Arteriosclerosis Thrombosis and Vascular Biology, 2012Co-Authors: Ben Caplin, Laura Dowsett, Mathew Delahaye, Alan D Salama, Anna Slaviero, David C. Wheeler, James Tomlinson, Zhen Wang, James LeiperAbstract:OBJECTIVE: Asymmetric dimethylarginine is an endogenous inhibitor of NO synthesis that may mediate cardiovascular disease. Alanine-glyoxylate aminotransferase-2 (AGXT2) has been proposed to degrade asymmetric dimethylarginine. We investigated the significance of AGXT2 in methylarginine metabolism in vivo and examined the effect of this enzyme on blood pressure. METHODS AND RESULTS: In isolated mouse kidney mitochondria, we show asymmetric dimethylarginine deamination under physiological conditions. We demonstrate increased asymmetric dimethylarginine, reduced NO, and hypertension in an AGXT2 knockout mouse. We provide evidence for a role of AGXT2 in methylarginine metabolism in humans by demonstrating an inverse relationship between renal (allograft) gene expression and circulating substrate levels and an association between expression and urinary concentrations of the product. Finally, we examined data from a meta-analysis of blood pressure genome-wide association studies. No genome-wide significance was observed, but taking a hypothesis-driven approach, there was a suggestive association between the T allele at rs37369 (which causes a valine-isoleucine substitution and altered levels of AGXT2 substrate) and a modest increase in diastolic blood pressure (P=0.0052). CONCLUSIONS: Although the effect of variation at rs37369 needs further study, these findings suggest that AGXT2 is an important regulator of methylarginines and represents a novel mechanism through which the kidney regulates blood pressure.
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alanine glyoxylate aminotransferase 2 metabolizes endogenous methylarginines regulates no and controls blood pressure
Arteriosclerosis Thrombosis and Vascular Biology, 2012Co-Authors: Ben Caplin, Laura Dowsett, Mathew Delahaye, Alan D Salama, Anna Slaviero, David C. Wheeler, James Tomlinson, Zhen Wang, James LeiperAbstract:Objective—Asymmetric dimethylarginine is an endogenous inhibitor of NO synthesis that may mediate cardiovascular disease. Alanine-glyoxylate aminotransferase-2 (AGXT2) has been proposed to degrade asymmetric dimethylarginine. We investigated the significance of AGXT2 in methylarginine metabolism in vivo and examined the effect of this enzyme on blood pressure. Methods and Results—In isolated mouse kidney mitochondria, we show asymmetric dimethylarginine deamination under physiological conditions. We demonstrate increased asymmetric dimethylarginine, reduced NO, and hypertension in an AGXT2 knockout mouse. We provide evidence for a role of AGXT2 in methylarginine metabolism in humans by demonstrating an inverse relationship between renal (allograft) gene expression and circulating substrate levels and an association between expression and urinary concentrations of the product. Finally, we examined data from a meta-analysis of blood pressure genome-wide association studies. No genome-wide significance was observed, but taking a hypothesisdriven approach, there was a suggestive association between the T allele at rs37369 (which causes a valine-isoleucine substitution and altered levels of AGXT2 substrate) and a modest increase in diastolic blood pressure (P=0.0052). Conclusion—Although the effect of variation at rs37369 needs further study, these findings suggest that AGXT2 is an important regulator of methylarginines and represents a novel mechanism through which the kidney regulates blood pressure. (Arterioscler Thromb Vasc Biol. 2012;32:2892-2900.)
Silke Brilloff - One of the best experts on this subject based on the ideXlab platform.
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kidney and liver are the main organs of expression of a key metabolic enzyme alanine glyoxylate aminotransferase 2 in humans
Atherosclerosis Supplements, 2019Co-Authors: Natalia Jarzebska, Renke Maas, Roman N. Rodionov, Silke Brilloff, Bernd Hohenstein, Normund Jabs, Sophia Georgi, Christian Zietz, Sabrina Montresor, Norbert WeissAbstract:Abstract Background The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that AGXT2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of AGXT2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. Material and methods We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. Results We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. Conclusions Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.
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diabetes linked transcription factor hnf4α regulates metabolism of endogenous methylarginines and β aminoisobutyric acid by controlling expression of alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Dmitri Burdin, Alexey A Kolobov, Anton V Demyanov, Alexey A. Soshnev, Nikolay Samusik, Silke Brilloff, Natalia Jarzebska, Chad Brocker, Jens Martenslobenhoffer, Maren MiethAbstract:Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of AGXT2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to AGXT2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in AGXT2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver AGXT2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of AGXT2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.
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a novel pathway for metabolism of the cardiovascular risk factor homoarginine by alanine glyoxylate aminotransferase 2
Scientific Reports, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Anton V Demyanov, Silke Brilloff, Natalia Jarzebska, James Leiper, Jens Martenslobenhoffer, Elisa Oppici, Anne Kolouschek, Renke MaasAbstract: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.
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Abstract 461: Hepatic Nuclear Factor 4 Alpha as a Regulator of Alanine: Glyoxylate Aminotransferase 2 Expression and Systemic Levels of Endogenous Methylarginines
Arteriosclerosis Thrombosis and Vascular Biology, 2016Co-Authors: Roman N. Rodionov, Dmitri Burdin, Alexey A Kolobov, Anton V Demyanov, Alexey A. Soshnev, Nikolay Samusik, Silke Brilloff, Natalia Jarzebska, Chad Brocker, Jens Martens-lobenhofferAbstract: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.
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role of alanine glyoxylate aminotransferase 2 in metabolism of asymmetric dimethylarginine in the settings of asymmetric dimethylarginine overload and bilateral nephrectomy
Nephrology Dialysis Transplantation, 2014Co-Authors: Roman N. Rodionov, Anja Kittel, Renke Maas, Silke Brilloff, Natalia Jarzebska, Jens Martenslobenhoffer, Bernd Hohenstein, Normund Jabs, Norbert Weiss, Stefanie M BodebogerAbstract:BACKGROUND Asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict complications and mortality in cardiovascular and renal diseases. Alanine:glyoxylate aminotransferase 2 (AGXT2) can metabolize both ADMA and SDMA; however, this metabolic pathway is still poorly understood. The goal of our study was to test the hypothesis that AGXT2 is compensatory upregulated in the settings of ADMA overload and bilateral nephrectomy. METHODS ADMA was infused for 3 days using osmotic minipumps in mice. Half of the mice underwent bilateral nephrectomy 24 h before the end of the infusion. RESULTS Infusion of ADMA caused a 3- to 4-fold increase in plasma and urine ADMA levels and a 2- to 3-fold increase in plasma and urine levels of the ADMA-specific metabolite of AGXT2 α-keto-δ-(N,N-dimethylguanidino)valeric acid (DMGV). Bilateral nephrectomy led to an ∼4-fold increase of plasma SDMA levels, but did not change plasma ADMA levels. Interestingly, plasma levels of DMGV were elevated 32-fold in the mice, which underwent bilateral nephrectomy. Neither bilateral nephrectomy nor ADMA infusion caused upregulation of AGXT2 expression or activity. CONCLUSIONS Our data demonstrate that short-term elevation of systemic levels of ADMA leads to a dramatic increase of DMGV formation without upregulation of AGXT2 expression or activity, which suggests that AGXT2-mediated pathway of ADMA metabolism is not saturated under normal conditions and may play a major role in the maintenance of ADMA homeostasis in the setting of local or systemic elevation of ADMA levels.