Arginine:glycine Amidinotransferase Research Articles

Reduced guanidinoacetate in plasma of patients with autosomal dominant Fanconi syndrome due to heterozygous P341L GATM variant and study of organoids towards treatment

AbstractAutosomal dominant Fanconi syndrome due to a GATM variant (GATM‐FS), causes accumulation of misfolded arginine‐glycine amidinotransferase (AGAT) in proximal renal tubules leading to cellular injury. GATM‐FS presents during childhood and progresses to end‐stage kidney disease (ESKD) in adults. We study creatine metabolism in two individuals of unrelated families with a known GATM variant and the effect of creatine supplementation in kidney organoids. Plasma and urine metabolites were measured by mass spectrometry. Brain creatine was assessed by magnetic resonance spectroscopy (MRS). Guanidinoacetate (GAA) synthesis by the AGAT mutant was measured in patient‐derived immortalized lymphocytes using stable isotopes of arginine and glycine. The effect of creatine on GATM expression was assessed in human kidney cells and organoids. Several family members from two unrelated families were diagnosed with Fanconi syndrome and had the c.1022C>T (p. P341L) variant in GATM. Two affected individuals in both families had moderately reduced plasma GAA levels. In comparison to wild‐type cells, GAA synthesis by patient‐derived GATMP341L+/− lymphoblastoid cell lines (LCL) was reduced, but not absent as in GATM cells from a patient with creatine deficiency syndrome. In vitro studies on human kidney organoids revealed reduced AGAT expression after treatment with creatine. Finally, we showed in one patient that creatine supplementation (5 g daily) substantially increased plasma creatine levels. We report low plasma and urine GAA in patients with autosomal dominant GATM‐FS and show that creatine downregulates AGAT in human kidney cells.

Therapeutic Potential and Prospects of L-arginine in Various Diseases and its Clinical Intervention

Abstract: The goal of this review is to explored the therapeutic application of L-Arginine (L-ARG) against various pathological illnesses, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), cardiovascular disorder, mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS), sickle cell anemia, tumour, epilepsy, erectile dysfunction therapy (ED), gestational hypertension (GH), and menopause issues. L-ARG is an important semi-essential alpha-amino acid that serves as a natural precursor for the synthesis of nitric oxide (NO). It is generally synthesized from proteinogenic amino acid proline through glutamine and glutamate. The degradation of L-ARG is a complex process due to the expression of multiple enzymes in the form of a substrate. The metabolism of L-ARG takes place in various multiple pathways, such as nitric oxide synthase, Arginine glycine amidinotransferase, and Arginine decarboxylase which results in the production of a diverse range of biochemical compounds, such as nitric oxide, polyamines, proline, glutamate, creanine, agmatine homoarginine, and urea. NO is a highly diffusible free radicle with a regulatory function in the heart and acts as an important vasodilator in intact endothelium. NO serves as an important neurotransmitter in the brain and a mediator of host defense in the immune system. L-ARG is also needed for ammonia detoxification, which is a very toxic chemical to the central nervous system. This review article focuses on the relevance of L-ARG in the prevention and treatment of a variety of illnesses.

ClinGen variant curation expert panel recommendations for classification of variants in GAMT, GATM and SLC6A8 for cerebral creatine deficiency syndromes

Cerebral creatine deficiency syndromes (CCDS) are inherited metabolic phenotypes of creatine synthesis and transport. There are two enzyme deficiencies, guanidinoacetate methyltransferase (GAMT), encoded by GAMT and arginine-glycine amidinotransferase (AGAT), encoded by GATM, which are involved in the synthesis of creatine. After synthesis, creatine is taken up by a sodium-dependent membrane bound creatine transporter (CRTR), encoded by SLC6A8, into all organs. Creatine uptake is very important especially in high energy demanding organs such as the brain, and muscle. To classify the pathogenicity of variants in GAMT, GATM, and SLC6A8, we developed the CCDS Variant Curation Expert Panel (VCEP) in 2018, supported by The Clinical Genome Resource (ClinGen), a National Institutes of Health (NIH)-funded resource. We developed disease-specific variant classification guidelines for GAMT-, GATM-, and SLC6A8-related CCDS, adapted from the American College of Medical Genetics/Association of Molecular Pathology (ACMG/AMP) variant interpretation guidelines. We applied specific variant classification guidelines to 30 pilot variants in each of the three genes that have variants associated with CCDS. Our CCDS VCEP was approved by the ClinGen Sequence Variant Interpretation Working Group (SVI WG) and Clinical Domain Oversight Committee in July 2022. We curated 181 variants including 72 variants in GAMT, 45 variants in GATM, and 64 variants in SLC6A8 and submitted these classifications to ClinVar, a public variant database supported by the National Center for Biotechnology Information. Missense variants were the most common variant type in all three genes. We submitted 32 new variants and reclassified 34 variants with conflicting interpretations. We report specific phenotype (PP4) using a points system based on the urine and plasma guanidinoacetate and creatine levels, brain magnetic resonance spectroscopy (MRS) creatine level, and enzyme activity or creatine uptake in fibroblasts ranging from PP4, PP4_Moderate and PP4_Strong. Our CCDS VCEP is one of the first panels applying disease specific variant classification algorithms for an X-linked disease. The availability of these guidelines and classifications can guide molecular genetics and genomic laboratories and health care providers to assess the molecular diagnosis of individuals with a CCDS phenotype.

Beyond protein synthesis: the emerging role of arginine in poultry nutrition and host-microbe interactions.

Arginine is a functional amino acid essential for various physiological processes in poultry. The dietary essentiality of arginine in poultry stems from the absence of the enzyme carbamoyl phosphate synthase-I. The specific requirement for arginine in poultry varies based on several factors, such as age, dietary factors, and physiological status. Additionally, arginine absorption and utilization are also influenced by the presence of antagonists. However, dietary interventions can mitigate the effect of these factors affecting arginine utilization. In poultry, arginine is utilized by four enzymes, namely, inducible nitric oxide synthase arginase, arginine decarboxylase and arginine: glycine amidinotransferase (AGAT). The intermediates and products of arginine metabolism by these enzymes mediate the different physiological functions of arginine in poultry. The most studied function of arginine in humans, as well as poultry, is its role in immune response. Arginine exerts immunomodulatory functions primarily through the metabolites nitric oxide (NO), ornithine, citrulline, and polyamines, which take part in inflammation or the resolution of inflammation. These properties of arginine and arginine metabolites potentiate its use as a nutraceutical to prevent the incidence of enteric diseases in poultry. Furthermore, arginine is utilized by the poultry gut microbiota, the metabolites of which might have important implications for gut microbial composition, immune regulation, metabolism, and overall host health. This comprehensive review provides insights into the multifaceted roles of arginine and arginine metabolites in poultry nutrition and wellbeing, with particular emphasis on the potential of arginine in immune regulation and microbial homeostasis in poultry.

Evidence of an intracellular creatine-sensing mechanism that modulates creatine biosynthesis via AGAT expression in human HAP1 cells

Cellular homeostasis of creatine (CT), integral part of the energy buffering and transducing system connecting intracellular sites of ATP production and utilization, comprises of mechanisms that increase CT, i.e., biosynthesis and cellular uptake, and CT-lowering processes, such as export and non-enzymatic conversion to creatinine. The biosynthesis of CT is controlled by negative feedback loop via suppression of the rate-limiting enzyme arginine:glycine amidinotransferase (AGAT). Although the regulatory mechanism involved is not well understood, AGAT suppression is successfully used in patients with guanidinoacetate methyltransferase (GAMT) deficiency to reduce the neurotoxic accumulation of the AGAT-mediated guanidinoacetate production by supplementing patients with CT. Utilizing the CT-dependent feedback loop for the upregulation of AGAT expression may well represent a therapeutic target for an additional CT deficiency syndrome, the CT transporter (CrT) defect, for which no effective treatment option is available so far. We have used CRISPR to tag the C-terminus of AGAT with a nanoluc luciferase (NLuc) reporter in HAP1 cells. A biphasic decay of AGAT-NLuc in response to increasing extracellular CT was observed, whereas the decrease in AGAT-NLuc expression was directly proportional to the rise in intracellular CT levels with an approximate IC50 of 1–2 mM. CRISPR generated HAP1 CrT null cells and HAP1 CrT null cells stably expressing a CrT-GFP fusion protein further demonstrated that the biphasic response to extracellular CT is mediated by a high-affinity (Km 9–10 µM) CrT dependent, saturable mechanism and a CrT independent, unsaturable uptake process. The direct response to intracellular CT suggests the existence of an intracellular CT sensing system enabling a dynamic cell response to changing CT concentration that is relevant for cellular CT homeostasis.

Aspects of human uterine creatine metabolism during the menstrual cycle and at term pregnancy†.

Creatine metabolism likely contributes to energy homeostasis in the human uterus, but whether this organ synthesizes creatine and whether creatine metabolism is adjusted throughout the menstrual cycle and with pregnancy are largely unknown. This study determined endometrial protein expression of creatine-synthesizing enzymes arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), creatine kinase (CKBB), and the creatine transporter (SLC6A8) throughout the menstrual cycle in fertile and primary infertile women. It also characterized creatine metabolism at term pregnancy, measuring aspects of creatine metabolism in myometrial and decidual tissue. In endometrial samples, AGAT, GAMT, SLC6A8, and CKBB were expressed in glandular and luminal epithelial cells. Except for SLC6A8, the other proteins were also located in stromal cells. Irrespective of fertility, AGAT, GAMT, and SLC6A8 high-intensity immunohistochemical staining was greatest in the early secretory phase of the menstrual cycle. During the proliferative phase, staining for SLC6A8 protein was greater (P = 0.01) in the primary infertile compared with the fertile group. Both layers of the term pregnant uterus contained creatine, phosphocreatine, guanidinoacetic acid, arginine, glycine, and methionine; detectable gene and protein expression of AGAT, GAMT, CKBB, and ubiquitous mitochondrial CK (uMt-CK); and gene expression of SLC6A8. The proteins AGAT, GAMT, CKBB, and SLC6A8 were uniformly distributed in the myometrium and localized to the decidual glands. In conclusion, endometrial tissue has the capacity to produce creatine and its capacity is highest around the time of fertilization and implantation. Both layers of the term pregnant uterus also contained all the enzymatic machinery and substrates of creatine metabolism.

The effects of a comparatively higher dose of 1000 mg/kg/d of oral L- or D-arginine on the L-arginine metabolic pathways in male Sprague-Dawley rats.

Oral L-arginine supplements are popular mainly for their nitric oxide mediated vasodilation, but their physiological impact is not fully known. L-arginine is a substrate of several enzymes including arginase, nitric oxide synthase, arginine decarboxylase, and arginine: glycine amidinotransferase (AGAT). We have published a study on the physiological impact of oral L- and D-arginine at 500 mg/kg/day for 4 wks in male Sprague-Dawley rats. We investigated the effects of oral L-arginine and D-arginine at a higher dose of 1000 mg/kg/d for a longer treatment duration of 16 wks in 9-week-old male Sprague-Dawley rats. We measured the expression and activity of L-arginine metabolizing enzymes, and levels of their metabolites in the plasma and various organs. L-arginine did not affect the levels of L-arginine and L-lysine in the plasma and various organs. L-arginine decreased arginase protein expression in the upper small intestine, and arginase activity in the plasma. It also decreased AGAT protein expression in the liver, and creatinine levels in the urine. L-arginine altered arginine decarboxylase protein expression in the upper small intestine and liver, with increased total polyamines plasma levels. Endothelial nitric oxide synthase protein was increased with D-arginine, the presumed metabolically inert isomer, but not L-arginine. In conclusion, oral L-arginine and D-arginine at a higher dose and longer treatment duration significantly altered various enzymes and metabolites in the arginine metabolic pathways, which differed from alterations produced by a lower dose shorter duration treatment published earlier. Further studies with differing doses and duration would allow for a better understanding of oral L-arginine uses, and evidence based safe and effective dose range and duration.

Guanidinoacetate (GAA) is a potent GABAA receptor GABA mimetic: Implications for neurological disease pathology.

Impairment of excretion and enzymatic processing of nitrogen, for example, because of liver or kidney failure, or with urea cycle and creatine synthesis enzyme defects, surprisingly leads to primarily neurologic symptoms, yet the exact mechanisms remain largely mysterious. In guanidinoacetate N-methyltransferase (GAMT) deficiency, the guanidino compound guanidinoacetate (GAA) increases dramatically, including in the cerebrospinal fluid (CSF), and has been implicated in mediating the neurological symptoms in GAMT-deficient patients. GAA is synthesized by arginine-glycine amidinotransferase (AGAT), a promiscuous enzyme that not only transfers the amidino group from arginine to glycine, but also to primary amines in, for example, GABA and taurine to generate γ-guanidinobutyric acid (γ-GBA) and guanidinoethanesulfonic acid (GES), respectively. We show that GAA, γ-GBA, and GES share structural similarities with GABA, evoke GABAA receptor (GABAA R) mediated currents (whereas creatine [methylated GAA] and arginine failed to evoke discernible currents) in cerebellar granule cells in mouse brain slices and displace the high-affinity GABA-site radioligand [3 H]muscimol in total brain homogenate GABAA Rs. While γ-GBA and GES are GABA agonists and displace [3 H]muscimol (EC50 /IC50 between 10 and 40 μM), GAA stands out as particularly potent in both activating GABAA Rs (EC50 ~6μM) and also displacing the GABAA R ligand [3 H]muscimol (IC50 ~3μM) at pathophysiologically relevant concentrations. These findings stress the role of substantially elevated GAA as a primary neurotoxic agent in GAMT deficiency and we discuss the potential role of GAA in arginase (and creatine transporter) deficiency which show a much more modest increase in GAA concentrations yet share the unique hyperexcitability neuropathology with GAMT deficiency. We conclude that orthosteric activation of GABAA Rs by GAA, and potentially other GABAA R mimetic guanidino compounds (GCs) like γ-GBA and GES, interferes with normal inhibitory GABAergic neurotransmission which could mediate, and contribute to, neurotoxicity.

Determination of equilibria constants of arginine:glycine amidinotransferase (AGAT)-catalyzed reactions using concentrations of circulating amino acids

Arginine:glycine amidinotransferase (AGAT) catalyzes mainly two reactions that generate 1) L-homoarginine (hArg) from L-arginine and L-lysine (Kharg) and 2) guanidinoacetate (GAA) and L-ornithine from L-arginine and glycine (Kgaa). Previously, we found that pharmacological treatment of Becker muscular dystrophy (BMD) patients with metformin or L-citrulline resulted in antidromic effects on serum hArg and GAA concentrations, seemingly acting as an inhibitor and effector of AGAT activity, respectively. Here, we used data of this study as a model to determine Kharg and Kgaa values by using the concentrations of the participating amino acids measured in serum samples of the BMD patients. The study aimed to prove the general utility of this approach to investigate effects of amino acids and drugs on AGAT-catalyzed reactions in vivo in humans.

Lysine and homoarginine are closely interrelated metabolites in the rat

L-Lysine (Lys) and L-arginine (Arg), but not L-homoarginine (hArg), are proteinogenic amino acids. In healthy humans, oral administration of hArg increased the plasma concentration of Lys, suggesting Lys as a metabolite of hArg. In humans and animals, hArg is biosynthesized from Arg and Lys by arginine:glycine amidinotransferase (AGAT). In vitro, recombinant human arginase and bovine liver arginase I hydrolyzed hArg to Lys, suggesting Lys as a metabolite of hArg. The aim of the present study was to investigate whether changes in blood concentrations of hArg and Lys in old rats fed for 4 months with varied controlled experimental diets could suggest interconversion of these amino acids. Blood samples (n = 253) were taken before (T0) and after 2 months (T2) and 4 months (T4) of the experiment. Plasma concentrations of Lys and hArg were determined by gas chromatography–mass spectrometry. The plasma hArg concentration markedly correlated with the plasma Lys concentration at all timepoints (r ≥ 0.7, P < 0.0001). Further analysis demonstrated that hArg and Lys are closely and specifically associated independently of experimental time/rat age and diet, suggesting that hArg and Lys are mutual metabolites in old rats. Based on the plasma concentration changes, the median yield of hArg from Lys was determined to be 0.17% at T0 and each 0.27% at T2 and T4. With a circulating concentration of about 3 µM, hArg a major metabolite of Lys in healthy humans. hArg supplementation is currently investigated as a cardioprotective means to improve impaired hArg synthesis. Present knowledge suggests that Lys rather than hArg supplementation may be even more favorable.

Laboratory Diagnosis of Cerebral Creatine Deficiency Syndromes by Determining Creatine and Guanidinoacetate in Plasma and Urine.

Cerebral creatine deficiency syndromes are caused by the dysfunctional creatine biosynthesis or transport and comprise three hereditary neurodevelopmental defects including arginine-glycine amidinotransferase (AGAT), guanidinoacetate methyltransferase (GAMT), and creatine transporter deficiencies. All conditions are characterized by seizures, intellectual disability, and behavioral abnormalities. Laboratory diagnosis of these disorders relies on the determination of creatine and guanidinoacetate concentrations in both plasma and urine. Here we describe a rapid quantitative UPLC/MS/MS method for the simultaneous determination of these analytes using a normal-phase HILIC column after analyte derivatization. The approach is suitable for neonatal screening follow-ups and monitoring of the treatment for creatine deficiency syndromes.

510 Late-Breaking: Dietary Glycine Supplementation During Growing and Finishing Phases Increases Tissue Concentrations of Total Creatine and Gene Expression of Creatine-synthetic Enzymes in Low-birthweight Pigs

Abstract Dietary glycine is required for maximum growth and development in animals by stimulating muscle protein synthesis and as a component of creatine. Creatine is synthesized from glycine, arginine, and S-adenosylmethionine by arginine:glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT). Sufficient creatine synthesis for growth requires adequate substrate supply. However, swine diets are deficient in glycine. Additionally, intrauterine growth restricted (IUGR) pigs have reduced glycine synthesis. This results in decreased creatine synthesis and lower total creatine content in tissues, leading to reduced cellular energy metabolism and diminished muscle protein accretion. This study was designed to test the hypothesis that dietary glycine supplementation in corn-and-soybean-meal-based diets would improve overall growth and skeletal muscle accretion in post-weaning IUGR pigs by increasing the expression of creatine-synthetic enzymes and tissue concentrations of total creatine. Fourteen IUGR pigs (birthweight = 0.98±0.03 kg, mean±SEM) and 20 normal birthweight pigs (birthweight = 1.44±0.02 kg, mean±SEM) were obtained at weaning for this study. Pigs from each birthweight group were randomly assigned to 1% glycine + 0.19% corn starch treatment group or 1.19% alanine group (isonitrogenous control) for the study (21 d to 188 d of age); tissues were collected at d 188. Data were analyzed by using 2-way ANOVA and the Duncan multiple comparison test. Glycine-supplemented IUGR pigs had greater tissue concentrations of creatine, creatinine, and creatine phosphate than control IUGR in all tissues measured (P&amp;lt; 0.05). Control IUGR pigs showed diminished activity and mRNA expression of creatine-synthetic enzymes (P &amp;lt; 0.05); this was mitigated by glycine supplementation as glycine supplemented IUGR pigs showed normal levels of enzyme activity and mRNA expression. Overall, results of this study indicate dietary glycine supplementation to IUGR pigs between weaning and market weight effectively restores creatine-synthetic enzyme activities and increase tissue concentrations of total creatine, leading to increased lean tissue growth. (Supported by USDA-NIFA)

Creatine supplementation and muscles: From metabolism to medical practice

Creatine has become the most popular dietary supplement in sport and exercise physiology. In humans creatine is synthesized by the kidneys, pancreas and liver and transported mainly into brain, skeletal and cardiac muscle. Phosphocreatine is a high-energy content molecule, essential for the ADP to ATP conversion during intensive physical activity. Creatine and phosphocreatine are crucial in the energy shuttle system of high-energy phosphates between the mitochondrial ATP production and the cytosolic ATP consumption. Creatine supplementation increases lean body mass acting on myogenic regulatory factors. During muscular recovery, creatine supplementation regulates the regeneration process by reduction of muscle damage-induced inflammation and oxidative stress, activation and proliferation of satellite cells and regulation of calcium transport in muscle. The effects of creatine supplementation on muscle physiology are beneficial in anaerobic/aerobic exercises. In several muscle disorders (muscular dystrophies, in idiopathic inflammatory myopathies) creatine improved functional performance, but apparently not in metabolic myopathies; in McArdle diseases it may even have paradoxical effects. More research is warranted to better understand the short and long-term effects and safety of creatine supplementation among adolescents or elderly, as well as in different types of muscle diseases; for the two enzymatic genetic defects of creatine biosynthesis – arginine: glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT), respectively – normal neurodevelopment has been achieved in early initiation of creatine therapy.

Value of Magnetic Resonance Spectroscopy for Diagnosis of Creatine Deficiency Syndrome

AbstractCreatine deficiency syndromes are congenital metabolic diseases characterized by decreased cerebral creatine levels as a result of disorders in creatine synthesis and transport. Therefore, magnetic resonance spectroscopy is a valuable tool for diagnosis. This disease can be explained by congenital disorders occurring in three forms at different stages of the creatine metabolic pathway. Two of disorders arise autosomal recessively in creatine biosynthesis, arginine-glycine amidinotransferase, and guanidinoacetate methyltransferase enzyme deficiency. The third disorder occurs as a result of an SLC6A8 variant in the form of creatine carrier protein deficiency. In this article, a patient with SLC6A8 carrier deficiency is presented.

Intrathecal and systemic alterations of L-arginine metabolism in patients after intracerebral hemorrhage

Alterations in the concentration of nitric oxide (NO) and L-arginine metabolites have been associated with the pathophysiology of different vascular diseases. Here, we describe striking changes in L-arginine metabolism after hemorrhagic stroke. Blood and cerebrospinal fluid (CSF) samples of patients with intracerebral hemorrhage (ICH) and/or intraventricular hemorrhage were collected over a ten-day period. Liquid chromatography-tandem mass spectrometry was used to quantify key substrates and products of L-arginine metabolizing enzymes as well as asymmetric (ADMA) and symmetric dimethylarginine (SDMA). Changes in the plasma were limited to early reductions in L-ornithine, L-lysine, and L-citrulline concentrations. Intrathecally, we observed signs of early NO synthase (NOS) upregulation followed by a decrease back to baseline accompanied by a rise in the level of its endogenous NOS-inhibitor ADMA. SDMA demonstrated increased levels throughout the observation period. For arginase, a pattern of persistently elevated activity was measured and arginine:glycine amidinotransferase (AGAT) appeared to be reduced in its activity at later time points. An early reduction in CSF L-arginine concentration was an independent risk factor for poor outcome. Together, these findings further elucidate pathophysiological mechanisms after ICH potentially involved in secondary brain injury and may reveal novel therapeutic targets.

Altered calcium handling in cardiomyocytes from arginine-glycine amidinotransferase-knockout mice is rescued by creatine.

The creatine kinase system facilitates energy transfer between mitochondria and the major ATPases in the heart. Creatine-deficient mice, which lack arginine-glycine amidinotransferase (AGAT) to synthesize creatine and homoarginine, exhibit reduced cardiac contractility. We studied how the absence of a functional CK system influences calcium handling in isolated cardiomyocytes from AGAT-knockouts and wild-type littermates as well as in AGAT-knockout mice receiving lifelong creatine supplementation via the food. Using a combination of whole cell patch clamp and fluorescence microscopy, we demonstrate that the L-type calcium channel (LTCC) current amplitude and voltage range of activation were significantly lower in AGAT-knockout compared with wild-type littermates. Additionally, the inactivation of LTCC and the calcium transient decay were significantly slower. According to our modeling results, these changes can be reproduced by reducing three parameters in knockout mice when compared with wild-type: LTCC conductance, the exchange constant of Ca2+ transfer between subspace and cytosol, and SERCA activity. Because tissue expression of LTCC and SERCA protein were not significantly different between genotypes, this suggests the involvement of posttranslational regulatory mechanisms or structural reorganization. The AGAT-knockout phenotype of calcium handling was fully reversed by dietary creatine supplementation throughout life. Our results indicate reduced calcium cycling in cardiomyocytes from AGAT-knockouts and suggest that the creatine kinase system is important for the development of calcium handling in the heart.NEW & NOTEWORTHY Creatine-deficient mice lacking arginine-glycine amidinotransferase exhibit compromised cardiac function. Here, we show that this is at least partially due to an overall slowing of calcium dynamics. Calcium influx into the cytosol via the L-type calcium current (LTCC) is diminished, and the rate of the sarcoendoplasmic reticulum calcium ATPase (SERCA) pumping calcium back into the sarcoplasmic reticulum is slower. The expression of LTCC and SERCA did not change, suggesting that the changes are regulatory.

P.139 Chemical models of dysfunctions of the brain 2-oxo acid dehydrogenases in vivo

l-Homoarginine (hArg) is biosynthesized from l-arginine (Arg) and l-lysine (Lys) by arginine:glycine amidinotransferase (AGAT). AGAT also catalyzes the formation of guanidinoacetate (GAA) from Arg and glycine (Gly). GAA is converted to creatine (N-methyl guanidinoacetate) by guanidinoacetate N-methyl-transferase (GAMT). Low circulating and excretory concentrations of hArg are associated with worse cardiovascular outcome and mortality. hArg is a poor substrate of nitric oxide synthase (NOS) and a weak inhibitor of arginase. The metabolism of hArg in humans is little investigated. Previously, we found that orally administered hArg (125 mg/day) increased the plasma concentration of hArg, but not of Arg, the substrate of NOS, in healthy subjects. We newly analyzed the plasma samples collected in that study for Lys and other amino acids. Repeated measures ANOVA revealed statistically significant differences between the groups (P = 0.008) with respect to plasma Lys concentration which increased by about 8% after a 4-week hArg supplementation. In vitro, recombinant human arginase and bovine liver arginase I were demonstrated by a specific and sensitive stable-isotope GC-MS assay to hydrolyze hArg to Lys. Our results suggest that Lys is a metabolite of hArg produced by the hydrolytic activity of arginase. Arginase may play a key role in hArg homeostasis in humans.

Reconstitution of the Ornithine Cycle with Arginine:Glycine Amidinotransferase to Engineer Escherichia coli into an Efficient Whole-Cell Catalyst of Guanidinoacetate.

Guanidino compounds can be synthesized by transamidination reactions using arginine as a guanidine group donor. The efficiency of guanidino biosynthesis is often affected by the supply of arginine and the inhibition of the coproduct ornithine. To alleviate this shortcoming, we designed a reconstituted ornithine cycle in Escherichia coli to engineer an efficient whole-cell catalyst for guanidinoacetate (GAA) production by introducing a heterogeneous arginine:glycine amidinotransferase (AGAT). To alleviate the inhibition of ornithine, a citrulline synthetic module was constructed and optimized by introducing a glutamine self-sufficient system. Then, to improve the pathway from citrulline to arginine, an aspartate self-sufficient system was introduced into the arginine synthetic module. By combining these modules (GAA, citrulline, and arginine synthetic modules), a reconstituted ornithine cycle was developed, which significantly improved the biocatalyst efficiency (3.9-fold increase). In the system, arginine was regenerated efficiently through the reconstituted ornithine cycle, which converted arginine from a substrate to a cofactor for the transamidination reaction, thereby relieving the ornithine inhibition. Moreover, the amidino group of GAA in this system was mainly supplied by carbon and nitrogen assimilation. After the engineering process, 8.61 g/L GAA (73.56 mM) with a productivity of 0.39 g/L/h was achieved in a 22 h bioconversion. To the best of our knowledge, this is the first time that GAA has been produced in E. coli. This reconstructed ornithine cycle could be used as a transamidination platform for amidino group supply and has potential applications in the biosynthesis of other guanidino compounds.

From broiler breeder hen feed to the egg and embryo: The molecular effects of guanidinoacetate supplementation on creatine transport and synthesis

Supplementation of broiler breeder hens with beneficial additives bears great potential for affecting nutrient deposition into the fertile egg. Guanidinoacetate (GAA) is the endogenous precursor of creatine that is used as a feed additive for improving cellular energy metabolism in animal nutrition. In the present study, we have investigated whether GAA supplementation in broiler breeder feed affects creatine deposition into the hatching egg and molecular mechanisms of creatine transport and synthesis within hens and their progeny. For this, broiler breeder hens of 47 wk of age were supplemented with 0.15% GAA for 15 wk, and samples from their tissues, hatching eggs and progeny were compared with those of control, nonsupplemented hens. A significant increase in creatine content was found within the yolk and albumen of hatching eggs obtained from the GAA group, compared with the control group. The GAA group exhibited a significant increased creatine transporter gene expression compared with the control group in their small intestines and oviduct. In GAA group progeny, a significant decrease in creatine transporter expression at embryonic day 19 and day of hatch was found, compared with control group progeny. At the day of hatch, creatine synthesis genes (arginine glycine amidinotransferase and guanidinoacetate N-methyltransferase) exhibited significant decrease in expression in the GAA group progeny compared with control group progeny. These results indicate that GAA supplementation in broiler breeder feed increases its absorbance and deposition into hatching eggs, subsequently affecting GAA and creatine absorbance and synthesis within broiler progeny.

Creatine is a Conditionally Essential Nutrient in Chronic Kidney Disease: A Hypothesis and Narrative Literature Review.

To accommodate the loss of the plethora of functions of the kidneys, patients with chronic kidney disease require many dietary adjustments, including restrictions on the intake of protein, phosphorus, sodium and potassium. Plant-based foods are increasingly recommended as these foods contain smaller amounts of saturated fatty acids, protein and absorbable phosphorus than meat, generate less acid and are rich in fibers, polyunsaturated fatty acids, magnesium and potassium. Unfortunately, these dietary recommendations cannot prevent the occurrence of many symptoms, which typically include fatigue, impaired cognition, myalgia, muscle weakness, and muscle wasting. One threat coming with the recommendation of low-protein diets in patients with non-dialysis-dependent chronic kidney disease (CKD) and with high-protein diets in patients with dialysis-dependent CKD, particularly with current recommendations towards proteins coming from plant-based sources, is that of creatine deficiency. Creatine is an essential contributor in cellular energy homeostasis, yet on a daily basis 1.6–1.7% of the total creatine pool is degraded. As the average omnivorous diet cannot fully compensate for these losses, the endogenous synthesis of creatine is required for continuous replenishment. Endogenous creatine synthesis involves two enzymatic steps, of which the first step is a metabolic function of the kidney facilitated by the enzyme arginine:glycine amidinotransferase (AGAT). Recent findings strongly suggest that the capacity of renal AGAT, and thus endogenous creatine production, progressively decreases with the increasing degree of CKD, to become absent or virtually absent in dialysis patients. We hypothesize that with increasing degree of CKD, creatine coming from meat and dairy in food increasingly becomes an essential nutrient. This phenomenon will likely be present in patients with CKD stages 3, 4 and 5, but will likely be most pronouncedly present in patients with dialysis-dependent CKD, because of the combination of lowest endogenous production of creatine and unopposed losses of creatine into the dialysate. It is likely that these increased demands for dietary creatine are not sufficiently met. The result of which, may be a creatine deficiency with important contributions to the sarcopenia, fatigue, impaired quality of life, impaired cognition, and premature mortality seen in CKD.