Systemic Effects of Homoarginine Supplementation on Arginine Metabolizing Enzymes in Rats with Heart Failure with Preserved Ejection Fraction.

A restoration of low homoarginine (hArg) levels in obese ZSF1 rats (O-ZSF1) before (S1-ZSF1) and after (S2-ZSF1) the manifestation of heart failure with preserved ejection fraction (HFpEF) did not affect the worsening of cardiac HFpEF characteristics. Here, potential regulation of key enzymes of arginine metabolism in other organs was analyzed. Arginase 2 (ARG2) was reduced >35% in the kidney and small intestine of hArg-supplemented rats compared to O-ZSF1. Glycine amidinotransferase (GATM) was 29% upregulated in the kidneys of S1-ZSF1. Dimethylarginine dimethylaminohydrolase 1 (DDAH1) levels were reduced >50% in the livers of O-ZSF1 but restored in S2-ZSF1 compared to healthy rats (L-ZSF1). In the skeletal muscle, iNOS was lower in O-ZSF1 and further decreased in S1-ZSF1 and S2-ZSF1 compared to L-ZSF1. iNOS levels were lower in the liver of the S2-ZSF1 group but higher in the kidneys of S1-ZSF1 compared to L-ZSF1. Supplementation with hArg in an in vivo HFpEF model resulted in the inhibition of renal ARG2 and an increase in GATM expression. This supplementation might contribute to the stabilization of intestinal iNOS and ARG2 imbalances, thereby enhancing barrier function. Additionally, it may offer protective effects in skeletal muscle by downregulating iNOS. In the conceptualization of hArg supplementation studies, the current disease progression stage as well as organ-specific enzyme regulation should be considered.

Homoarginine and creatine deficiency do not exacerbate murine ischaemic heart failure.

AIMS: Low levels of homoarginine and creatine are associated with heart failure severity in humans, but it is unclear to what extent they contribute to pathophysiology. Both are synthesized via L-arginine:glycine amidinotransferase (AGAT), such that AGAT-/- mice have a combined creatine and homoarginine deficiency. We hypothesized that this would be detrimental in the setting of chronic heart failure. METHODS AND RESULTS: Study 1: homoarginine deficiency-female AGAT-/- and wild-type mice were given creatine-supplemented diet so that both had normal myocardial creatine levels, but only AGAT-/- had low plasma homoarginine. Myocardial infarction (MI) was surgically induced and left ventricular (LV) structure and function assessed at 6-7 weeks by in vivo imaging and haemodynamics. Study 2: homoarginine and creatine-deficiency-as before, but AGAT-/- mice were given creatine-supplemented diet until 1 week post-MI, when 50% were changed to a creatine-free diet. Both groups therefore had low homoarginine levels, but one group also developed lower myocardial creatine levels. In both studies, all groups had LV remodelling and dysfunction commensurate with the development of chronic heart failure, for example, LV dilatation and mean ejection fraction <20%. However, neither homoarginine deficiency alone or in combination with creatine deficiency had a significant effect on mortality, LV remodelling, or on any indices of contractile and lusitropic function. CONCLUSIONS: Low levels of homoarginine and creatine do not worsen chronic heart failure arguing against a major causative role in disease progression. This suggests that it is unnecessary to correct hArg deficiency in patients with heart failure, although supra-physiological levels may still be beneficial.

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.

Homoarginine in the cardiovascular system: Pathophysiology and recent developments.

Upcoming experimental and epidemiological data have identified the endogenous non-proteinogenic amino acid L-homoarginine (L-hArg) not only as a novel biomarker for cardiovascular disease but also as being directly involved in the pathogenesis of cardiac dysfunction. The association of low L-hArg levels with adverse cardiovascular events and mortality has proposed the idea of nutritional supplementation to rescue pathways inversely associated with cardiovascular health. Subsequent clinical and experimental studies contributed significantly to our knowledge of potential effects on the cardiorenal axis, acting either as a biomarker or a cardiovascular active agent. In this review article, we provide a comprehensive summary of the L-hArg metabolism, pathophysiological aspects, and current developments in the field of experimental and clinical evidence in favor of protective cardiovascular effects. Establishing a reliable biomarker to identify patients at high risk to die of cardiovascular disease represents one of the main goals for tackling this disease and providing individual therapeutic guidance.

The Amino Acid Homoarginine Inhibits Atherogenesis by Modulating T-Cell Function.

BACKGROUND: Amino acid metabolism is crucial for inflammatory processes during atherogenesis. The endogenous amino acid homoarginine is a robust biomarker for cardiovascular outcome and mortality with high levels being protective. However, the underlying mechanisms remain elusive. We investigated the effect of homoarginine supplementation on atherosclerotic plaque development with a particular focus on inflammation. METHODS: Female ApoE-deficient mice were supplemented with homoarginine (14 mg/L) in drinking water starting 2 weeks before and continuing throughout a 6-week period of Western-type diet feeding. Control mice received normal drinking water. Immunohistochemistry and flow cytometry were used for plaque- and immunological phenotyping. T cells were characterized using mass spectrometry-based proteomics, by functional in vitro approaches, for example, proliferation and migration/chemotaxis assays as well as by super-resolution microscopy. RESULTS: Homoarginine supplementation led to a 2-fold increase in circulating homoarginine concentrations. Homoarginine-treated mice exhibited reduced atherosclerosis in the aortic root and brachiocephalic trunk. A substantial decrease in CD3+ T cells in the atherosclerotic lesions suggested a T-cell-related effect of homoarginine supplementation, which was mainly attributed to CD4+ T cells. Macrophages, dendritic cells, and B cells were not affected. CD4+ T-cell proteomics and subsequent pathway analysis together with in vitro studies demonstrated that homoarginine profoundly modulated the spatial organization of the T-cell actin cytoskeleton and increased filopodia formation via inhibition of Myh9 (myosin heavy chain 9). Further mechanistic studies revealed an inhibition of T-cell proliferation as well as a striking impairment of the migratory capacities of T cells in response to relevant chemokines by homoarginine, all of which likely contribute to its atheroprotective effects. CONCLUSIONS: Our study unravels a novel mechanism by which the amino acid homoarginine reduces atherosclerosis, establishing that homoarginine modulates the T-cell cytoskeleton and thereby mitigates T-cell functions important during atherogenesis. These findings provide a molecular explanation for the beneficial effects of homoarginine in atherosclerotic cardiovascular disease.

Short-Term Supplementation of Sodium Nitrate vs. Sodium Chloride Increases Homoarginine Synthesis in Young Men Independent of Exercise.

The aim of the study was to investigate the effects of short-term oral administration of inorganic nitrate (NaNO3; n = 8) or placebo (NaCl; n = 9) (each 0.1 mmol/kg body weight/d for 9 days) on plasma amino acids, creatinine, and oxidative stress in healthy young men. At baseline, the plasma concentrations of amino acids did not differ between the groups. At the end of the study, the plasma concentrations of homoarginine (hArg; by 24%, p = 0.0001), citrulline and ornithine (Cit/Orn; by 16%, p = 0.015), and glutamine/glutamate (Gln/Glu; by 6%, p = 0.0003) were higher in the NaNO3 group compared to the NaCl group. The plasma concentrations of sarcosine (Sarc; by 28%, p < 0.0001), tyrosine (by 14%, p = 0.0051), phenylalanine (by 8%, p = 0.0026), and tryptophan (by 8%, p = 0.0047) were lower in the NaNO3 group compared to the NaCl group. These results suggest that nitrate administration affects amino-acid metabolism. The arginine/glycine amidinotransferase (AGAT) catalyzes two reactions: (1) the formation of l-homoarginine (hArg) and l-ornithine (Orn) from l-arginine (Arg) and l-lysine (Lys): Arg + Lys <−> hArg + Orn, with equilibrium constant Kharg; (2) the formation of guanidinoacetate (GAA) and Orn from Arg and glycine (Gly): Arg + Gly <−> GAA + Orn, with equilibrium constant Kgaa. The plasma Kgaa/KhArg ratio was lower in the NaNO3 group compared to the NaCl group (1.57 vs. 2.02, p = 0.0034). Our study suggests that supplementation of inorganic nitrate increases the AGAT-catalyzed synthesis of hArg and decreases the N-methyltransferase-catalyzed synthesis of GAA, the precursor of creatine. To our knowledge, this is the first study to demonstrate elevation of hArg synthesis by inorganic nitrate supplementation. Remarkably, an increase of 24% corresponds to the synthesis capacity of one kidney in healthy humans. Differences in the association between plasma concentrations of amino acids in the NaNO3 and NaCl groups suggest changes in amino-acid homeostasis. Plasma concentrations of the oxidative stress marker malondialdehyde (MDA) did not change after supplementation of NaNO3 or NaCl over the whole exercise time range. Plasma nitrite concentration turned out to be a more discriminant marker of NaNO3 ingestion than plasma nitrate (area under the receiver operating characteristic curve: 0.951 vs. 0.866, p < 0.0001 each).

Reference Interval for Serum L-Homoarginine Determined with Enzyme-Linked Immunosorbent Assay in the Population-Based Study of Health in Pomerania.

BACKGROUND: Low levels of the endogenous amino acid L-homoarginine are a risk factor for cardiovascular morbidity and mortality. For individual risk prediction, commercially available test systems are mandatory. This study aims at formulating sex- and age-specific reference intervals of serum L-homoarginine determined with an ELISA. METHODS: We determined reference intervals for serum L-homoarginine stratified by age and sex in a sample of 1285 healthy participants of the Study of Health in Pomerania (SHIP)-TREND cohort after exclusion of participants with cardiovascular diseases, diabetes mellitus, hypertension, metabolic syndrome, elevated liver enzymes, chronic kidney disease stages III or IV, or body mass index >25 kg/m2. Serum L-homoarginine was determined applying a commercially available ELISA. RESULTS: The reference cohort included 836 women (median age 41, 25th and 75th percentiles are 32 and 50 years) and 449 men (median age 38, 25th, and 75th percentiles are 30 and 49 years). The median serum concentration of L-homoarginine was 1.93 (25th 1.49; 75th 2.60) µmol/L in women and 2.02 (25th 1.63; 75th 2.61) µmol/L in men (P = 0.04). The reference intervals (2.5th to 97.5th percentile) were 0.89-5.29 µmol/L for women and 1.09-3.76 µmol/L for men. The L-homoarginine serum concentration declined over age decades in both sexes and a notable interaction with sex hormone intake in women was observed. CONCLUSIONS: The novelty of our study is that we determined reference intervals specific for the L-isomer being lower than those previously reported for homoarginine in SHIP and thus might be helpful in identifying individuals suitable for oral L-homoarginine supplementation.

Influence of homoarginine on creatine accumulation and biosynthesis in the mouse.

Organisms obtain creatine from their diet or by de novo synthesis via AGAT (L-arginine:glycine amidinotransferase) and GAMT (Guanidinoacetate N-methyltrasferase) in kidney and liver, respectively. AGAT also synthesizes homoarginine (hArg), low levels of which predict poor outcomes in human cardiovascular disease, while supplementation maintains contractility in murine heart failure. However, the expression pattern of AGAT has not been systematically studied in mouse tissues and nothing is known about potential feedback interactions between creatine and hArg. Herein, we show that C57BL/6J mice express AGAT and GAMT in kidney and liver respectively, whereas pancreas was the only organ to express appreciable levels of both enzymes, but no detectable transmembrane creatine transporter (Slc6A8). In contrast, kidney, left ventricle (LV), skeletal muscle and brown adipose tissue must rely on creatine transporter for uptake, since biosynthetic enzymes are not expressed. The effects of creatine and hArg supplementation were then tested in wild-type and AGAT knockout mice. Homoarginine did not alter creatine accumulation in plasma, LV or kidney, whereas in pancreas from AGAT KO, the addition of hArg resulted in higher levels of tissue creatine than creatine-supplementation alone (P < 0.05). AGAT protein expression in kidney was downregulated by creatine supplementation (P < 0.05), consistent with previous reports of end-product repression. For the first time, we show that hArg supplementation causes a similar down-regulation of AGAT protein (P < 0.05). These effects on AGAT were absent in the pancreas, suggesting organ specific mechanisms of regulation. These findings highlight the potential for interactions between creatine and hArg that may have implications for the use of dietary supplements and other therapeutic interventions.

Effects of homoarginine supplementation on heart and skeletal muscle of rats with heart failure with preserved ejection fraction.

AIM: Heart failure with preserved ejection fraction (HFpEF) is associated with left ventricular stiffness, impaired diastolic relaxation, and severe exercise intolerance. Decreased homoarginine (hArg) levels are an independent predictor of mortality in cardiovascular disease and correlate with impaired exercise performance. We recently reported alterations in arginine, hArg, and related amino acids in obese ZSF1 rats (O-ZSF1), with a HFpEF phenotype. Although low hArg is associated with diastolic dysfunction in humans, potential effects of hArg supplementation were not tested yet. METHODS AND RESULTS: At an age of 6 weeks, 12 O-ZSF1 were randomized into two groups: (1) O-ZSF1 rats supplemented with hArg in their drinking water (sO-ZSF1) or (2) O-ZSF1 rats receiving no hArg supplementation (O-ZSF1). At an age of 32 weeks, effects of primary prevention by hArg supplementation on echocardiographic, histological, and functional parameters of heart and skeletal muscle were determined. Lean ZSF1 rats (L-ZSF1) served as controls. hArg supplementation did not prevent impairment of diastolic relaxation (E/e': O-ZSF1 21 ± 3 vs. sO-ZSF1 22 ± 3, P = 0.954, L-ZSF1 18 ± 5) but resulted in more cardiac fibrosis (histological collagen staining: +57% in sO-ZSF1 vs. O-ZSF1, P = 0.027) and increased collagen gene expression (Col1a1: +48% in sO-ZSF1 vs. O-ZSF1, P = 0.026). In contrary, right ventricular function was preserved by hArg supplementation (TAPSE (mm): O-ZSF1 1.2 ± 0.3 vs. sO-ZSF1 1.7 ± 0.3, P = 0.020, L-ZSF1 1.8 ± 0.4). Musculus soleus maximal specific muscle force (N/cm2 ) in O-ZSF1 (30.4 ± 0.8) and sO-ZSF1 (31.9 ± 0.9) was comparable but significantly reduced compared with L-ZSF1 (36.4 ± 0.7; both P < 0.05). Maximal absolute muscle force (g) (O-ZSF1: 177.6 ± 7.8, sO-ZSF1: 187.8 ± 5.0, L-ZSF1: 181.5 ± 7.9, all P > 0.05) and cross-sectional fibre area (arbitrary units) (O-ZSF1: 1697 ± 57, sO-ZSF1: 1965 ± 121, L-ZSF1: 1691 ± 104, all P > 0.05) were not altered. CONCLUSIONS: Preservation of physiological hArg level in HFpEF may not be suited to prevent alterations in left ventricular and skeletal muscle function and structure. However, hArg supplementation may be beneficial for right ventricular function especially in pulmonary hypertension in HFpEF. We may speculate that clinically observed decreased hArg level are not the cause but the consequence of a yet unrecognized pathomechanism that underpins HFpEF.

Comparative evaluation of the effect of L-Arginine and L-Homoarginine supplementation on reproductive physiology in ewes.

L-Arginine (LA) is a well-known amino acid involved in vital physiological processes. However, the physiological function of its methylated form known as L-Homoarginine (LHA), is not well understood. The aim of this study was to study the comparative effects of LA and LHA treatment on selected metabolites and parameters of reproductive physiology in non-pregnant ewes. Twelve Deccani ewes were selected and randomly distributed into 3 groups: Control, LA treated group and LHA group. Blood samples were collected for the evaluation of blood indices and hormone levels. Overall, nitrite, ovary weight, surface follicle number, estrogen, insulin like growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF) showed an increase in the LA and LHA treated animals. On the other side, the levels of progesterone, inteleukin-1 beta (IL-1ß), IL-6, tumor necrosis factor-alpha (TNF-α) were found to be decreased in LA and LHA group. Ovary size was not affected by LA and LHA treatment groups. In comparison with LA, the ovary weight, surface follicle number, estrogen levels and IL-6 expression were higher in LHA treated animals. The expression of VEGF indicated improved angiogenesis in the treated animals. Further, the expression of heat shock protein-27 (HSP-27) and HSP-70 were differentially modulated by LA and LHA. This data reinforces the beneficial role of LA and its metabolites LHA on the ovarian physiology and functionality and also reveals the potent role of LHA as an alternative to LA treatment in enhancing the reproductive ability in non-pregnant ewes.

Population kinetics of homoarginine and optimized supplementation for cardiovascular risk reduction.

Homoarginine is an endogenous amino acid whose levels are reduced in patients with renal, cardio- and cerebrovascular disease. Moreover, low homoarginine concentrations independently predict morbidity and mortality in these patients. Besides endogenous synthesis, homoarginine is also a constituent of the human diet. The objective of the present study was to analyze the kinetics of orally supplemented homoarginine in human plasma by means of a pharmacometric approach. We developed a pharmacometric model to evaluate different dosing regimens, especially the regimen of 125 mg once weekly, based on a previous clinical study (n = 20). The model was adapted to account for differences in baseline homoarginine plasma concentrations between healthy and diseased individuals. A novel dosing regimen of 25 mg once daily led to higher attainment of homoarginine reference concentrations using clinical trial simulations. With 25 mg/day, the trough concentration of only 6% of the older and 3.8% of the younger population was predicted to be below the target concentration of 2.0-4.1 µmol/L. In synopsis, the new dosing regimen recapitulates the kinetics of homoarginine in healthy individuals optimally.

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.

Arginine:Glycine Amidinotransferase Is Essential for Creatine Supply in Mice During Chronic Hypoxia.

Objective: Chronic hypoxia induces pulmonary and cardiovascular pathologies, including pulmonary hypertension (PH). L-arginine:glycine amidinotransferase (AGAT) is essential for homoarginine (hArg) and guanidinoacetate synthesis, the latter being converted to creatine by guanidinoacetate methyltransferase. Low hArg concentrations are associated with cardiovascular morbidity and predict mortality in patients with PH. We therefore aimed to investigate the survival and cardiac outcome of AGAT knockout (Agat -/-) mice under hypoxia and a possible rescue of the phenotype. Methods: Agat -/- mice and wild-type (WT) littermates were subjected to normoxia or normobaric hypoxia (10% oxygen) for 4 weeks. A subgroup of Agat -/- mice was supplemented with 1% creatine from weaning. Survival, hematocrit, blood lactate and glucose, heart weight-to-tibia length (HW/TL) ratio, hArg plasma concentration, and Agat and Gamt expression in lung, liver, and kidneys were evaluated. Results: After 6 h of hypoxia, blood lactate was lower in Agat -/--mice as compared to normoxia (p < 0.001). Agat -/- mice died within 2 days of hypoxia, whereas Agat -/- mice supplemented with creatine and WT mice survived until the end of the study. In WT mice, hematocrit (74 ± 4 vs. 55 ± 2%, mean ± SD, p < 0.001) and HW/TL (9.9 ± 1.3 vs. 7.3 ± 0.7 mg/mm, p < 0.01) were higher in hypoxia, while hArg plasma concentration (0.25 ± 0.06 vs. 0.38 ± 0.12 µmol/L, p < 0.01) was lower. Agat and Gamt expressions were differentially downregulated by hypoxia in lung, liver, and kidneys. Conclusion: Agat and Gamt are downregulated in hypoxia. Agat-/- mice are nonviable in hypoxia. Creatine rescues the lethal phenotype, but it does not reduce right ventricular hypertrophy of Agat-/- mice in hypoxia.

Homoarginine- and Creatine-Dependent Gene Regulation in Murine Brains with l-Arginine:Glycine Amidinotransferase Deficiency.

l-arginine:glycine amidinotransferase (AGAT) and its metabolites homoarginine (hArg) and creatine have been linked to stroke pathology in both human and mouse studies. However, a comprehensive understanding of the underlying molecular mechanism is lacking. To investigate transcriptional changes in cerebral AGAT metabolism, we applied a transcriptome analysis in brains of wild-type (WT) mice compared to untreated AGAT-deficient (AGAT-/-) mice and AGAT-/- mice with creatine or hArg supplementation. We identified significantly regulated genes between AGAT-/- and WT mice in two independent cohorts of mice which can be linked to amino acid metabolism (Ivd, Lcmt2), creatine metabolism (Slc6a8), cerebral myelination (Bcas1) and neuronal excitability (Kcnip3). While Ivd and Kcnip3 showed regulation by hArg supplementation, Bcas1 and Slc6a8 were creatine dependent. Additional regulated genes such as Pla2g4e and Exd1 need further evaluation of their influence on cerebral function. Experimental stroke models showed a significant regulation of Bcas1 and Slc6a8. Together, these results reveal that AGAT deficiency, hArg and creatine regulate gene expression in the brain, which may be critical in stroke pathology.

Muscle phenotype of AGAT- and GAMT-deficient mice after simvastatin exposure.

Statin-induced myopathy affects more than 10 million people worldwide. But discontinuation of statin treatment increases mortality and cardiovascular events. Recently, L-arginine:glycine amidinotransferase (AGAT) gene was associated with statin-induced myopathy in two populations, but the causal link is still unclear. AGAT is responsible for the synthesis of L-homoarginine (hArg) and guanidinoacetate (GAA). GAA is further methylated to creatine (Cr) by guanidinoacetate methyltransferase (GAMT). In cerebrovascular patients treated with statin, lower hArg and GAA plasma concentrations were found than in non-statin patients, indicating suppressed AGAT expression and/or activity (n = 272, P = 0.033 and P = 0.039, respectively). This observation suggests that statin-induced myopathy may be associated with AGAT expression and/or activity in muscle cells. To address this, we studied simvastatin-induced myopathy in AGAT- and GAMT-deficient mice. We found that simvastatin induced muscle damage and reduced AGAT expression in wildtype mice (myocyte diameter: 34.1 ± 1.3 µm vs 21.5 ± 1.3 µm, P = 0.026; AGAT expression: 1.0 ± 0.3 vs 0.48 ± 0.05, P = 0.017). Increasing AGAT expression levels of transgenic mouse models resulted in rising plasma levels of hArg and GAA (P < 0.01 and P < 0.001, respectively). Simvastatin-induced motor impairment was exacerbated in AGAT-deficient mice compared with AGAT-overexpressing GAMT-/- mice and therefore revealed an effect independent of Cr. But Cr supplementation itself improved muscle strength independent of AGAT expression (normalized grip strength: 55.8 ± 2.9% vs 72.5% ± 3.0%, P < 0.01). Homoarginine supplementation did not affect statin-induced myopathy in AGAT-deficient mice. Our results from clinical and animal studies suggest that AGAT expression/activity and its product Cr influence statin-induced myopathy independent of each other. The interplay between simvastatin treatment, AGAT expression and activity, and Cr seems to be complex. Further clinical pharmacological studies are needed to elucidate the underlying mechanism(s) and to evaluate whether supplementation with Cr, or possibly GAA, in patients under statin medication may reduce the risk of muscular side effects.

l-Homoarginine supplementation prevents diabetic kidney damage.

l-homoarginine is an endogenous, non-proteinogenic amino acid that has emerged as a new player in health and disease. Specifically, low l-homoarginine levels are associated with cardiovascular diseases, stroke, and reduced kidney function. However, the role of l-homoarginine in the pathogenesis of diabetic nephropathy (DN) is not known. Experiments were conducted in 6-week-old Ins2Akita mice supplemented with l-homoarginine via drinking water or mini osmotic pump for 12 weeks. Both plasma and kidney l-homoarginine levels were significantly reduced in diabetic mice compared to nondiabetic controls. Untreated Ins2Akita mice showed significant increases in urinary albumin excretion, histological changes, glomerular macrophage recruitment, the inflammatory cytokine KC-GRO/CXCL1, and urinary thiobarbituric acid reactive substances (TBARS) excretion as an indicator of oxidative stress, along with a significant reduction in kidney nitrate + nitrite levels compared to control mice at 18 weeks of age. In contrast, l-homoarginine supplementation for 12 weeks in Ins2Akita mice, via either drinking water or mini osmotic pump, significantly reduced albuminuria, renal histological changes, glomerular macrophage recruitment, KC-GRO/CXCL1 levels, urinary TBARS excretion, and largely restored kidney nitrate + nitrite levels. These data demonstrate that l-homoarginine supplementation attenuates specific features of DN in mice and could be a potential new therapeutic tool for treating diabetic patients.

Homoarginine Supplementation Prevents Left Ventricular Dilatation and Preserves Systolic Function in a Model of Coronary Artery Disease.

Background Homoarginine ( hA rg) has been shown to be cardioprotective in a model of ischemic heart failure; however, the mechanism remains unknown. hA rg can inhibit tissue-nonspecific alkaline phosphatase ( TNAP ), an enzyme that promotes vascular calcification. We hypothesized that hA rg will exert beneficial effects by reducing calcification in a mouse model of coronary artery disease associated with TNAP overexpression and hypercholesterolemia. Methods and Results TNAP was overexpressed in the endothelium in mice homozygous for a low-density lipoprotein receptor mutation (wicked high cholesterol [ WHC ] allele). WHC and WHC -endothelial TNAP mice received placebo or hA rg supplementation (14 mg/L in drinking water) starting at 6 weeks of age simultaneously with an atherogenic diet. Outcomes were compared between the groups after 4 to 5 weeks on treatment. Experiments were performed in males, which presented a study limitation. As expected, WHC -endothelial TNAP mice on the placebo had increased mortality (median survival 27 days, P<0.0001), increased coronary calcium and lipids ( P<0.01), increased left ventricular end-diastolic diameter ( P<0.0001), reduced ejection fraction ( P<0.05), and increased myocardial fibrosis ( P<0.0001) compared with WHC mice. Contrary to our hypothesis, hA rg neither inhibited TNAP activity in vivo nor reduced coronary artery calcification and atherosclerosis in WHC -endothelial TNAP mice; however, compared with the placebo, hA rg prevented left ventricular dilatation ( P<0.01), preserved ejection fraction ( P<0.05), and reduced myocardial fibrosis ( P<0.001). Conclusions The beneficial effect of hA rg supplementation in the setting of calcified coronary artery disease is likely due to its direct protective actions on the myocardial response to the ischemic injury and not to the inhibition of TNAP activity and calcification.

Evidence by GC-MS that lysine is an arginase-catalyzed metabolite of homoarginine in vitro and in vivo in humans.

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.

Effects of single and combined metformin and L-citrulline supplementation on L-arginine-related pathways in Becker muscular dystrophy patients: possible biochemical and clinical implications.

The L-arginine/nitric oxide synthase (NOS) pathway is considered to be altered in muscular dystrophy such as Becker muscular dystrophy (BMD). We investigated two pharmacological options aimed to increase nitric oxide (NO) synthesis in 20 male BMD patients (age range 21-44 years): (1) supplementation with L-citrulline (3 × 5 g/d), the precursor of L-arginine which is the substrate of neuronal NO synthase (nNOS); and (2) treatment with the antidiabetic drug metformin (3 × 500 mg/d) which activates nNOS in human skeletal muscle. We also investigated the combined use of L-citrulline (3 × 5 g/d) and metformin (3 × 500 mg/d). Before and after treatment, we measured in serum and urine samples the concentration of amino acids and metabolites of L-arginine-related pathways and the oxidative stress biomarker malondialdehyde (MDA). Compared to healthy subjects, BMD patients have altered NOS, arginine:glycine amidinotransferase (AGAT) and guanidinoacetate methyltransferase (GAMT) pathways. Metformin treatment resulted in concentration decrease of arginine and MDA in serum, and of homoarginine (hArg) and guanidinoacetate (GAA) in serum and urine. L-Citrulline supplementation resulted in considerable increase of the concentrations of amino acids and creatinine in the serum, and in their urinary excretion rates. Combined use of metformin and L-citrulline attenuated the effects obtained from their single administrations. Metformin, L-citrulline or their combination did not alter serum nitrite and nitrate concentrations and their urinary excretion rates. In conclusion, metformin or L-citrulline supplementation to BMD patients results in remarkable antidromic changes of the AGAT and GAMT pathways. In combination, metformin and L-citrulline at the doses used in the present study seem to abolish the biochemical effects of the single drugs in slight favor of L-citrulline.

Cognitive performance of 20 healthy humans supplemented with L-homoarginine for 4 weeks.

l-homoarginine (l-hArg) is an endogenous non-proteinogenic amino acid. Low l-hArg concentrations are associated with increased all-cause mortality, fatal strokes, and worse outcome after stroke. On the other hand, oral supplementation with l-hArg in mice improved neurological deficits and preserved cardiac function in experimental models of stroke and heart failure, respectively. Recently, oral supplementation with 125 mg daily l-hArg capsules in healthy volunteers demonstrated increased l-hArg plasma levels. Therefore, oral l-hArg supplementation could represent a potential treatment for patients with cerebrovascular disease. In addition to vascular physiology, animal studies have suggested that l-hArg might play a role in synapse function, neurotransmitter metabolism and cognitive training. However the direct influence of l-hArg on cognitive function has not been studied so far. In this study, cognitive performance in healthy humans was analyzed concerning memory, learning, and attention following supplementation with placebo or l-hArg for 4 weeks. Our results did not reveal any effects on cognition, neither impairment nor improvement, upon l-hArg supplementation. Therefore, potential l-hArg treatment is not expected to cause any acute neurocognitive or behavioral side effects.