Enhancing kidney DDAH-1 expression by adenovirus delivery reduces ADMA and ameliorates diabetic nephropathy.

Endothelial dysfunction, characterized by reduced bioavailability of nitric oxide and increased oxidative stress, is a hallmark characteristic in diabetes and diabetic nephropathy (DN). High levels of asymmetric dimethylarginine (ADMA) are observed in several diseases including DN and are a strong prognostic marker for cardiovascular events in patients with diabetes and end-stage renal disease. ADMA, an endogenous endothelial nitric oxide synthase (NOS3) inhibitor, is selectively metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Low DDAH levels have been associated with cardiac and renal dysfunction, but its effects on DN are unknown. We hypothesized that enhanced renal DDAH-1 expression would improve DN by reducing ADMA and restoring NOS3 levels. DBA/2J mice injected with multiple low doses of vehicle or streptozotocin were subsequently injected intrarenally with adenovirus expressing DDAH-1 (Ad-h-DDAH-1) or vector control [Ad-green fluorescent protein (GFP)], and mice were followed for 6 wk. Diabetes was associated with increased kidney ADMA and reduced kidney DDAH activity and DDAH-1 expression but had no effect on kidney DDAH-2 expression. Ad-GFP-treated diabetic mice showed significant increases in albuminuria, histological changes, glomerular macrophage recruitment, inflammatory cytokine and fibrotic markers, kidney ADMA levels, and urinary thiobarbituric acid reactive substances excretion as an indicator of oxidative stress, along with a significant reduction in kidney DDAH activity and kidney NOS3 mRNA compared with normal mice. In contrast, Ad-h-DDAH-1 treatment of diabetic mice reversed these effects. These data indicate, for the first time, that DDAH-1 mediates renal tissue protection in DN via the ADMA-NOS3-interaction. Enhanced renal DDAH-1 activity could be a novel therapeutic tool for treating patients with diabetes.

Proposals for Upper Limits of Safe Intake for Methionine, Histidine, and Lysine in Healthy Humans.

Based on research presented during the 10th Amino Acid Assessment Workshop, no observed adverse effect levels (NOAELs) for supplemental methionine at 46 mg/(kg·d) (∼3.2 g/d), for supplemental histidine at 8.0 g/d, and for supplemental lysine at 6.0 g/d have been proposed. These NOAELs are relevant to healthy adults and are applicable only to high-purity amino acids administered in fortified foods or dietary supplements. Because individuals are exposed to the above supplemental amino acids in the context of complex combinations of essential amino acids or individually in dietary supplements for various physiologic benefits, such as body fat reduction, skin conditioning, mental energy increase, or herpes simplex treatments, the above safety recommendations will make an important contribution to regulatory and nutritional practices.

Distinct roles of arginases 1 and 2 in diabetic nephropathy.

Diabetes is the leading cause of end-stage renal disease, resulting in a significant health care burden and loss of economic productivity by affected individuals. Because current therapies for progression of diabetic nephropathy (DN) are only moderately successful, identification of underlying mechanisms of disease is essential to develop more effective therapies. We showed previously that inhibition of arginase using S-(2-boronoethyl)-l-cysteine (BEC) or genetic deficiency of the arginase-2 isozyme was protective against key features of nephropathy in diabetic mouse models. However, those studies did not determine whether all markers of DN were dependent only on arginase-2 expression. The objective of this study was to identify features of DN that are associated specifically with expression of arginase-1 or -2. Elevated urinary albumin excretion rate and plasma urea levels, increases in renal fibronectin mRNA levels, and decreased renal medullary blood flow were associated almost completely and specifically with arginase-2 expression, indicating that arginase-2 selectively mediates major aspects of diabetic renal injury. However, increases in renal macrophage infiltration and renal TNF-α mRNA levels occurred independent of arginase-2 expression but were almost entirely abolished by treatment with BEC, indicating a distinct role for arginase-1. We therefore generated mice with a macrophage-specific deletion of arginase-1 (CD11bCre /Arg1fl/fl ). CD11bCre /Arg1fl/fl mice had significantly reduced macrophage infiltration but had no effect on albuminuria compared with Arg1fl/fl mice after 12 wk of streptozotocin-induced diabetes. These results indicate that selective inhibition of arginase-2 would be effective in preventing or ameliorating major features of diabetic renal injury.

Arginase-2 mediates renal ischemia-reperfusion injury.

Novel therapeutic interventions for preventing or attenuating kidney injury following ischemia-reperfusion injury (IRI) remain a focus of significant interest. Currently, there are no definitive therapeutic or preventive approaches available for ischemic acute kidney injury (AKI). Our objective is to determine 1) whether renal arginase activity or expression is increased in renal IRI, and 2) whether arginase plays a role in development of renal IRI. The impact of arginase activity and expression on renal damage was evaluated in male C57BL/6J (wild type) and arginase-2 (ARG2)-deficient (Arg2-/- ) mice subjected to bilateral renal ischemia for 28 min, followed by reperfusion for 24 h. ARG2 expression and arginase activity significantly increased following renal IRI, paralleling the increase in kidney injury. Pharmacological blockade or genetic deficiency of Arg2 conferred kidney protection in renal IRI. Arg2-/- mice had significantly attenuated kidney injury and lower plasma creatinine and blood urea nitrogen levels after renal IRI. Blocking arginases using S-(2-boronoethyl)-l-cysteine (BEC) 18 h before ischemia mimicked arginase deficiency by reducing kidney injury, histopathological changes and kidney injury marker-1 expression, renal apoptosis, kidney inflammatory cell recruitment and inflammatory cytokines, and kidney oxidative stress; increasing kidney nitric oxide (NO) production and endothelial NO synthase (eNOS) phosphorylation, kidney peroxisome proliferator-activated receptor-γ coactivator-1α expression, and mitochondrial ATP; and preserving kidney mitochondrial ultrastructure compared with vehicle-treated IRI mice. Importantly, BEC-treated eNOS-knockout mice failed to reduce blood urea nitrogen and creatinine following renal IRI. These findings indicate that ARG2 plays a major role in renal IRI, via an eNOS-dependent mechanism, and that blocking ARG2 activity or expression could be a novel therapeutic approach for prevention of AKI.

FoxO4 promotes early inflammatory response upon myocardial infarction via endothelial Arg1.

RATIONALE: Inflammation in post-myocardial infarction (MI) is necessary for myocyte repair and wound healing. Unfortunately, it is also a key component of subsequent heart failure pathology. Transcription factor forkhead box O4 (FoxO4) regulates a variety of biological processes, including inflammation. However, its role in MI remains unknown. OBJECTIVE: To test the hypothesis that FoxO4 promotes early post-MI inflammation via endothelial arginase 1 (Arg1). METHODS AND RESULTS: We induced MI in wild-type and FoxO4(-/-) mice. FoxO4(-/-) mice had a significantly higher post-MI survival, better cardiac function, and reduced infarct size. FoxO4(-/-) hearts had significantly fewer neutrophils, reduced expression of cytokines, and competitive nitric oxide synthase inhibitor Arg1. We generated conditional FoxO4 knockout mice with FoxO4 deleted in cardiac mycoytes or endothelial cells. FoxO4 endothelial cell-specific knockout mice showed significant post-MI improvement of cardiac function and reduction of neutrophil accumulation and cytokine expression, whereas FoxO4 cardiac mycoyte-specific knockout mice had no significant difference in cardiac function and post-MI inflammation from those of control littermates. FoxO4 binds the Foxo-binding site in the Arg1 promoter and activates Arg1 transcription. FoxO4 knockdown in human aortic endothelial cells upregulated nitric oxide on ischemia and suppressed monocyte adhesion that can be reversed by ectopic-expression of Arg1. Furthermore, chemical inhibition of Arg1 in wild-type mice had similar cardioprotection and reduced inflammation after MI as FoxO4 inactivation and administration of nitric oxide synthase inhibitor to FoxO4 KO mice reversed the beneficial effects of FoxO4 deletion on post-MI cardiac function. CONCLUSIONS: FoxO4 activates Arg1 transcription in endothelial cells in response to MI, leading to downregulation of nitric oxide and upregulation of neutrophil infiltration to the infarct area.

Arginine Metabolism Revisited.

Mammalian arginine metabolism is complex due to the expression of multiple enzymes that utilize arginine as substrate and to interactions or competition between specific enzymes involved in arginine metabolism. Moreover, cells may contain multiple intracellular arginine pools that are not equally accessible to all arginine metabolic enzymes, thus presenting additional challenges to more fully understanding arginine metabolism. At the whole-body level, arginine metabolism ultimately results in the production of a biochemically diverse range of products, including nitric oxide, urea, creatine, polyamines, proline, glutamate, agmatine, and homoarginine. Included in this group of compounds are the methylated arginines (e.g., asymmetric dimethylarginine), which are released upon degradation of proteins containing methylated arginine residues. Changes in arginine concentration also can regulate cellular metabolism and function via a variety of arginine sensors. Although much is known about arginine metabolism, elucidation of the physiologic or pathophysiologic roles for all of the pathways and their metabolites remains an active area of investigation, as exemplified by current findings highlighted in this review.

Proposals for Upper Limits of Safe Intake for Arginine and Tryptophan in Young Adults and an Upper Limit of Safe Intake for Leucine in the Elderly.

On the basis of research presented during the 9th Amino Acid Assessment Workshop, a No Observed Adverse Effect Level (NOAEL) for diet-added arginine (added mostly in the form of dietary supplements) of 30 g/d and an upper limit of safe intake (ULSI) for diet-added tryptophan (added mostly in the form of dietary supplements) of 4.5 g/d have been proposed. Both recommendations apply to healthy young adults. The total dietary leucine ULSI proposed for elderly individuals is 500 mg · kg-1 · d-1 All 3 recommendations are relevant only to high-quality amino acid-containing products with specifications corresponding to those listed in the US Pharmacopeia Because the above amino acids are extensively utilized as dietary supplements for various real or perceived benefits, such as vasodilation, spermatogenesis, sleep, mood regulation, or muscle recovery, the above safety recommendations will have an important impact on regulatory and nutritional practices.

Arginase inhibition: a new treatment for preventing progression of established diabetic nephropathy.

Our previous publication showed that inhibition of arginase prevents the development of diabetic nephropathy (DN). However, identification of targets that retard the progression of established DN-which is more clinically relevant-is lacking. Therefore, we tested the hypothesis that arginase inhibition would prevent the progression of established DN. Effects of arginase inhibition were compared with treatment with the angiotensin-converting enzyme inhibitor captopril, a current standard of care in DN. Experiments were conducted in Ins2(Akita) mice treated with the arginase inhibitor S-(2-boronoethyl)-l-cysteine (BEC) or captopril starting at 6 wk of age for 12 wk (early treatment) or starting at 12 wk of age for 6 wk (late treatment). Early and late treatment with BEC resulted in protection from DN as indicated by reduced albuminuria, histological changes, kidney macrophage infiltration, urinary thiobarbituric acid-reactive substances, and restored nephrin expression, kidney nitrate/nitrite, kidney endothelial nitric oxide synthase phosphorylation, and renal medullary blood flow compared with vehicle-treated Ins2(Akita) mice at 18 wk of age. Interestingly, early treatment with captopril reduced albuminuria, histological changes, and kidney macrophage infiltration without affecting the other parameters, but late treatment with captopril was ineffective. These findings highlight the importance of arginase inhibition as a new potential therapeutic intervention in both early and late stages of diabetic renal injury.

Shaping the murine macrophage phenotype: IL-4 and cyclic AMP synergistically activate the arginase I promoter.

Arginase I is a marker of murine M2 macrophages and is highly expressed in many inflammatory diseases. The basis for high arginase I expression in macrophages in vivo is incompletely understood but likely reflects integrated responses to combinations of stimuli. Our objective was to elucidate mechanisms involved in modulating arginase I induction by IL-4, the prototypical activator of M2 macrophages. IL-4 and 8-bromo-cAMP individually induce arginase I, but together they rapidly and synergistically induce arginase I mRNA, protein, and promoter activity in murine macrophage cells. Arginase I induction by IL-4 requires binding of the transcription factors STAT6 and C/EBPß to the IL-4 response element of the arginase I gene. Chromatin immunoprecipitation showed that the synergistic response involves binding of both transcription factors to the IL-4 response element at levels significantly greater than in response to IL-4 alone. The results suggest that C/EBPß is a limiting factor for the level of STAT6 bound to the IL-4 response element. The enhanced binding in the synergistic response was not due to increased expression of either STAT6 or C/EBPß but was correlated primarily with increased nuclear abundance of C/EBPß. Our findings also suggest that induction of arginase I expression is stochastic; that is, differences in induction reflect differences in probability of transcriptional activation and not simply differences in rate of transcription. Results of the present study also may be useful for understanding mechanisms underlying regulated expression of other genes in macrophages and other myeloid-derived cells in health and disease.

Microenvironments in tuberculous granulomas are delineated by distinct populations of macrophage subsets and expression of nitric oxide synthase and arginase isoforms.

Macrophages in granulomas are both antimycobacterial effector and host cell for Mycobacterium tuberculosis, yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial NO synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared with nongranulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, whereas epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68, and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1, and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with greater eNOS expression and ratio of iNOS/Arg1 expression in epithelioid macrophages as compared with cells in the lymphocyte cuff. iNOS, Arg1, and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas, whereas the inner regions were more likely to contain macrophages with proinflammatory, presumably bactericidal, phenotypes. Together, these data support the concept that granulomas have organized microenvironments that balance antimicrobial anti-inflammatory responses to limit pathology in the lungs.

Diabetic nephropathy is resistant to oral L-arginine or L-citrulline supplementation.

Our recent publication showed that pharmacological blockade of arginases confers kidney protection in diabetic nephropathy via a nitric oxide (NO) synthase (NOS)3-dependent mechanism. Arginase competes with endothelial NOS (eNOS) for the common substrate L-arginine. Lack of L-arginine results in reduced NO production and eNOS uncoupling, which lead to endothelial dysfunction. Therefore, we hypothesized that L-arginine or L-citrulline supplementation would ameliorate diabetic nephropathy. DBA mice injected with multiple low doses of vehicle or streptozotocin (50 mg/kg ip for 5 days) were provided drinking water with or without L-arginine (1.5%, 6.05 g·kg(-1)·day(-1)) or L-citrulline (1.66%, 5.73 g·kg(-1)·day(-1)) for 9 wk. Nonsupplemented diabetic mice showed significant increases in albuminuria, blood urea nitrogen, glomerular histopathological changes, kidney macrophage recruitment, kidney TNF-α and fibronectin mRNA expression, kidney arginase activity, kidney arginase-2 protein expression, and urinary oxidative stress along with a significant reduction of nephrin and eNOS protein expression and kidney nitrite + nitrate compared with normal mice after 9 wk of diabetes. Surprisingly, L-arginine or L-citrulline supplementation in diabetic mice did not affect any of these parameters despite greatly increasing kidney and plasma arginine levels. These findings demonstrate that chronic L-arginine or L-citrulline supplementation does not prevent or reduce renal injury in a model of type 1 diabetes.

The central role of arginine catabolism in T-cell dysfunction and increased susceptibility to infection after physical injury.

OBJECTIVE: To explore the hypothesis that decreased arginine availability by myeloid-derived suppressor cells (MDSCs) is a cause of T-cell dysfunction after physical injury (PI). BACKGROUND: Arginine is an essential amino acid for normal T-cell function whose availability becomes limited after PI. MDSCs expressing arginase 1 are induced by PI. T-cell dysfunction after PI seems to increase the risk of infection but the mechanisms that cause it are unclear. METHODS: PI was created using a standard laparotomy model. Phenotypical and functional alterations in T cells were evaluated in vivo. MDSCs expressing arginase 1 were measured by flow cytometry. Infection after PI was created by intraperitoneal injection of Listeria monocytogenes. N-Hydroxy-Nor-L-arginine (Nor-NOHA) was used as an arginase inhibitor. The effect of arginine depletion on T-cell function and susceptibility to infection was assessed through adoptive transfer of MDSC or injection of arginase into noninjured mice. RESULTS: PI caused a decrease in intracellular arginine in T cells, loss of the T-cell receptor (TCR) CD3-ζ chain, inhibition of in vivo T-cell proliferation, memory, and cytotoxicity. PI exponentially increased bacterial growth and mortality to L. monocytogenes. T-cell dysfunction and increased infection were reversed by arginase inhibitor Nor-NOHA but were reproduced by adoptively transferring MDSC or injecting arginase 1 to noninjured mice. CONCLUSIONS: Arginine availability is decreased after PI coinciding with an induction of MDSC expressing arginase 1. Decreased arginine may inhibit T-cell function and increase susceptibility to infection after injury.

Retinoic acid promotes the development of Arg1-expressing dendritic cells for the regulation of T-cell differentiation.

Arginase I (Arg1), an enzyme expressed by many cell types including myeloid cells, can regulate immune responses. Expression of Arg1 in myeloid cells is regulated by a number of cytokines and tissue factors that influence cell development and activation. Retinoic acid, produced from vitamin A, regulates the homing and differentiation of lymphocytes and plays important roles in the regulation of immunity and immune tolerance. We report here that optimal expression of Arg1 in DCs requires retinoic acid. Induction of Arg1 by retinoic acid is directly mediated by retinoic acid-responsive elements in the 5' noncoding region of the Arg1 gene. Arg1, produced by DCs in response to retinoic acid, promotes the generation of FoxP3(+) regulatory T (Treg) cells. Importantly, blocking the retinoic acid receptor makes DCs hypo-responsive to known inducers of Arg1 such as IL-4 and GM-CSF in Arg1 expression. We found that intestinal CD103(+) DCs that are known to produce retinoic acid highly express Arg1. Our results establish retinoic acid as a key signal in expression of Arg1 in DCs.

Arginase inhibition mediates renal tissue protection in diabetic nephropathy by a nitric oxide synthase 3-dependent mechanism.

Recently, we showed that pharmacological blockade or genetic deficiency of arginase-2 confers kidney protection in diabetic mouse models. Here, we tested whether the protective effect of arginase inhibition is nitric oxide synthase 3 (eNOS) dependent in diabetic nephropathy. Experiments were conducted in eNOS-knockout and their wild-type littermate mice using multiple low doses of vehicle or streptozotocin, and treated with continuous subcutaneous infusion of vehicle or the arginase inhibitor S-(2-boronoethyl)-L-cysteine by an osmotic pump. Inhibition of arginases for 6 weeks in diabetic wild-type mice significantly attenuated albuminuria, the increase in plasma creatinine and blood urea nitrogen, histopathological changes, kidney fibronectin and TNF-α expression, kidney macrophage recruitment, and oxidative stress compared with vehicle-treated diabetic wild-type mice. Arginase inhibition in diabetic eNOS-knockout mice failed to affect any of these parameters, but reduced kidney macrophage recruitment and kidney TNF-α expression compared with vehicle-treated diabetic eNOS-knockout mice. Furthermore, diabetic wild-type and eNOS-knockout mice exhibited increased kidney arginase-2 protein, arginase activity, and ornithine levels. Thus, arginase inhibition mediates renal tissue protection in diabetic nephropathy by an eNOS-dependent mechanism and has an eNOS-independent effect on kidney macrophage recruitment.

Adenosine promotes alternative macrophage activation via A2A and A2B receptors.

Adenosine has been implicated in suppressing the proinflammatory responses of classically activated macrophages induced by Th1 cytokines. Alternative macrophage activation is induced by the Th2 cytokines interleukin (IL)-4 and IL-13; however, the role of adenosine in governing alternative macrophage activation is unknown. We show here that adenosine treatment of IL-4- or IL-13-activated macrophages augments the expression of alternative macrophage markers arginase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), and macrophage galactose-type C-type lectin-1. The stimulatory effect of adenosine required primarily A(2B) receptors because the nonselective adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) increased both arginase activity (EC(50)=261.8 nM) and TIMP-1 production (EC(50)=80.67 nM), and both pharmacologic and genetic blockade of A(2B) receptors prevented the effect of NECA. A(2A) receptors also contributed to the adenosine augmentation of IL-4-induced TIMP-1 release, as both adenosine and NECA were less efficacious in augmenting TIMP-1 release by A(2A) receptor-deficient than control macrophages. Of the transcription factors known to drive alternative macrophage activation, CCAAT-enhancer-binding protein ß was required, while cAMP response element-binding protein and signal transducer and activator of transcription 6 were dispensable in mediating the effect of adenosine. We propose that adenosine receptor activation suppresses inflammation and promotes tissue restitution, in part, by promoting alternative macrophage activation.

LXRα regulates macrophage arginase 1 through PU.1 and interferon regulatory factor 8.

RATIONALE: Activation of liver X receptors (LXRs) inhibits the progression of atherosclerosis and promotes regression of existing lesions. In addition, LXRα levels are high in regressive plaques. Macrophage arginase 1 (Arg1) expression is inversely correlated with atherosclerosis progression and is markedly decreased in foam cells within the lesion. OBJECTIVE: To investigate LXRα regulation of Arg1 expression in cultured macrophages and atherosclerotic regressive lesions. METHODS AND RESULTS: We found that Arg1 expression is enhanced in CD68+ cells from regressive versus progressive lesions in a murine aortic arch transplant model. In cultured macrophages, ligand-activated LXRα markedly enhances basal and interleukin-4-induced Arg1 mRNA and protein expression as well as promoter activity. This LXRα-enhanced Arg1 expression correlates with a reduction in nitric oxide levels. Moreover, Arg1 expression within regressive atherosclerotic plaques is LXRα-dependent, as enhanced expression of Arg1 in regressive lesions is impaired in LXRα-deficient CD68+ cells. LXRα does not bind to the Arg1 promoter but instead promotes the interaction between PU.1 and interferon regulatory factor (IRF)8 transcription factors and induces their binding of a novel composite element. Accordingly, knockdown of either IRF8 or PU.1 strongly impairs LXRα regulation of Arg1 expression in macrophage cells. Finally, we demonstrate that LXRα binds the IRF8 locus and its activation increases IRF8 mRNA and protein levels in these cells. CONCLUSIONS: This work implicates Arg1 in atherosclerosis regression and identifies LXRα as a novel regulator of Arg1 and IRF8 in macrophages. Furthermore, it provides a unique molecular mechanism by which LXRα regulates macrophage target gene expression through PU.1 and IRF8.

Regulation of macrophage arginase expression and tumor growth by the Ron receptor tyrosine kinase.

M1 activation of macrophages promotes inflammation and immunity to intracellular pathogens, whereas M2 macrophage activation promotes resolution of inflammation, wound healing, and tumor growth. These divergent phenotypes are characterized, in part, by the expression of inducible NO synthase and arginase I (Arg1) in M1 versus M2 activated macrophages, respectively. In this study, we demonstrate that the Ron receptor tyrosine kinase tips the balance of macrophage activation by attenuating the M1 phenotype while promoting expression of Arg1 through a Stat6-independent mechanism. Induction of the Arg1 promoter by Ron is mediated by an AP-1 site located 433 bp upstream of the transcription start site. Treatment of primary macrophages with macrophage stimulating protein, the ligand for Ron, induces potent MAPK activation, upregulates Fos, and enhances binding of Fos to the AP-1 site in the Arg1 promoter. In vivo, Arg1 expression in tumor-associated macrophages (TAMs) from Ron(-/-) mice was significantly reduced compared with that in TAMs from control animals. Furthermore, we show that Ron is expressed specifically by Tie2-expressing macrophages, a TAM subset that exhibits a markedly skewed M2 and protumoral phenotype. Decreased Arg1 in TAMs from Ron(-/-) mice was associated with reduced syngeneic tumor growth in these animals. These findings indicate that Ron induces Arg1 expression in macrophages through a previously uncharacterized AP-1 site in the Arg1 promoter and that Ron could be therapeutically targeted in the tumor microenvironment to inhibit tumor growth by targeting expression of Arg1.

Arginases and arginine deficiency syndromes.

PURPOSE OF REVIEW: Many physiologic and pathophysiologic processes are modulated by arginine availability, which can be regulated by arginase. An understanding of the conditions that result in elevated arginase activity as well as the consequences of arginine deficiency is essential for design of effective nutritional support for disease. This review will emphasize recent findings regarding effects of plasma arginase and arginine deficiencies in disease. RECENT FINDINGS: Elevations in plasma arginase, derived primarily from hemolysis of red blood cells or liver damage, that are associated with arginine deficiency have been identified in an increasing number of diseases and conditions. Arginine insufficiency not only can activate a stress kinase pathway that impairs function of T lymphocytes but it also can inhibit the mitogen-activated protein kinase signaling pathway required for macrophage production of cytokines in response to bacterial endotoxin/lipopolysaccharide. SUMMARY: There are at least two broad categories of arginine deficiency syndromes, involving either T-cell dysfunction or endothelial dysfunction, depending on the disease context in which arginine deficiency occurs. There is limited information regarding the safety and efficacy of supplementation with arginine or its precursor citrulline in ameliorating arginine deficiency in specific diseases, indicating the need for further studies.

A proposal for an upper limit of leucine safe intake in healthy adults.

Based on recent research, an upper limit of safe intake (ULSI) for leucine is proposed for healthy adults: 0.53 g/(kg·d). Because leucine has been used as a dietary supplement for many years in people practicing exercise and sport, further study with long-term exposure to leucine in this specific subpopulation should be performed to eventually adjust the ULSI.

A large-scale chemical screen for regulators of the arginase 1 promoter identifies the soy isoflavone daidzeinas a clinically approved small molecule that can promote neuronal protection or regeneration via a cAMP-independent pathway.

An ideal therapeutic for stroke or spinal cord injury should promote survival and regeneration in the CNS. Arginase 1 (Arg1) has been shown to protect motor neurons from trophic factor deprivation and allow sensory neurons to overcome neurite outgrowth inhibition by myelin proteins. To identify small molecules that capture Arg1's protective and regenerative properties, we screened a hippocampal cell line stably expressing the proximal promoter region of the arginase 1 gene fused to a reporter gene against a library of compounds containing clinically approved drugs. This screen identified daidzein as a transcriptional inducer of Arg1. Both CNS and PNS neurons primed in vitro with daidzein overcame neurite outgrowth inhibition from myelin-associated glycoprotein, which was mirrored by acutely dissociated and cultured sensory neurons primed in vivo by intrathecal or subcutaneous daidzein infusion. Further, daidzein was effective in promoting axonal regeneration in vivo in an optic nerve crush model when given intraocularly without lens damage, or most importantly, when given subcutaneously after injury. Mechanistically, daidzein requires transcription and induction of Arg1 activity for its ability to overcome myelin inhibition. In contrast to canonical Arg1 activators, daidzein increases Arg1 without increasing CREB phosphorylation, suggesting its effects are cAMP-independent. Accordingly, it may circumvent known CNS side effects of some cAMP modulators. Indeed, daidzein appears to be safe as it has been widely consumed in soy products, crosses the blood-brain barrier, and is effective without pretreatment, making it an ideal candidate for development as a therapeutic for spinal cord injury or stroke.