L-arginine is a substrate for the family of nitric oxide (NO) synthase (NOS) enzymes that generate NO, a key chemical involved in normal endothelial function and, hence, cardiovascular health. Reduced NO bioavailability is central to the endothelial dysfunction that underlies vascular complications of diabetes . The cofactor 6R-5,6,7,8-tetrahydrobiopterin (BH4) is crucial for proper functioning of all NOS isoforms, including the endothelial NOS (eNOS). When levels of BH4 are insufficient or the ratio of BH4 to its oxidized 7,8-dihydrobiopterin (BH2) form (BH4/BH2) falls, eNOS is said to be ‘uncoupled’ from the oxidation of L-arginine and superoxide (O2 −) is produced rather than NO . This O2 − can react with and rapidly inactivate any NO that is formed, further reducing NO bioavailability. L-arginine is also the substrate for the arginase enzyme, which converts arginine to ornithine and limits NO bioavailability in endothelial cells through increased arginine consumption . Under physiological conditions, formation of reactive oxygen species (ROS) (such as O2 −) and their elimination are delicately balanced in the vascular wall. However, in disease states like diabetes, enhanced activity of pro-oxidant enzymes and/or reduced activity of antioxidant enzymes result in oxidative stress, leading to endothelial cell dysfunction.
There is widespread interest in L-arginine biochemistry because it is involved in multiple metabolic processes. However, much of the recent interest stems from the central role of NO in vascular homeostasis and the realization that L-arginine is the source of the nitrogen atom in the biosynthesis of NO.
Arginine bioavailability/arginase activity
Individuals with diabetes face an increased risk for cardiovascular disease, and it is endothelial dysfunction brought about by reduced NO bioavailability that is associated with this increased risk . Treatment of the multiple modifiable risk factors for complications in patients with type 2 diabetes (e.g., hyperglycemia, dyslipidemia, and hypertension) results in reduced macrovascular events . Plasma arginine levels have been reported to be low in diabetic patients, likely due to increased arginase activity in these individuals . Tripolt et al. [7▪] report data from a subgroup of the multifactorial treatment of Cardiovascular Risk in Patients with Diabetes Mellitus Type 2: Identification of Treatment Non-Responders – the CARDIONOR Study – which is an ongoing study aimed at investigating early markers of progressive atherosclerosis in type 2 diabetic patients. They show that intensified risk factor management significantly improved the global arginine bioavailability ratio [GABR, defined as [L-arginine]/([L-ornithine]+[L-citrulline])], which is proposed to be a new cardiovascular surrogate parameter that is reduced in diabetic individuals . The GABR, as well as the arginine to ornithine ratio (indirect measure of arginase activity), is inversely correlated with intima–media thickness [7▪], another surrogate parameter for cardiovascular outcome, and with biochemical markers of endothelial dysfunction . Interestingly, in a group of 2236 patients recruited within the LUdwigshafen RIsk and Cardiovascular Health Study, those with type 2 diabetes had a significantly lower GABR than individuals without diabetes . The GABR may provide a noninvasive assessment of endothelial function, as it not only accounts for the substrate arginine but also for the metabolic products citrulline and ornithine, overcoming the poor prognostic value of L-arginine levels alone , but this requires verification.
Sourij et al.  found that the GABR was not superior to the arginine to ornithine ratio in predicting cardiovascular outcome, presumably because the prognostic importance of these ratios is driven mainly by the arginase pathway (only ornithine levels were associated with cardiovascular outcome). Endothelial cells contain two isoforms of arginase: arginase I (located in the cytoplasm) and arginase II (located in the mitochondria) . Two recent studies demonstrate the involvement of arginase I in vascular dysfunction of diabetic patients [10▪] and mice . Increased protein expression of arginase I was found in coronary arterioles from diabetic patients, and expression colocalized with eNOS in endothelial cells [10▪]. This close localization of arginase to eNOS supports the concept of competition of enzymes for the L-arginine substrate and the demonstration that increased arginase I activity leads to reduced availability of L-arginine for eNOS in coronary arteries of diabetic rats . Examination of subcellular localization of arginase, eNOS, and their common substrate L-arginine may be more pertinent to the issue of reciprocal regulation of eNOS by arginase than a comparison of the K m for these enzymes [1–20 mmol/l for arginase compared to 1–5 μmol/l for eNOS], which would not provide an appropriate measure of relative rates of reactions of these two enzymes . Different intracellular pools of L-arginine are proposed to exist, with at least one accessible to eNOS and arginase but not exchangeable with extracellular L-arginine. The relative L-arginine consumption rates for arginase and eNOS vary on the basis of the molar ratios of eNOS/arginase, with arginase activity being favored as the eNOS/arginase molar ratio falls, and are likely influenced by local substrate concentrations .
Unfortunately, recent studies have failed to elucidate the underlying mechanism(s) leading to selective upregulation of arginase I in vessels of diabetic individuals. Romero et al.  suggest that arginase II may contribute to the diabetes-induced arginase I expression/activity in diabetic mice, as arginase II knockout mice exhibited less elevation of arginase activity and arginase I expression than wild-type mice. Beleznai et al. [10▪], on the contrary, postulate that insulin resistance may lead to reduced insulin-induced suppression of expression/activity of enzymes in the urea synthesis pathway, including arginase, but this remains to be tested. Hyperglycemia is known to increase arginase expression in cultured endothelial cells. Chandra et al.  demonstrate that peroxynitrite and hydrogen peroxide, ROS upregulated in diabetes, increase the expression/activity of arginase in endothelial cells through protein kinase C-mediated activation of the RhoA/Rho kinase pathway. Interestingly, arginase I expression was increased, whereas that of arginase II was not affected.
Under normal conditions, synthesis of L-arginine from L-citrulline recycling represents 5–15% of L-arginine production, making endogenously produced L-citrulline a more efficient NO donor than exogenously supplied L-arginine that may be metabolized by arginase . An alternative mechanism to explain reduced levels of arginine in diabetes is the demonstration that insulin drives recycling of citrulline to arginine to support NO production via the stimulation of argininosuccinate synthase (ASS1) in endothelial cells and reduced ASS1 may contribute to endothelial dysfunction [15▪]. Indeed, overexpression of ASS1 in cultured endothelial cells increased NO production with no change in eNOS protein expression or phosphorylation, suggesting that ASS1 may actively participate in regulating endothelial NO production by supplying L-arginine to eNOS [16▪]. ASS1 phosphorylation at Ser-328 supports the calcium-dependent stimulation of eNOS in endothelial cells and is mediated by protein kinase Cα [17▪▪]. An integral role for the subsequent argininosuccinate lyase (ASL) step in endogenous arginine production and later NO production was also recently identified [18▪▪]. A proposed multiprotein complex, including ASS1, ASL, and eNOS, functions to regulate NO production from both endogenously synthesized and exogenously supplied L-arginine. It remains to be tested whether a diabetes-driven decrease in ASS1 expression would affect formation of this complex and subsequent NO production in vascular cells.
L-arginine supplementation as a therapeutic approach
Support for the use of L-arginine supplementation as a therapeutic treatment in diabetes is inconsistent (likely due to variation in concentrations utilized and length of treatment), but some recent studies have demonstrated beneficial effects. For example, 2 months of oral supplementation with L-arginine (3 × 2 g/day) significantly increased NO concentration and total antioxidant status in diabetic patients (matching levels in normal, healthy volunteers), without affecting fasting glucose concentration or hemoglobin A1c levels . This finding is in contrast to earlier studies showing that long-term oral administration of L-arginine enhanced insulin sensitivity and glucose metabolism while improving endothelial function in type 2 diabetic patients [20,21]. Recently, Monti et al. [22▪▪] examined whether long-term L-arginine therapy could prevent or delay the onset of diabetes in patients with impaired glucose tolerance and metabolic syndrome. They found that L-arginine (6.4 g/day) in conjunction with structured lifestyle intervention did not significantly reduce the incidence of diabetes, but did significantly increase regression to normal glucose tolerance, presumably due to improvement in insulin sensitivity shown in these patients [22▪▪] and in several diabetic and obese rat models . The ability of L-arginine to attenuate the many deleterious effects of methylglyoxal, a reactive metabolite of glucose (upregulated three to four fold in diabetic patients) and major precursor for the formation of advanced glycation end products (AGE), may underlie some of the beneficial effects of L-arginine supplementation in diabetic individuals . Both D-arginine and L-arginine could attenuate the increased arginase I and II expression (but not arginase activity), oxidative stress, endothelial dysfunction, and AGE formation induced by methylglyoxal and high glucose, suggesting an eNOS-independent scavenging of methylglyoxal. Indeed, L-arginine can bind and inactivate methylglyoxal, preventing methylglyoxal-induced pancreatic β cell dysfunction as well as reduced adipose tissue glucose uptake , potentially explaining the improved insulin sensitivity of patients in the Monti study [22▪▪].
We have shown that oral L-arginine supplementation can increase transcription of guanosine triphosphate (GTP) cyclohydrolase I, the first enzyme in the de-novo synthesis of BH4 , increasing BH4 and NO production while also reducing weight loss and plasma glucose in type 1 diabetic rats . BH4, one of the most potent antioxidants in the cell, plays a crucial role not only in increasing the rate of NO synthesis by eNOS but also in reducing the formation of O2 − in endothelial cells . Increasing levels of both the eNOS substrate, L-arginine, and its critical cofactor, BH4, are necessary to ensure adequate endothelial NO synthesis.
The function of all NOS isoforms is directly associated with NOS–BH4 stoichiometry. BH4 is bound at the interface between two NOS monomers, where it stabilizes the active dimer and participates in L-arginine oxidation through the N-hydroxyl-L-arginine intermediate and in the subsequent generation of NO. However, BH4 is also highly redox sensitive and, thus, can be readily oxidized, reducing its intracellular availability.
Nitric oxide synthase-dependent 6R-5,6,7,8-tetrahydrobiopterin effects on vascular function
Although oxidative stress has been proposed to be responsible for diminished NO bioavailability in diabetics, the vast majority of studies utilizing standard antioxidant therapy in humans have failed to significantly improve NO-mediated dilation of vessels. In contrast, acute studies with high levels of BH4 alone or BH4 combined with L-arginine treatment showed improved vascular function in diabetic patients [27,28] and prompted the initiation of clinical studies to determine whether oral BH4 supplementation could provide a therapeutic approach to improve endothelial function. Unfortunately, several recent clinical trials involving oral supplementation of BH4 for various types of cardiovascular disease failed to show significant improvement in clinical endpoints, including dilation or NOS coupling in ex-vivo vessels [29,30]. However, one of these trials [31▪▪], involving patients with coronary artery disease, provided some mechanistic insight into the limitations of oral BH4 supplementation as a therapeutic approach to treating cardiovascular disease in humans. Oral BH4 was able to increase BH4 levels in plasma and saphenous vein (but not internal mammary artery) relative to placebo-treated control patients, but BH4-treated patients exhibited a concomitant increase in plasma levels of BH2 (which lacks eNOS cofactor activity and competitively inhibits eNOS), and no significant improvement in the BH4/BH2 ratio, conversion of L-arginine to L-citrulline, or superoxide production [31▪▪]. This study suggests that the ability of BH4 to recouple NOS in patients with diabetes and/or cardiovascular disease may be limited by BH4 oxidation, BH2 accumulation, and failure to increase BH4/BH2 ratios. Indeed, raising endogenous BH2 levels in the presence of adequate BH4 levels was sufficient to cause eNOS uncoupling and increased O2 − production in rats in vivo . Thus, alternative strategies are likely required to target BH4-dependent endothelial function in vascular disease states, such as diabetes, potentially by increasing endogenous BH4 stores without changing BH2 levels in endothelial cells. This approach has been validated in studies utilizing statins, which increase GTP cyclohydrolase I activity in endothelial cells and increase vascular BH4 bioavailability in atherosclerosis patients [33▪].
The recycling of BH2 to BH4 is under the control of dihydrofolate reductase (DHFR) and its activity is critical for maintaining the BH4/BH2 ratio and, thus, eNOS coupling . This may be particularly important under pathological conditions that favor formation of BH2 in contrast to normal physiological conditions in which the de novo pathway for BH4 synthesis may be sufficient to maintain cellular BH4 levels. Youn et al. [34▪] show that oral administration of folic acid, which can recouple eNOS in angiotensin II-induced hypertensive mice via restoration of endothelial DHFR content and activity, completely attenuates eNOS uncoupling in streptozotocin-induced diabetic mice via restoration of DHFR function. Folic acid supplementation or DHFR overexpression reduced O2 − production and restored NO-dependent endothelial function in these diabetic mice, demonstrating the importance of DHFR activity in regulating eNOS uncoupling in vivo. Further support for the importance of the BH2 reductase activity of DHFR in maintaining BH4 levels in vivo was the finding that DHFR activity is required to maintain eNOS coupling under conditions of genetic BH4 deficiency . Thus, net cellular NO bioavailability likely reflects the balance between de novo BH4 synthesis, loss of BH4 by oxidation to BH2, and regeneration of BH4 by DHFR [34▪].
Both BH2 and BH4 are transported into cultured endothelial cells and the majority of uptake occurs via the equilibrative nucleoside transporter 2 (ENT2) in cultured rat aortic endothelial cells [36,37▪]. The ENT2 transporter is predominantly localized on the apical surface, suggesting a mechanism for preferential uptake of BH2 from plasma by endothelial cells [37▪]. Interestingly, accumulation of BH4 in cultured cells continued linearly, whereas that of BH2 reached a plateau within 10 min, suggesting that BH2 accumulation was counterbalanced by conversion to BH4 via DHFR [37▪]. The relationship between plasma BH4 and endothelial cell BH4 is not clear, and this complicates assessment of BH4 in target tissues in vivo. ENT2 may transport BH2 into endothelial cells for BH4 production via the DHFR salvage pathway, but in cases in which DHFR activity is too low, the bidirectional nature of the ENT2 transporter may move more of the BH2 out of the cell and into the plasma . Hence, higher plasma levels of BH2 may be indicative of the greater oxidative stress within the endothelial cell, but this remains to be verified.
Nitric oxide synthase-independent effects of 6R-5,6,7,8-tetrahydrobiopterin
BH4 also exhibits vasoprotective effects that are independent of NOS. BH4 is a growth factor for endothelial cells  and endothelial progenitor cells , and thus may play a role in wound healing in diabetes. BH4 is also a powerful antioxidant with scavenging capabilities that help preserve NO bioavailability . Recently, BH4 was shown to protect soluble guanylate cyclase against oxidative inactivation that prevents NO binding and renders this enzyme insensitive to activation by NO .
It is well accepted that uncoupled eNOS switches production of NO to production of O2 −, but the precise molecular mechanisms that underlie this switch are not entirely clear. Recently, it was demonstrated that the S-glutathionylation of critical thiols reversibly uncouples eNOS, and the resultant O2 − generation at the reductase domain of S-glutathionylated eNOS is not suppressed by N-nitro-L-arginine methyl ester or calcium removal. This S-glutathionylation uncoupling mechanism would, thus, be different from that induced by lack of substrate L-arginine, increases in competitive methylarginines, or alteration in NOS-bound BH4 [42,43]. Binding of methylarginines to eNOS, in addition to inhibiting NO formation, stimulates O2 − production and uncouples eNOS . ROS can lead to accumulation of methylarginines, further uncoupling eNOS and generating positive feedback for more ROS . The O2 − produced by uncoupled eNOS can generate thiyl radical formation at Cys-908 in eNOS, which in turn can react with reduced glutathione to bring about eNOS S-glutathionylation, a unique mechanism for redox regulation of eNOS . The oxidant stress associated with diabetes may trigger S-glutathionylation of eNOS, inducing endothelial dysfunction, but, at the same time, this post-translational modification of eNOS may actually prevent irreversible oxidation of cellular components by limiting formation of peroxynitrite from NO and O2 − . Interestingly, sequestration of eNOS by caveolin-1 has been described as another means of controlling eNOS-derived O2 − production and the effect of caveolin-1 on the Akt/eNOS axis is differentially regulated by the cellular biopterin status .
NO bioavailability is a major determinant of vascular homeostasis and is reduced in diabetes. New information is emerging to elucidate how eNOS activity and, hence, NO production is dynamically regulated in endothelial cells normally and during diabetes (Fig. 1) . Basic science is driving efforts to identify targets for intervening in the uncoupling of eNOS as well as successfully augmenting BH4 levels, and, at the same time, suppressing BH2 increases in order to achieve the optimal therapeutic benefit of BH4 supplementation. Clinical trials need to continue to determine the optimal concentrations and combinations of L-arginine, BH4, and/or antioxidants to combat the oxidative stress driving down NO production and to address the impact of arginase on cardiovascular outcomes in humans.
This work was supported in part by grants from the National Institutes of Health (R21 HL093689) and American Heart Association (11GRNT7930004) to C.J.M.
Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
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Additional references related to this topic can also be found in the Current World Literature section in this issue (p. 116).
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