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Nutrient Metabolism

Dietary L-Arginine Supplementation Enhances Endothelial Nitric Oxide Synthesis in Streptozotocin-Induced Diabetic Rats¹

Ripla Kohli^*, Cynthia J. Meininger, Tony E. Haynes^*, Wene Yan^*, Jon T. Self^* and Guoyao Wu^*,,2

^* Faculty of Nutrition and Department of Animal Science, Texas A&M University, College Station, TX 77843 and Department of Medical Physiology and Cardiovascular Research Institute, Texas A&M University System Health Science Center, College Station, TX 77843-1114

²To whom correspondence should be addressed. E-mail: g-wu{at}tamu.edu.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
This study tested the hypothesis that dietary arginine supplementation increases endothelial tetrahydrobiopterin (BH₄) availability for nitric oxide (NO) synthesis in diabetic rats. Streptozotocin-induced diabetic rats either were given unrestricted access to a casein-based diet (Expt. 1) or were pair-fed the diet on the basis of the food intake per kg of body weight of nondiabetic rats (Expt. 2). Beginning 1 d after vehicle or streptozotocin injection, arginine-HCl (1.51%) or alanine (isonitrogenous control, 2.55%) was added daily to the drinking water for nondiabetic rats, whereas concentrations were adjusted (0.43% arginine-HCl and 0.73% alanine) in the drinking water for diabetic rats (which consumed more water) to ensure isonitrogenous provision. At 2 wk after the initiation of arginine supplementation, coronary endothelial cells and plasma were obtained for the measurement of NO synthesis and metabolites. In both experiments, plasma and endothelial concentrations of N^G-monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine increased, but those of arginine as well as endothelial BH₄ availability and NO synthesis decreased in diabetic rats, compared with nondiabetic rats. In both diabetic and nondiabetic rats, arginine supplementation increased plasma concentrations of arginine and insulin, endothelial concentrations of arginine and BH₄, and endothelial NO synthesis, but did not affect plasma and endothelial concentrations of methylarginines or plasma concentrations of homocysteine. Dietary arginine supplementation or provision of a BH₄ precursor normalized endothelial NO synthesis in diabetic rats. Arginine supplementation did not affect plasma glucose levels in nondiabetic rats, but reduced body weight loss and plasma glucose levels in diabetic rats. Thus, dietary L-arginine supplementation stimulates endothelial NO synthesis by increasing BH₄ provision, which is beneficial for vascular function and glucose homeostasis in diabetic subjects.

KEY WORDS: • arginine • nitric oxide • methylarginines • endothelial cells • diabetes

One of the hallmarks of diabetes mellitus is endothelial dysfunction, which may result from a deficiency of nitric oxide (NO),³ the endothelium-derived relaxing factor (1). The cardiovascular complications associated with this deficiency represent a major risk factor associated with the high morbidity and mortality of diabetic patients (2,3). L-Arginine is the physiological nitrogen-containing substrate for NO synthesis by NO synthases (3). Interestingly, both diabetic rats (4,5) and human patients (6,7) have markedly decreased plasma concentrations of arginine. Therefore, in recent years, interest in using L-arginine to prevent or ameliorate endothelial dysfunction in diabetics has grown (8,9). Both clinical and experimental studies have shown beneficial effects of L-arginine administration in improving vascular function in diabetic subjects (10–12). However, the mechanism for the action of arginine in enhancing endothelial NO synthesis remains unknown.

Tetrahydrobiopterin (BH₄), an essential factor for NO synthase, plays a crucial role in regulating endothelial NO synthesis (13–15). We recently found that impaired endothelial NO synthesis in diabetic BioBreeding (BB) rats was due to a deficiency of BH₄ owing to reduced expression of GTP-cyclohydrolase I, the first and rate-controlling enzyme in de novo synthesis of BH₄ (15). Our in vitro studies further demonstrated that increasing extracellular L-arginine concentration enhanced GTP-cyclohydrolase I expression, thereby increasing BH₄ availability for NO production in cultured endothelial cells (16). However, it is not known whether dietary arginine supplementation increases BH₄ availability for NO generation in endothelial cells of normal or diabetic subjects. Also, it is not known whether increased endothelial synthesis of NO, a major regulator of glucose utilization by skeletal muscle (2), is associated with improved glucose homeostasis in diabetes.

Thus, we hypothesized that dietary L-arginine supplementation increases BH₄ availability for endothelial NO synthesis in diabetics. The major objective of the present study was to test this hypothesis using rats with streptozotocin (STZ)-induced diabetes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Chemicals. Hexokinase, glucose-6-phosphate dehydrogenase, and nitrate reductase were purchased from Roche. Joklik’s modified minimal essential medium, Dulbecco’s phosphate-buffered saline, Dulbecco’s modified Eagle’s medium, Basal Medium Eagle, and penicillin/streptomycin/amphotericin B were obtained from GIBCO-BRL. Collagenase type-II was purchased from Worthington Biochemical. Heparin sodium was purchased from Elkins-Sinn, and 2,3-diaminonaphthalene, dithioerythritol, amino acids, and STZ were procured from Sigma.

    Animals. Male Sprague-Dawley rats (65 d old; Harland) were fed a casein-based diet (Research Diets) (17). This diet contained 20% casein, 0.3% DL-methionine, 15% cornstarch, 50% sucrose, 5% cellulose, 5% corn oil, 3.5% salt mix, 1% vitamin mix, and 0.2% choline bitartrate, and provided 16.812 MJ gross energy/kg (17). Arginine and alanine content of the diet were 0.64% and 0.46%, respectively. For healthy young and adult rats, this diet was not deficient in arginine (17). Two experimental series were conducted, in which STZ-diabetic rats either were given unrestricted access to the casein-based diet (Expt. 1) or were pair-fed the diet on the basis of the food intake per kg of body weight of nondiabetic rats (Expt. 2). Food intake, and therefore dietary intake of all nutrients (including amino acids, antioxidant vitamins, and antioxidant minerals that affect BH₄ availability) differed between nondiabetic and STZ-diabetic rats that had unrestricted access to feed (18). To overcome this confounding problem, rats were pair-fed in Expt. 2 to ensure similar intake of all nutrients. Because this study involved nitrogen metabolism and animal growth, an isonitrogenous control for arginine supplementation was necessary for the sound interpretation of results. Alanine was chosen for isonitrogenous control from among the nonessential amino acids on the basis of the following considerations. First, alanine is not a toxic substance, antioxidant, neutrotransmitter, or a substrate for citrulline/arginine synthesis (19,20). Second, alanine metabolism does not consume BH₄, and this neutral amino acid does not disturb acid-base balance (20). Third, animals have a high capacity to catabolize alanine (21). Fourth, in our preliminary study, we found that the addition of 0.75% alanine to drinking water did not affect food intake, water consumption, body weight loss, plasma glucose concentration, endothelial BH₄ availability, or endothelial NO synthesis in STZ-diabetic rats, compared with no alanine supplementation. Thus, dietary supplementation of alanine did not impair glucose homeostasis or adversely affect STZ-diabetic rats. This research was approved by the Texas A&M University Animal Care and Use Committee.

In Expt. 1, 70-d-old rats (n = 8 per treatment group) received an intravenous injection either of STZ (50 mg/kg body wt) to induce diabetes or of vehicle solution (50 mmol/L sodium citrate, pH 4.5) (22). At 1 d after the injection of STZ or vehicle solution, the nondiabetic rats were given drinking water containing either 1.51% L-arginine-HCl (equivalent to 1.25% arginine) or 2.55% L-alanine (isonitrogenous control). Concentrations in the drinking water supplied to the diabetic rats were adjusted daily (0.43% arginine-HCl and 0.73% alanine on average) to ensure isonitrogenous intake, because their water consumption was 3.5 times that of the nondiabetic rats. Drinking water was changed daily. Throughout the course of the study, all rats had free access to drinking water and the casein-based diet. Arginine-HCl, rather than free arginine base, was chosen to prevent any problems associated with alkalinity of the drinking water and acid-base imbalance in the body (8). The dose of arginine was chosen because our preliminary study showed it to normalize plasma arginine concentrations in STZ-diabetic rats. At 1 and 2 d after injection of STZ or vehicle solution and again after 14 d of arginine supplementation, tail venous blood samples (0.1 mL) were obtained from unanaesthetized rats between 0830 and 0900, using a heparinized microhematocrit tube (23). The plasma was analyzed for glucose and amino acids. At the end of the 14-d arginine supplementation period, the rats were anesthetized by intraperitoneal administration of pentobarbital sodium (140 mg/kg body wt) (24), and cardiac blood samples (5 mL) were collected for analysis of plasma insulin, homocysteine, and methylarginines, whereas coronary endothelial cells were isolated for metabolic studies (see below).

Experiment 2 was conducted as described for Expt. 1, except that diabetic rats were individually pair-fed the casein-based diet on the basis of the food intake per kg of body weight of nondiabetic rats. After 14 d of arginine supplementation, blood samples (tail vein and heart) and coronary endothelial cells were obtained, as described for Expt. 1.

    Isolation of coronary endothelial cells. Coronary endothelial cells were isolated from the hearts of diabetic and nondiabetic rats using collagenase, as we previously described (24). Cells were suspended in 1 mL of Basal Medium Eagle containing 5 mmol/L D-glucose and 0.5 mmol/L L-glutamine. The endothelial identity of the collected cells was confirmed by the uptake of modified low-density lipoprotein (24,25).

    Synthesis of NO in freshly isolated endothelial cells. Endothelial cells (1.5 x 10⁶) were rinsed 3 times with 1 mL of Basal Medium Eagle containing 0.2 mmol/L L-arginine, 0.5 mmol/L L-glutamine, 5 mmol/L D-glucose, 100,000 U/L penicillin, 100 mg/L streptomycin and 0.25 mg/L amphotericin B. Cells were preincubated at 37°C for 0.5 h in 0.5 mL of Basal Medium Eagle, then incubated at 37°C for 6 h in 0.5 mL of fresh Basal Medium Eagle. Concentrations (mmol/L) of other amino acids (all L-isomers except for glycine) in the Basal Medium Eagle were as follows: alanine, 0.4; asparagine, 0.05; aspartate, 0.05; glutamate, 0.1; cystine, 0.05; glycine, 0.3; histidine, 0.05; isoleucine, 0.2; leucine, 0.2; lysine, 0.2; methionine, 0.1; phenylalanine, 0.1; proline, 0.2; serine, 0.2; threonine, 0.2; tryptophan, 0.1; tyrosine, 0.1; and valine, 0.2. The Basal Medium Eagle also contained 0 or 10 µmol/L sepiapterin [a substrate for BH₄ synthesis via the salvage pathway (26)] to determine whether increasing BH₄ availability would normalize endothelial NO synthesis in diabetic rats. The concentration of sepiapterin was chosen on the basis of our previous studies with endothelial cells from diabetic BB rats (15). At the end of the 6-h incubation period, media and cells were analyzed for nitrite plus nitrate (an indicator of NO production) and BH₄, respectively, using HPLC methods (27,28). In all experiments, medium incubated without cells was used as the control.

    Measurement of plasma glucose, insulin, and amino acids. Plasma glucose concentration was assayed by an enzymatic method involving hexokinase and glucose-6-phosphate dehydrogenase (24). Insulin was analyzed using a radioimmunoassay kit (rat insulin kit) from Linco. Plasma concentrations of amino acids, except for homocysteine and methylarginines, were measured using an HPLC method, as previously described (29).

Plasma total homocysteine was measured as described by Hyland and Bottiglieri (30) with modifications to improve the sensitivity of detection and facilitate automation on the HPLC apparatus. The plasma sample (150 µL) was mixed with 10 µL of 10% ß-mercaptoethanol, and the whole solution was loaded into a 10-kD Millipore Ultrafree-MC filter. The filtrate (50 µL) was mixed with 50 µL of 28 mmol/L ß-mercaptoethanol. After 5 min, 25 µL of 50 mmol/L iodoacetic acid was added to the assay mixture, and 100 µL of this solution was mixed with 1.2% benzoic acid (in 40 mmol/L sodium borate, pH 9.5). The assay mixture (25 µL) was derivatized in an autosampler (Model 712 WISP; Waters) with 25 µL of 30 mmol/L o-phthaldialdehyde, and 25 µL of the solution was injected into a Phenosphere ODS (1) column (4.6 x 250 mm, i.d. 5 µm; Phenomenex). Separation of homocysteine was satisfactorily achieved under isocratic conditions (1.3 mL/min) with Solvent A (50 mmol/L sodium acetate and 8% acetonitrile, pH 6.8). Fluorescence was monitored with excitation at 335 nm and emission at 455 nm using a Waters Model 474 fluorescence detector (gain setting at 100). Retention time for homocysteine was 4.2 min. At 4 min after the homocysteine derivative was eluted, the column was washed (1.3 mL/min) with 30% Solvent A and 70% Solvent B (HPLC-grade acetonitrile) for 2 min and then with 100% Solvent A for 13 min to prepare the column for the next injection. The recovery of homocysteine from plasma and the Millipore Ultrafree-MC filter was 94%, as determined with known amounts of standard. Homocysteine in samples was quantified using Millenium-32 Software (Waters) on the basis of standards (2 to 10 µmol/L) treated in the same manner as samples.

Plasma concentrations of N^G-monomethylarginine (NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) were analyzed using the HPLC methods of Bode-Böger et al. (31) and Teerlink et al. (32), with modifications to improve sample cleanup, eliminate the need for column heating, and facilitate automation on the HPLC apparatus. The plasma sample (0.2 mL) was deproteinized with 0.1 mL of 1.5 mol/L HClO₄, then 0.05 mL of 2 mol/L K₂CO₃ was added. The whole neutralized extract plus 0.7 mL of Dulbecco’s phosphate-buffered saline was loaded into a cation-exchange solid-phase extraction column (1 mL, 30 mg; Waters) equilibrated with 0.1 mol/L HCl. The column was washed with 1 mL of 0.1 mol/L HCl and then with 1 mL of 100% HPLC-grade methanol. Methylarginines were eluted with 1 mL of 29.5% ammonium hydroxide:water:methanol (10:40:50, by vol); the solvent was removed in a Model RC10.10 centrifugal evaporator (Jouan), and the residue was suspended in 0.2 mL of HPLC-grade H₂O. An aliquot (10 µL) of the suspension was mixed with 150 µL of HPLC-grade H₂O and 10 µL of 1.2% benzoic acid (in 40 mmol/L sodium borate, pH 9.5). This solution (15 µL) was derivatized in an autosampler (Model 712 WISP) with 15 µL of 30 mmol/L o-phthaldialdehyde (29), and 15 µL of the derivatized mixture was injected into a Nucleosil 100–5 C₆H₅ column (4.6 x 250 mm; Macherey-Nagel). Separation of methylarginines was satisfactorily achieved under isocratic conditions (0.7 mL/min) using a solution consisting of 33.3% methanol and 0.96% sodium citrate (pH 6.8) (running time is 60 min). Fluorescence was monitored with excitation at 340 nm and emission at 455 nm using a Waters Model 474 fluorescence detector (gain setting at 100). Retention times for NMMA, SDMA, and ADMA were 18.8, 23.6, and 26.4 min, respectively. The recovery of NMMA, ADMA, and SDMA from the solid-phase extraction column was 98, 86, and 97%, respectively, as determined with known amounts of standards. Methylarginines in samples were quantified using Millenium-32 Software on the basis of standards (0.5 to 5 µmol/L) treated in the same manner as samples.

    Analysis of BH_4, arginine, and methylarginines in freshly isolated endothelial cells. Arginine and BH₄ in endothelial cells (1.5 x 10⁶) were determined using HPLC methods, as previously described (15,33). For methylarginine analysis, endothelial cells (3 x 10⁶) were lysed with 0.2 mL of 1.5 mol/L HClO₄, then 0.1 mL of 2 mol/L K₂CO₃ was added. The neutralized cell lysates were centrifuged at 10,000 x g for 1 min, and an aliquot (0.2 mL) of the supernatant was used for sample cleanup and determination of NMMA, ADMA, and SDMA concentrations, as described above.

    Calculation and statistical analysis. Intracellular concentrations of BH_4, arginine, and methylated arginines were calculated on the basis of the mean cell volume of rat coronary endothelial cells (0.348 µL/10⁶ cells), which was measured using ³H₂O (34). Intracellular water content was similar (P > 0.05) among endothelial cells from alanine- and arginine-supplemented nondiabetic and diabetic rats. Combustion energy values of arginine (3738 kJ/mol) and alanine (1577 kJ/mol) (35), respectively, were used to calculate gross energy intake from arginine and alanine in drinking water. Values presented in the text are means ± SEM. Data were statistically analyzed by two-way (Tables 1, 2, 3, 4, 5, 6) or three-way (Table 7) ANOVA, using SAS statistical software (SAS Institute). Differences between means were determined by the Student-Newman-Keul’s multiple comparison test. Values of P 0.05 were considered significant.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

    Body weight. Body weight was recorded daily throughout the study period (Table 3). In Expt. 1, the mean body weight gain for nondiabetic rats was 35 g, but the STZ-diabetic rats that were not subjected to arginine treatment lost 46 g during the 14-d treatment period (P < 0.01). Dietary arginine supplementation did not affect body weight in nondiabetic rats (P > 0.05), but reduced the body weight loss of diabetic rats by 67% (P < 0.01), compared with that of corresponding rats subjected to alanine treatment. In Expt. 2, the mean body weight gain for nondiabetic rats was 34 g, but the STZ-diabetic rats that were not subjected to arginine treatment lost 167 g over the 14-d treatment period (P < 0.01); arginine supplementation reduced the body weight loss of diabetic rats by 36% (P < 0.01), compared with alanine supplementation.

    Plasma glucose and insulin. All rats treated with the STZ injection developed diabetes within 24 h of administration, as determined on the basis of glucosuria, ketosis, hyperglycemia (plasma glucose, 17 to 20 mmol/L), and body weight loss. In Expt. 1, at 1 d after beginning arginine treatment, plasma concentrations of glucose did not differ (P > 0.05) between alanine- and arginine treated nondiabetic rats (8.24 ± 0.74 vs 8.17 ± 0.63 mmol/L, n = 8), or between alanine- and arginine-treated diabetic rats (20.3 ± 1.51 vs. 18.7 ± 1.74 mmol/L, n = 8). In Expt. 2, at 1 d after beginning arginine, plasma concentrations of glucose did not differ (P > 0.05) between alanine- and arginine-treated nondiabetic rats (8.38 ± 0.82 vs. 7.91 ± 0.41 mmol/L, n = 8), or between alanine- and arginine-treated diabetic rats (19.8 ± 1.74 vs. 16.9 ± 1.90 mmol/L, n = 8). However, in both experiments, at 14 d after the onset of diabetes, dietary arginine supplementation markedly reduced (P < 0.01) plasma concentrations of glucose (Table 3). In Expts. 1 and 2, plasma glucose levels fell (P < 0.01) by 38% and 54%, respectively, in arginine-treated diabetic rats, compared with alanine-treated diabetic rats (Table 3). In contrast, arginine treatment did not affect plasma concentrations of glucose in nondiabetic rats (P > 0.05). As expected, rats with STZ-induced diabetes exhibited low concentrations of plasma insulin (P < 0.01), regardless of food intake. Arginine treatment increased (P < 0.01) plasma concentrations of insulin in both nondiabetic and diabetic rats, compared with rats subjected to alanine treatment.

    Plasma amino acids. Plasma concentrations of arginine, glutamine, ornithine and homocysteine in STZ-diabetic rats decreased (P < 0.01) and those of NMMA, ADMA, and SDMA increased (P < 0.01), compared with nondiabetic rats, regardless of food intake (Table 4). Furthermore, plasma concentrations of arginine, citrulline, and ornithine were higher (P < 0.01), and those of alanine were lower (P < 0.01) in arginine-treated diabetic and nondiabetic rats, compared with the corresponding alanine-treated rats (Table 5). Arginine treatment reduced (P < 0.01) plasma glutamine concentration in nondiabetic rats, and normalized plasma glutamine concentration in diabetic rats. In contrast, arginine treatment did not affect (P > 0.05) plasma concentrations of methylarginines, homocysteine, and other amino acids (including glutamate, lysine, histidine, and branched-chain amino acids) in either nondiabetic or diabetic rats (data not shown).

    Intracellular arginine and methylarginines. The effects of diabetes and arginine supplementation on concentrations of arginine and methylarginines in endothelial cells were summarized (Table 6). Intracellular concentrations of arginine decreased (P < 0.01), but those of NMMA, ADMA, and SDMA increased (P < 0.01) in diabetic rats, compared with nondiabetic rats, in both Expt. 1 and Expt. 2. Dietary supplementation of arginine increased (P < 0.01) intracellular concentrations of arginine in both nondiabetic and diabetic rats, but did not affect (P > 0.05) those of NMMA, ADMA, and SDMA, regardless of food intake.

    BH₄ concentration and NO production in endothelial cells. The effects of diabetes and arginine supplementation on endothelial BH₄ availability and in vitro NO synthesis were summarized (Table 7). In both Expt. 1 and Expt. 2, intracellular BH₄ concentration and NO production decreased in the endothelial cells of diabetic rats (P < 0.01), compared with those of nondiabetic rats. Dietary arginine supplementation for 14 d markedly increased (P < 0.01) BH₄ concentration and NO production in the endothelial cells of both diabetic and nondiabetic rats, regardless of food intake. The addition of 10 µmol/L of sepiapterin to the incubation medium increased (P < 0.01) both BH₄ concentration and NO synthesis in the endothelial cells of both nondiabetic and diabetic rats. It is important to note that restoring the intracellular BH₄ level with sepiapterin normalized endothelial NO synthesis in diabetic rats to levels similar to those in nondiabetic rats.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The STZ-diabetic rat is a well-characterized animal model of type I diabetes mellitus for metabolic studies (4,24) and evaluation of vascular function (3,36,37). Dietary arginine supplementation did not affect the food or water intake of the studied rats (Table 1); therefore, the effects of arginine treatment on STZ-diabetic rats in Expts. 1 and 2 were independent of the dietary intake of other nutrients. To our knowledge, this is the first report on the effects of dietary arginine supplementation on endothelial concentrations of arginine, methylarginines, and BH₄ as well as on in vitro endothelial NO synthesis in normal and diabetic animals.

A novel and important finding of this study is that daily oral administration of L-arginine-HCl (1.4 g arginine/kg body wt) for 14 d reduced plasma glucose concentration and the loss of body weight in STZ-diabetic rats, regardless of food intake (Table 3). Notably, daily oral administration of L-arginine-HCl (0.62 g arginine/kg body wt) to diabetic hamsters (38) or intraperitoneal administration of arginine (10 µmol) to rats with alloxan-induced diabetes (39) reduced plasma glucose levels by up to 65%. The underlying mechanisms for these results are unknown, but may involve an increase in insulin release. In the STZ-diabetic rat model, not all pancreatic ß-cells are destroyed, and the remaining cells can secrete a quantity of insulin sufficient to keep the animals alive for up to 2 mo (36,37). Arginine stimulates the secretion of insulin (an anabolic hormone) by pancreatic ß-cells (40). Thus, dietary L-arginine supplementation increases plasma concentrations of insulin in nondiabetic and diabetic rats (Table 3), which would promote net protein synthesis and glucose utilization in skeletal muscle (20). The available evidence suggests that the action of insulin and arginine involves the following mechanisms. First, both insulin and arginine stimulate NO production by endothelial cells (16,41), which would contribute to increases in blood flow and, therefore, glucose and amino acid uptake by skeletal muscle in vivo (42). Second, NO itself stimulates glucose transport and oxidation by skeletal muscle (43). Third, physiological concentrations of NO may inhibit muscle proteolysis (40).

The results of the present study confirm previous findings that plasma concentrations of arginine are reduced in diabetic rats (4,5) and humans (6,7). Because the intestinal synthesis of citrulline, the exclusive endogenous source of arginine (44), did not differ between STZ-diabetic and nondiabetic rats (18), the relative deficiency of arginine in STZ-diabetic rats may result from an increase in arginine catabolism and/or a decrease in the extraintestinal conversion of citrulline to arginine. Whatever the mechanism, dietary arginine supplementation increased both arginine concentration (Table 5) and in vitro NO synthesis in endothelial cells (Table 6) in both diabetic and nondiabetic rats. Changes in arginine concentration in the endothelial cells closely matched those in plasma, supporting a role for membrane transport in the regulation of intracellular arginine availability (44). Although the intracellular concentration of L-arginine (0.48 mmol/L) in diabetic rats was substantially lower than that in nondiabetic rats (Table 6), it was still much higher than the K_m value (2.9 µmol/L) of endothelial NO synthase for L-arginine (45). This finding implies that endothelial NO synthase was saturated with intracellular L-arginine in both diabetic and nondiabetic rats and that arginine was not a limiting substrate for endothelial NO synthesis by the enzyme. However, dietary L-arginine supplementation did markedly increase in vitro NO production in the coronary endothelial cells of both diabetic and nondiabetic rats (Table 7). Remarkably, the arginine treatment normalized endothelial NO synthesis in STZ-diabetic rats to the values for nondiabetic rats (Table 7). Our results support the view that intracellular or extracellular arginine concentrations are critical for endothelial NO production (46). In addition, these findings provide a metabolic explanation for the beneficial effect of dietary arginine supplementation on cardiovascular function in diabetic patients (9–12).

Because NMMA and ADMA are competitive inhibitors of NO synthase and SDMA inhibits arginine transport by animal cells (44), interest in their role in vascular function has grown (47–49). Consistent with studies of alloxan-induced diabetic rabbits (50), concentrations of NMMA, ADMA, and SDMA in the plasma and endothelial cells of STZ-diabetic rats increased, compared with those of nondiabetic rats (Table 4). Recent studies suggest that reduced expression of dimethylarginine dimethylaminohydrolase, the enzyme that degrades ADMA, contributes to an elevated concentration of ADMA in the endothelial cells (47). In contrast, plasma concentrations of homocysteine [an inhibitor of NO synthesis (46)] decreased in STZ-diabetic rats, as reported by Ratnam et al. (51). This result can be explained by increased expression of hepatic cystathionine ß-synthase, a key enzyme in the transsulfuration pathway that irreversibly converts homocysteine to cysteine (51). Thus, homocysteine is not likely to contribute to the reduced NO synthesis in the endothelial cells of STZ-diabetic rats. In our recent study involving 24-h incubation of freshly isolated rat coronary endothelial cells in Basal Medium Eagle containing 0.2 mmol/L of arginine and physiological concentrations of other amino acids, we found that the addition of 2 µmol/L of NMMA or ADMA to the medium decreased NO synthesis by 7%, compared with 1 µmol/L of NMMA or ADMA (unpublished data). This result suggests that the increase in plasma concentration of NMMA from 1.1 to 1.65 µmol/L and of ADMA from 0.8 to 1.2 µmol/L is not a major factor in the marked decrease in endothelial NO synthesis in STZ-diabetic rats.

Tetrahydrobiopterin plays a crucial role in regulating endothelial NO synthesis (13–15). It is an essential cofactor for NO synthase, where BH₄ functions as a one-electron donor to a heme-dioxyenzyme intermediate (26). We recently discovered that BH₄ concentrations were markedly reduced in the endothelial cells of spontaneously diabetic BB rats (an animal model of type I diabetes mellitus) due to decreased expression of GTP-cyclohydrolase I protein (15), the first and rate-controlling enzyme for de novo synthesis of BH₄ (26). Endothelial BH₄ was also deficient in STZ-diabetic rats (Table 7), which is consistent with the recent report that STZ-diabetic tissues (e.g., liver and kidney) exhibit reduced expression of GTP-cyclohydrolase I protein (52). Notably, dietary arginine supplementation increased in vivo BH₄ concentrations in the endothelial cells of both diabetic and nondiabetic rats (Table 7). This result extends our recent in vitro observation that arginine stimulated BH₄ synthesis in endothelial cells by enhancing the expression of GTP-cyclohydrolase I protein (16). Remarkably, restoring BH₄ levels with sepiapterin [a precursor of BH₄ via the salvage pathway (26)], like dietary arginine supplementation, normalized NO synthesis in the endothelial cells of STZ-diabetic rats (Table 7), as we previously reported for diabetic BB rats (15). Functionally, increasing arginine or BH₄ availability prevents endothelial dysfunction in STZ-diabetic rats (53,54) and humans (11,55) via an NO-dependent mechanism. Our findings indicate that dietary L-arginine supplementation enhanced endothelial NO generation via an increase in BH₄ availability in diabetic rats. Collectively, these results support the view that a deficiency of BH₄ is the metabolic basis for impaired endothelial NO generation in diabetes (15,56).

Dietary arginine supplementation may represent a potentially useful strategy for the management of diabetes. Arginine is a stable amino acid in an aqueous solution, and is not destroyed by sterilization conditions (e.g., high temperature and high pressure) (40). In addition, arginine is nontoxic, and its administration is generally safe for both humans and animals (8,9). Although arginine cannot replace insulin in the treatment of patients with type I diabetes, this amino acid may be used along with insulin therapy to increase insulin secretion by the remaining pancreatic ß-cells and improve insulin sensitivity in tissues via enhanced production of NO (42). This may help reduce the doseage and frequency of insulin therapy for patients with type I diabetes, while improving protein balance and endothelial function. In patients with type II diabetes, the pancreas is exhausted from the overproduction of insulin to overcome the insulin resistance of tissues, and most clinicians do not use drugs or agents that stimulate insulin secretion to treat this type of diabetes (57). In contrast to STZ-diabetic rats, dietary arginine supplementation did not affect plasma insulin levels in Zucker diabetic fatty rats, an animal model of type II diabetes (58). Similarly, arginine treatment did not increase the plasma concentration of insulin in patients with type II diabetes (12). However, arginine supplementation may be a promising method of improving cardiovascular function in patients with type II diabetes by increasing endothelial NO synthesis. In support of this view, recent studies indicate that intravenous infusion of arginine (30 g over 30 min) reduces blood pressure and improves hemodynamic function in patients with type II diabetes (11,12). Thus, dietary arginine supplementation may enhance insulin sensitivity and attenuate or even prevent insulin resistance, thereby eliminating the need for the additional insulin therapy that is often required with sulfonylurea [a drug used for the treatment of type II diabetes for > 40 y (57)].

In conclusion, treatment of STZ-diabetic rats with dietary arginine supplementation reduced body weight loss, improved glucose homeostasis, increased plasma and endothelial concentrations of arginine, and enhanced endothelial BH₄ availability for NO synthesis. These findings provide a biochemical basis for the beneficial effect of dietary arginine supplementation in preventing endothelial dysfunction in diabetic subjects.

	ACKNOWLEDGMENTS

 
The authors thank Frances Mutscher for office support.

	FOOTNOTES

 
¹ Supported in part by the American Heart Association of Texas, grant 0255878Y, and the Juvenile Diabetes Research Foundation, grant 1–2002-228.

³ Abbreviations used: ADMA, asymmetric dimethylarginine; BB, BioBreeding; BH₄, tetrahydrobiopterin; NMMA, N^G-monomethylarginine; NO, nitric oxide; SDMA, symmetric dimethylarginine; STZ, streptozotocin.

Manuscript received 14 August 2003. Initial review completed 5 September 2003. Revision accepted 13 November 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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