Six healthy males participated in the study (mean (± SEM), age 25 (2) yr; BMI 25.9 (1.8) kg·m−2), which was approved by the University of Nottingham Medical School Ethics Committee. Subjects were fully informed of the nature and purpose of the investigation and gave their written consent to participate. All subjects reported that they performed moderate daily activity (e.g., 20–30 min walking to/from their place of work) but were not involved in any exercise training or regular sporting activity. Subjects’ V̇O2peak, determined at a preliminary visit, was 47.5 (3.5) mL·kg−1·min−1
On six separate occasions, each subject reported to the laboratory in the morning after an overnight fast. Visits were separated by at least 1 wk. The evening meal before each visit was identical for each subject. Upon arrival, subjects rested supine for 30 min while measurements of heart rate (HR), blood pressure (BP), and forearm blood flow (BF) were recorded. A cannula was inserted into a vein on the dorsal surface of the hand, which was heated to arterialize the venous drainage of the hand (12). After a 15-min equilibration period, an arterialized venous blood sample was collected from the cannula for subsequent analysis. Immediately after this baseline measurement period, subjects were required to perform one of three “activities” in a randomized manner:
a) One hour lying semirecumbent on an experimental bed (Rest).
b) One hour of repeated bouts of squatting exercise, carrying a backpack containing weight equivalent to 25% of their body mass (Resistance). Each squat required the subject to bend his leg at the knee and lower the weight until the axis of the hips was parallel with the axis of the knees, followed by a return to the standing position. Subjects performed 20 repetitions of the squatting exercise in a 1-min period followed by 3 min of rest in a standing position, whereupon they repeated the squatting exercise. All subjects completed 15 bouts of this work/rest cycle in the 1-h period. Supervision was provided throughout the exercise period and HR was constantly monitored using a telemetric heart rate monitor (Polar PE3000, Polar, Kempele, Finland).
c) One-hour cycling exercise (Cycling). Each subject cycled at a cadence of 70 rpm against a workload predetermined to elicit an oxygen consumption approximately 70% of his peak aerobic capacity. During exercise, subjects ingested 200 mL of water every 15 min to maintain euhydration. HR was monitored by a three-lead ECG throughout the exercise period.
Each activity was designed to modify subsequent glucose disposal: resting responses were considered as a control treatment, cycling exercise was anticipated to result in relatively lower blood glucose concentrations (suggesting greater disposal), and resistance exercise was expected to result in relatively higher blood glucose concentrations (suggesting reduced disposal) compared with cycling.
After each activity, subjects rested supine for 30 min while measurements of HR, BP, and BF were recorded, and samples of blood and expired air were collected. Subjects then ingested 250 mL of a sugar-free fruit drink containing, in a double-blind, randomized manner, either 10-g glycine (placebo) or 10-g L-arginine (Ajinomoto Co., Kawasaki City, Japan). This was immediately followed by ingestion of 378 mL of a CHO solution containing 70-g simple sugars (LucozadeTM, GlaxoSmithKline, Uxbridge, UK). During the subsequent 3-h postingestion period, blood samples were collected every 20 min, expired air samples were collected during the last 15 min of each 30 min period, and HR, BP, and BF were recorded during the last 8 min of each 30-min period.
Blood collection and analysis.
Arterialized-venous blood samples were obtained from the hand via a three-way tap system connected to the cannula. Cannula patency was maintained throughout the experiment by an isotonic saline drip (0.9% sodium chloride BP, Baxter Healthcare Ltd., Thetford, UK). Upon collection, a blood sample (∼ 100 μL) was immediately introduced into an automated analyzer (Hemocue AB, Ångelholm, Sweden) for measurement of glucose concentration. The remainder of the sample (5 mL) was allowed to clot, centrifuged at 3000 rpm for 10 min, and the serum used to measure insulin concentration, using a commercially available radio-immunoassay diagnostic kit (Diagnostic Products Corporation, Los Angeles, CA).
Expired air collection and analysis.
Subjects rested on a bed with their head completely covered by a plastic canopy. The canopy consisted of a domed hood with an inlet for atmospheric air and an outlet from which expired air was collected and analyzed by an on-line gas analysis system (GEM, Europa Scientific Ltd., UK). Before each test, the analysis system was calibrated for accuracy of oxygen and carbon dioxide measurement using certified calibration gases and atmospheric air. The system generated reports of expired air analysis for every 60 s of sampling. From these values, the contributions of different substrates to oxidative metabolism were estimated using standard methods (1).
Forearm blood flow measurement.
Forearm blood flow was measured using venous occlusion plethysmography (27). A high-pressure arterial occlusion cuff was wrapped around the subject’s wrist, and a lower-pressure venous occlusion cuff was wrapped around the upper arm. To measure changes in limb circumference, a calibrated mercury-in-rubber strain gauge of an appropriate size was attached around the forearm between the cuffs and connected to a signal amplifier and microcomputer running chart-recording software (Chart V 3.4 for Apple Macintosh). For each blood flow measurement, the arterial occlusion cuff was inflated to a pressure exceeding arterial systolic pressure to prevent any shift of blood away from the hand into the limb. After 1 min of inflation, to avoid introduction of artifacts to the measurements, the low-pressure venous occlusion cuff was inflated above venous pressure, but lower than systolic pressure, to allow arterial in-flow. Arterial flow was monitored for 8 s, then the low-pressure cuff was deflated to allow venous out-flow of blood. The chart-recording software recorded the resulting signals, which were used to calculate the rate of blood flow, based upon the calibration factor of the gauge.
The data presented as area under curve (AUC) were calculated from the glucose and insulin concentrations of each subject over the measurement period. A curve of best fit was constructed for each series of subject data using a least squares method. A third-order polynomial equation was used to predict the curve of best fit, and the AUC was found by calculating a definite integral from a lower to an upper value for each consecutive data point and totalling all area values in the measurement period. All AUC analysis was performed using graph analysis software (Kaleidagraph V 3.0.2, Synergy Software, Reading, PA).
Comparison of data across and within experimental groups was assessed for statistical significance using ANOVA. Significant differences revealed by ANOVA were scrutinised further using Scheffe’s F-tests. These statistical tests were conducted using commercially available statistical analysis software (StatView, SAS Institute Inc., Cary, NC).
Effects of exercise on blood glucose.
Blood glucose concentration decreased significantly after resistance exercise before L-arginine ingestion (Fig. 1b; P < 0.05), and after cycling exercise (Fig. 1c; P < 0.001) before both placebo and L-arginine ingestion. After CHO ingestion, blood glucose concentration increased significantly in all conditions (all P < 0.001). Glucose concentration peaked 40 min after CHO ingestion after all activities (Table 1) and gradually returned toward resting concentration by the end of the measurement period. Peak glucose concentrations after Resistance and Cycling exercise were significantly lower than those during the resting condition (P < 0.05;Table 1).
Effects of L-arginine on blood glucose.
These responses were no different after L-arginine ingestion compared with placebo ingestion (Fig. 1, a–c). The area under blood glucose concentration/time curve (glucose AUC) was no different after L-arginine ingestion compared with placebo ingestion for all activities (Table 1).
Effects of exercise on serum insulin.
Serum insulin significantly increased in response to CHO ingestion after all activities (all P < 0.05;Fig. 2), peaked within 20–40 min, and had returned to resting concentrations by the end of the measurement period (P > 0.05 at 180 min compared with basal, all conditions). No difference in peak insulin concentration was observed between activities.
Effects of L-arginine on serum insulin.
No differences in peak insulin concentration were seen within any activity when L-arginine was ingested compared with placebo. No significant difference was observed between insulin concentrations after L-arginine or placebo ingestion at any time point during the measurement period. The area under the insulin concentration/time curve (insulin AUC) over the postingestion period after each activity was no different after L-arginine ingestion than when placebo was ingested (Table 1)
Effects of exercise on CHO oxidation.
Total CHO oxidation over the 3-h postingestion period (Fig. 3) was significantly lower after resistance (P < 0.01) and cycling exercise (P < 0.001) compared with CHO oxidation at rest.
Effects of L-arginine on CHO oxidation.
Within any activity, there was no significant difference in CHO oxidation when L-arginine was consumed compared with placebo ingestion, although a consistent tendency for less CHO oxidation was apparent after L-arginine ingestion (Fig. 3).
Effects of exercise on forearm blood flow.
As increased L-arginine availability has been shown to induce peripheral blood flow, forearm blood flow was measured. Forearm blood flow did not change throughout the course of the experiment when subjects were rested or after they had performed resistance exercise (Fig. 4, a and b). Forearm blood flow increased significantly above the preexercise rate after cycling (P < 0.01) and declined during the 3 h of recovery (Fig. 4c).
Effects of L-arginine on forearm blood flow.
Forearm blood flow responses were no different compared with placebo after L-arginine ingestion for any activity (Fig. 4, a–c).
Effects of exercise on hemodynamic function.
Systolic and diastolic blood pressure decreased significantly after resistance and cycling exercise (all P < 0.05) but remained constant during the remainder of the 3-h measurement period.
Effects of L-arginine on hemodynamic function.
No difference in systolic or diastolic blood pressure was observed after L-arginine ingestion compared with placebo ingestion after any activity. No effect of L-arginine treatment upon HR was observed after any activity.
All subjects reported that one of the test drinks (subsequently known to contain L-arginine) had an unpleasant flavor, some reported it leaving a “plastic” aftertaste. One subject reported gastrointestinal discomfort and diarrhea during the day after two of the three occasions that L-arginine was ingested. No subjects reported problems associated with ingestion of the placebo drink.
The aim of this investigation was to determine whether 10 g of L-arginine, when administered orally, could influence the fate of CHO ingested at the same time. Previous investigations have demonstrated that intravenous administration of L-arginine augments glucose-stimulated insulin release (10), increases peripheral blood flow (16), and increases insulin-mediated whole-body glucose disposal (21). The present results suggest that the amount of L-arginine given was insufficient to cause a significant increase in circulating insulin or any enhancement of peripheral blood flow and, consequently, did not significantly improve muscle glucose delivery either at rest or after different modes of exercise. Dietary intake of arginine is approximately 5 g·d−1 in a normal Western omnivorous diet (26). Consumption of a meal containing approximately 25 g L-arginine produced higher plasma insulin concentrations than a meal containing a normal daily L-arginine intake (3). However, such a large amount of L-arginine is unpalatable (personal observation) and can result in gastrointestinal distress (15). Hence, we chose to use a lower quantity of arginine in the current investigation but one that was still greater than the normal daily intake.
The patterns of appearance and disappearance of blood glucose after CHO ingestion with L-arginine were no different from placebo conditions within each activity. These results are reflected by the serum insulin responses (peak insulin and insulin AUC) to CHO and L-arginine ingestion, which were similar to placebo conditions. This demonstrates that the ingestion of 10 g of L-arginine was insufficient to exert any influence upon insulin-stimulated glucose disposal.
Preliminary evidence (13) reports the glucose and insulin responses of healthy subjects receiving L-arginine (1 mmol·kg−1 lean body mass), glucose (25 g) or L-arginine + glucose orally (the amount of L-arginine administered was approximately the same as the 10-g dose used in the current investigation). The authors showed that L-arginine alone stimulated a modest rise in blood glucose concentration but did not stimulate insulin release. When L-arginine was provided with glucose, it decreased peak blood glucose concentration, compared with glucose ingestion alone, but prolonged the rise in blood glucose over the postingestion period. From their evidence, the authors proposed that L-arginine delayed glucose absorption and/or affected endogenous glucose production or clearance from the blood. Although the current investigation has no comparative data for glucose only ingestion, it could be suggested that no impairment of glucose absorption was apparent. Comparison of the peak glucose concentration and glucose AUC after the ingestion of glycine (which is not known to have any effect upon glucose or insulin metabolism) and glucose with that after L-arginine and glucose showed no differences. It cannot be discounted, however, that both L-arginine and glycine might both have similar negative effects upon CHO absorption, but this seems unlikely given the rapid and similar magnitude of increase of blood glucose concentration under all conditions.
L-arginine is also known to stimulate glucagon release (2), which in turn stimulates glycogenolysis in the liver (14). It is possible that the stimulation of glucagon release by L-arginine elevated endogenous glucose production, contributing to the trend toward a slight increase in peak glucose concentration seen in subjects when rested and after cycling exercise. The glucagon response to L-arginine may, however, only have been small in the present study, as it is diminished relative to increases in circulating glucose concentration (20).
The information provided by total CHO oxidation gives an indication of the fate of the ingested CHO, which can be utilized for energy immediately, stored in muscle or liver as glycogen (23), or become incorporated into fat storage as acetyl-CoA or glycerol (6). A previous study from this laboratory showed that during postexercise provision of a similar amount of CHO to that used in the present study (1 g·kg−1 body mass), 30% of the CHO load was extracted by the liver before it was made available to skeletal muscle (7). The degree of CHO oxidation is less when the drive for CHO storage is high. This is demonstrated in the present study by the lesser CHO oxidation after both forms of exercise compared with that in rested subjects (Fig. 3). There was no significant difference in CHO oxidation between treatments after any activity, although a consistent tendency for less CHO oxidation (and presumably greater CHO storage) after L-arginine treatment was apparent. It is possible that, to a small extent, the carbon skeleton of L-arginine was used for glycogen resynthesis; however, the amount of CHO storage suggested by this small difference is unlikely to represent any significant improvement in postexercise substrate replenishment.
The results show that there was no effect of the L-arginine ingestion upon forearm blood flow in rested subjects or after resistance exercise. Examination of the decrease in blood flow from preingestion levels through to the end of the measurement period revealed no effect of L-arginine ingestion upon blood flow compared with the response to placebo. The vasodilatory effect of L-arginine is partly mediated by endogenous insulin release (16), which in the present study was no different between L-arginine and placebo treatments. Nitric oxide is also involved in vasodilation, but despite L-arginine being the unique precursor to NO, no evidence exists that L-arginine availability is limiting to NO production or that increasing its availability produces a consequent increase in NO formation (19). Therefore, it can be suggested that the lack of an effect of L-arginine ingestion upon forearm blood flow was most probably because of the lack of a stimulatory effect on insulin release.
The majority of investigations that have administered L-arginine intravenously to humans have used doses that produce plasma arginine concentrations in the millimolar range (16,20,25). Plasma arginine concentration after ingestion of food containing approximately 25 g L-arginine was elevated by less than 400 μmol·L−1 to a concentration of approximately 600 μmol·L−1 (3). It has recently been demonstrated that only 70% of an oral dose of L-arginine is available for use by the body (5). These authors also demonstrated that a 6-g dose of L-arginine was insufficient to influence BP or total peripheral resistance (TPR) when given orally or intravenously. It is feasible, therefore, to suggest that the dosage of L-arginine given in the present study did not raise plasma arginine concentration sufficiently to produce a glycogenic effect. It would be of interest to determine the minimum amount of ingested L-arginine necessary to produce these desired effects. The amount of L-arginine ingested in the present study was near the limit of palatability, however, and one subject experienced gastrointestinal intolerance during two of the days that L-arginine was ingested. These findings, coupled with a potential loss of water and sodium in the urine after prolonged L-arginine feeding as previously observed (3), all suggest that including high amounts of L-arginine in a CHO beverage would not be a practical method of improving postexercise CHO replenishment.
In conclusion, it is unlikely that the addition of a tolerable amount of arginine to a CHO-containing beverage will improve postexercise glucose transport into muscle and subsequent glycogen resynthesis.
This research was supported by Ajinomoto Co. Ltd.
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