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FINAL ACCEPTED VERSION

L-ARGININE INFUSION INCREASES GLUCOSE CLEARANCE DURING

PROLONGED EXERCISE IN HUMANS

G.K. McConell1,2, N. N. Huynh2, R.S. Lee-Young1, B. J. Canny2, G. D. Wadley1.

1Department of Physiology, The University of Melbourne, Parkville, Victoria, 3010,

Australia; 2Department of Physiology, Monash University, Clayton, Victoria, 3800,

Australia.

Short title: L-arginine and glucose disposal during exercise.

Correspondence: Dr. Glenn McConell,Department of Physiology,The University of Melbourne,Parkville, Victoria,3010, Australia.PH: 61-3-8344-5844FAX: 61-3-8344-5818Email: mcconell@unimelb.edu.au

Articles in PresS. Am J Physiol Endocrinol Metab (August 16, 2005). doi:10.1152/ajpendo.00263.2005

Copyright © 2005 by the American Physiological Society.

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ABSTRACT

Nitric oxide synthase (NOS) inhibition has been shown in humans to attenuate

exercise-induced increases in muscle glucose uptake. We examined the effect of

infusing the NO precursor L-arginine (L-Arg) on glucose kinetics during exercise in

humans. Nine endurance-trained males cycled for 120 min at 72±1% VO2 peak

followed immediately by a 15 min “all-out” cycling performance bout. A [6,6-

2H]glucose tracer was infused throughout exercise and either saline alone (Control,

CON) or saline containing L-Arg HCl (L-Arg, 30 g at 0.5g/min) was co-infused in a

double blind, randomized order during the last 60 min of exercise. L-Arg augmented

the increases in glucose Ra, glucose Rd and glucose clearance rate (L-Arg: 16.1±1.8

ml.min-1.kg-1, CON: 11.9±0.7 ml.min-1.kg-1 at 120 min. P<0.05) during exercise with

a net effect of reducing plasma glucose concentration during exercise. L-Arg

infusion had no significant effect on plasma insulin concentration but attenuated the

increase in non-esterified fatty acid and glycerol concentrations during exercise. L-

Arg infusion had no effect on cycling exercise performance. In conclusion, L-Arg

infusion during exercise significantly increases skeletal muscle glucose clearance in

humans. Since plasma insulin concentration was unaffected by L-Arg infusion,

greater NO production may have been responsible for this effect.

Keywords: Nitric oxide, glucose kinetics, contraction.

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INTRODUCTION

Skeletal muscle glucose uptake is increased by translocation of the GLUT-4 glucose

transporter from intracellular vesicles to the plasma membrane (23). Both insulin and

muscle contraction increase skeletal muscle GLUT-4 translocation and glucose

uptake, but the mechanisms involved differ (23, 36, 37, 49). The factors regulating

glucose uptake into skeletal muscle during exercise are unclear with calcium,

calcium/calmodulin-dependent protein kinase, protein kinase C, AMP-activated

protein kinase (AMPK) and nitric oxide (NO) all implicated (23, 49).

We have evidence in humans that NO may be playing an essential role in the

regulation of skeletal muscle glucose uptake during exercise (9, 34). We found

during cycling exercise in young, healthy individuals that femoral artery infusion of a

NO synthase (NOS) inhibitor reduced leg glucose uptake during exercise by 40-50%

without influencing leg blood flow (LBF), blood pressure or arterial plasma insulin

concentration (9). In a follow up study we found that NOS inhibition reduced leg

glucose uptake during exercise to a greater extent in people with type 2 diabetes than

in matched controls (34). Although our results in humans are clear, studies in rats

examining the effect of NOS inhibition on contraction-stimulated glucose uptake

have yielded conflicting results (3, 17, 26, 51, 53, 57).

Aside from NOS inhibition, another way to examine the potential importance of NO

in skeletal muscle glucose disposal during exercise in humans is to examine the effect

of increasing the availability of L-arginine which is the substrate of NOS. In skeletal

muscle the major form of NOS expressed is the alternatively spliced neuronal form

(nNOSµ) (54)(Bradley et al. unpublished observations) which is associated with the

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sarcolemma. There is a small amount of eNOS expressed in skeletal muscle which,

in humans, appears to be confined to the endothelium of blood vessels (19, 20). We

hypothesise that L-arginine enters skeletal muscle where it is converted to NO and L-

citrulline by nNOSµ. Skeletal muscle NOS activity is increased during treadmill

exercise in rats (50) and electrical stimulation increases NO production in primary rat

skeletal muscle cell culture (55) and in isolated rat skeletal muscle (4). Exercise

appears to increase NO production in humans based on increases in urinary

nitrate/nitrite and cGMP levels in humans (7). L-arginine infusion also increases

plasma and urinary nitrate/nitrite content and cGMP concentrations in humans (5, 6,

27) suggesting increased NO production.

We postulate that any effects of L-arginine infusion on glucose kinetics during

exercise, if they exist, will most likely occur independent of changes in

haemodynamics, in particular muscle blood flow. Intravenous infusion of L-arginine

during exercise in healthy individuals (11) and patients with coronary heart disease

(45) has no affect on heart rate or blood pressure during exercise. In addition,

infusing L-arginine through a microdialysis probe into skeletal muscle has no effect

on human skeletal muscle blood flow during exercise (25) and 5 min of L-arginine

infusion into the femoral artery has no effect on leg blood flow during leg exercise in

humans (9). Further, NOS inhibition has no affect on leg blood flow during leg

exercise in humans (9, 18, 34, 46). Therefore, it appears that although NO clearly

effects blood flow at rest (46), neither increases (via L-Arg) or decreases (via NOS

inhibition) in NO production have any effect on blood flow during exercise in

humans.

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In humans, L-arginine infusion at rest results in a ~2-fold increase in plasma insulin

concentration (6, 45). It is not known if L-arginine infusion increases plasma insulin

levels during prolonged exercise in humans (i.e. under conditions when increases in

plasma catecholamines inhibit insulin secretion). It is important to determine this

since insulin is additive on skeletal muscle glucose uptake during exercise (16). Two

weeks of oral L-arginine-L-aspartate supplementation had no effect on plasma insulin

concentration during ~3 hr of running in endurance-trained athletes (15).

No study has examined the effect of L-arginine infusion on prolonged exercise

performance in humans. It is possible that if L-arginine infusion increases glucose

uptake into muscle during prolonged exercise it may also improve exercise capacity.

All previous studies examining the effects of L-arginine on exercise capacity have

only examined short intense incremental exercise (8-15 min VO2 max test). Dietary

L-arginine supplementation increases aerobic capacity (~8-9% increase in VO2 max)

in hypercholesterolemic and normal mice (38). In humans results have been

contradictory (60), but on balance it would appear that chronic oral L-arginine

supplementation improves VO2 max exercise capacity in patients with cardiovascular

diseases (12, 13, 48). Chronic oral L-arginine supplementation appears to have no

effect on VO2 max test exercise time in healthy individuals (1). In addition, acute L-

arginine infusion appears to have no effect on VO2 max test exercise time in all

human studies, including in patients with chronic heart failure (32) and patients with

hypercholesterolaemia (61).

Therefore, the first aim of this study was to determine whether L-arginine infusion

during exercise increases glucose disposal in humans. The second aim was to

determine whether L-arginine infusion influences prolonged exercise performance in

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healthy humans. We hypothesized that L-arginine infusion during exercise would

increase the rate of glucose disappearance but not significantly influence exercise

performance during exercise in healthy humans. We chose to use endurance-trained

participants in this study to minimize the possibility of adaptive changes between the

two experimental trials (CON and L-Arg). In addition, in order to detect an exercise

performance effect of a particular treatment it is known to be most appropriate to

employ exercise-trained participants who are more reproducible in their exercise

performance (29).

METHODS

PARTICIPANTS

Nine endurance-trained males provided informed written consent to participate in this

study which was approved by the Monash University Standing Committee for

Research on Humans and the Human Research Ethics Committee of The University

of Melbourne and conducted in accordance with the Declaration of Helsinki. The

participant’s age, weight and height were 28 ± 2 yr, 73.7± 2.8 kg and 180 ± 1 cm,

respectively (mean ± SEM). The participants cycled on average 283 ± 35 km.wk-1.

EXPERIMENTAL PROCEDURES

PRELIMINARY TESTING AND DIET CONTROL

Peak pulmonary oxygen consumption during cycling (VO2 peak) was determined

using a graded exercise test to volitional exhaustion on an ergometer (Lode,

Groningen, The Netherlands) and averaged 4.65 ± 0.23 l.min-1 (63.0 ± 1.9 ml.kg-

1.min-1). On a separate day, participants completed a familiarization trial where they

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cycled for 120 min at 236 ± 12 W (72±1% VO2 peak), immediately followed by

completion of as much work as possible in 15 min with the ergometer placed in the

“linear mode”. In this ‘linear mode’ participants are able increase the power output

by increasing their cadence (revolutions per minute; rpm) (power output (watts) =

‘linear factor’ x (rpm)2). On average the participants completed 249 ± 14 kJ during

the 15 min. This amount of work (249 ± 14 kJ) was then completed as quickly as

possible following the 120 min of exercise at 72 ± 1% VO2peak in the experimental

trials and comprised the performance measurement (time trial (TT)). Approximately

one week later the first of the two experimental trials was undertaken.

The day before each experimental trial, exercise and diet were standardized.

Approximately 24 hr prior to each experimental trial participants were required to

come to the laboratory to exercise for 1 hour at 70% VO2 peak (standard ride), after

which participants were asked to abstain from exercise for the remainder of the day.

Following the standard ride participants were provided with a diet (food and drinks)

for the day (i.e. breakfast, lunch, dinner and snacks). This diet was designed to

provide the participants with ~ 199 KJ/kg body weight of energy, ~ 66% of

carbohydrates, ~ 15% proteins and ~ 19% fats and was prepared according to the

participant’s body weight. Standardized exercise and diet were implemented to

replicate the pre-trial metabolic and hormonal levels that affect carbohydrate

metabolism (41). The participants were required to consume their last meal by 11pm.

Participants were also asked to maintain a similar exercise regime during the week

preceding each experimental trial.

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EXPERIMENTAL TRIALS

The participants (overnight fasted) reported to the laboratory in the morning having

abstained from exercise, alcohol and caffeine for 24 hr. One catheter was inserted

into an antecubital forearm vein for infusion of a stable isotope glucose tracer ([6,6-

2H]glucose, Cambridge Isotope Laboratories, MA) and another into the contralateral

forearm for blood sampling. A blood sample was obtained then a bolus of 44.4 ± 0.6

µmol.kg-1 of the tracer was administered prior to a 2 hr pre-exercise constant infusion

(0.63 ± 0.03 µmol.kg-1.min-1), which was continued throughout exercise (i.e. until the

end of the time trial).

Participants then cycled for 120 min at 72 ± 1% VO2peak (236 ± 12 W; 59 ± 2% of

peak power output during VO2 peak test). Blood was sampled at -120 min, -30 min, -

10 min, at 0 min then every 15 min of exercise and at the end of the performance ride.

Plasma insulin, glycerol and NEFA concentrations were not measured between the

onset and 60 min of exercise since the treatment (L-arginine infusion vs saline) did

not start until 75 min and therefore there was no reason to expect any differences

between the two trials during the first 60 min of exercise. At the commencement of

exercise 8 ml/kg body weight of water was ingested followed by 2 ml/kg every 15

min until 120 min to ensure the participant’s hydration levels were similar between

each trial. Expired air was collected into Douglas bags during the last 3 min of each

15 min of exercise (until 120 min of exercise). Heart rate was monitored throughout

exercise (Polar Favor, Oulu, Finland). Rating of perceived exertion was assessed

using the Borg scale (ranging from 6: easiest to 19: hardest) (8). In a double blind,

randomized cross-over design, participants were co-infused intravenously after 75

min of exercise with either 30g of L-arginine Hydrochloride (Opthalmic Laboratories

for Pharmalab, Brookvale, NSW, Australia) mixed with saline (L-Arg; 0.5 g/min, i.v.)

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or a placebo (CON) treatment (0.9 % Saline, Baxter Healthcare P/L, Toongabbie,

NSW, Australia). Infusion of 30g of L-arginine into humans over 30-60 min results

in plasma L-arginine concentration increasing from approximately 0.1 mM to ~ 6.2-

7.2 mM (5, 27). Immediately following the initial 120 min of exercise, the ergometer

was placed into the “linear” mode function and the participants completed 249 ± 14

kJ as quickly as possible (performance ride). To standardize motivational factors no

encouragement was given to the participants during the performance rides of the

experimental trials and the participants were only permitted to see the accumulating

kilojoules and not the clock or the instantaneous power output or RPM. The last

Douglas bag collection period was completed by 120 min of exercise just prior to

commencement of the performance ride so that participants could concentrate on their

performance. Since it has been shown that infusion of 30g of L-arginine at rest can

result in decreases in blood pressure, the participants were instructed to lie down for

30 min after each experimental trial. No participant complained of feeling light

headed during this recovery period.

ANALYTICAL TECHNIQUES

BLOOD ANALYSIS

Plasma glucose and lactate were determined using an automated glucose oxidase and

L-lactate oxidase method, respectively (YSI 2300 Stat, Yellow Springs, OH), plasma

non-esterified fatty acids (NEFA) by an enzymatic colorimetric procedure (NEFA-C

test, Wako, Osaka, Japan), plasma glycerol by an enzymatic fluorometric method (14)

and plasma insulin using a human insulin specific radioimmunoassay kit (Linco

Research, Inc, St.Charles, MO). Glucose kinetics at rest and during exercise were

estimated using a modified one-pool, non-steady-state model as proposed by Steele et

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al. (56) which has been validated by Radziuk et al. (47). We assumed 0.65 as the

rapidly mixing portion of the glucose pool and estimated the apparent glucose space

as 25% of body weight. Rates of plasma glucose appearance (Ra) and glucose

disappearance (Rd) were determined from the changes in % enrichment of [6,6-

2H]glucose and the plasma glucose concentration. The glucose clearance rate

(glucose CR) was calculated by dividing the glucose Rd by the plasma glucose

concentration (indicates the glucose disposal per unit of plasma glucose). During

cycling exercise at ~60% of VO2 max workload, over 95% of tracer-determined

glucose Rd is oxidized (30).

STATISTICAL ANALYSIS

Results were analysed using two factor repeated measures analysis of variance

(ANOVA). Because the treatment was not initiated until 75 min of exercise, the

ANOVA was partitioned and analyzed to include data up to and including 75 min and

data from 75 min onwards (including 75 min). As was expected no significant

differences were found between the two trials during the first 75 min of exercise. If

the ANOVA revealed a significant trial by time interaction, specific differences

between mean values were located using the Fisher’s least significance difference

test. Performance times were compared using Student’s paired t-test. All data are

presented as means ± SEM. The level of significance was set at P < 0.05.

RESULTS

Respiratory Measures, rating of perceived exertion, heart rate and exercise

performance

During the first 75 min of “steady-state” exercise (before commencement of saline/L-

arg infusion) there were no significant differences (p>0.05) in oxygen consumption,

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carbon dioxide production, respiratory exchange ratio (RER), heart rate (HR), or

rating of perceived exertion (RPE) between the two treatments (data not shown). In

addition, there were no significant differences (p>0.05) in oxygen consumption,

carbon dioxide production (data not shown), RER, HR, or RPE between the two

treatments during the last 30 min of “steady-state” exercise after the saline/L-Arg

infusion was commenced (Table 1).

Immediately following the 120 min of “steady-state” exercise the participants

completed 249 ± 14 kJ as quickly as possible (the amount of work completed in 15

min in the familiarization ride). This “time trial” was used to determine the exercise

capacity (exercise performance). The time taken to complete the work did not differ

significantly between the two trials (P=0.62, Table 1). Accordingly, there was no

significant difference in the mean power output between the two time trials (CON:

292 ± 20 watts, L-Arg: 298 ± 19 watts, P=0.62). In addition, there were no

significant differences between trials in the HR (CON: 176 ± 4 b.min-1, L-Arg: 171 ±

4 b.min-1, P=0.19) or RPE (CON: 18.1 ± 0.4 Borg scale units, L-Arg: 18.3 ± 0.3 Borg

scale units, P=0.44) at the end of the performance ride.

Glucose Kinetics

Plasma glucose concentration decreased during the second hour of the steady state

exercise in both trials and was significantly lower in the L-Arg trial at 105 min and

120 min (Figure 1A). Plasma glucose increased significantly (P<0.05) during the

performance ride in both trials and did not differ significantly (P>0.05) between trials

at the end of exercise.

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Glucose Ra, glucose Rd and glucose CR increased during exercise in both trials

(Figure 2A-C). Each of glucose Ra, glucose Rd and glucose CR were significantly

higher in L-Arg during the last 30 min of the steady state exercise. During the

performance ride glucose Ra, glucose Rd and glucose CR increased in both trials and

at the end of exercise there were no significant differences between the trials in any of

these parameters (P>0.05; Figure 2A-C).

Plasma insulin, non-esterified fatty acids (NEFA), glycerol and lactate

Plasma insulin decreased during exercise in both trials with no significant difference

between the two trials at any time point (Figure 1B). Plasma glycerol concentration

increased during exercise in both trials but was significantly lower during the latter

stages of exercise in L-Arg (Figure 3A). Plasma NEFA increased during the second

hour of exercise in both trials (Figure 3B). Plasma NEFA was significantly lower at

105 min and 120 min of exercise in L-Arg compared with CON. There was a small

significant increase in plasma lactate concentration during the first 120 min of

exercise in both trials with no difference between trials (data not shown). Plasma

lactate then increased greatly during the performance ride in both trials with the

concentration at the end of exercise being not significantly different between the two

trials (CON: 7.8 ± 1.0 mmol.l-1, L-Arg: 6.8 ± 0.7 mmol.l-1).

DISCUSSION

The main finding of this study was that L-arginine infusion during exercise

augmented the normal exercise-induced increases in glucose disposal in humans.

One interpretation of this finding is that L-arginine infusion increased NO production,

which increased the level of glucose utilization by the contracting muscles, probably

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via increased GLUT-4 translocation. Taken with our earlier findings where NOS

inhibition reduced leg glucose uptake during cycling exercise (9, 34), our results

reinforce the hypothesis that NO is a central regulator of skeletal muscle glucose

uptake during exercise in humans.

Our studies are novel since no other groups have examined the role of NO in glucose

uptake during exercise in humans. In rats it has been universally found that NO

donors increase basal skeletal muscle glucose uptake (3, 17, 26, 63), but the effects of

NOS inhibition on contraction-stimulated glucose uptake are variable (3, 17, 26, 51,

53, 57). The rat studies are difficult to compare with our human studies because, in

general, both in vivo exercise and in vitro contraction rat studies involving NOS

inhibition assessed glucose transport/uptake 30 min or more after the series of

contractions or treadmill exercise. One exception is a study by Rottman et al. (53)

which reported that glucose uptake measured during rat treadmill exercise was not

reduced by 3 days of ingestion of a NOS inhibitor.

Since local muscle infusion of L-arginine (25), femoral artery infusion of L-arginine

(9) and femoral artery infusion of a NOS inhibitor (9, 34, 46) have no affect on blood

flow during exercise in humans, we feel that it is likely that L-arginine infusion

increased glucose Rd during exercise by increasing GLUT-4 translocation to the

plasma membrane rather than increasing skeletal muscle blood flow. Therefore it

appears that irrespective of whether NO production is increased by L-Arg infusion, or

decreased by NOS inhibition, muscle blood flow is unchanged during exercise in

humans. It should be kept in mind, however, that during exercise NO may affect the

proportion of the blood flow that enters the nutritive (capillary) vessels compared

with that entering the non-nutritive vessels (25, 59, 62). To clarify the precise

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mechanism(s) whereby NO influences skeletal muscle glucose uptake during

exercise, human studies investigating the effect of L-arginine infusion or NOS

inhibition on both GLUT-4 translocation and nutritive flow during exercise are

warranted.

L-arginine infusion attenuated the increase in plasma glycerol during exercise (Figure

3A) implying that lipolysis was reduced by L-arginine infusion. Indeed, plasma

NEFA concentrations were also lower during exercise in L-Arg (Figure 3B). Studies

have consistently shown that NO donors attenuate the effect of catecholamines to

activate lipolysis in adipose tissue and fat cells (21, 35). Therefore, since plasma

catecholamines increase during exercise (39), it is possible that L-arginine infusion

increased NO levels which then inhibited the catecholamine stimulation of lipolysis.

In humans, NO gas, as well as the NO donor, nitroglycerine, reduced glycerol release

from isolated adipocytes in vitro (2). In addition, NOS inhibition in vivo increased

lipolysis in subcutaneous adipose tissue (2) and augmented isoproterenol- and

epinephrine-induced glycerol release from both fat and muscle in humans (31). It

appears therefore that NO is important in modulating and ameliorating sympathetic

effects in peripheral tissues (31). We are not aware of any evidence that L-arginine

directly inhibits lipolysis.

It is possible, but unlikely, that the small but significant attenuation of the increase in

plasma NEFA concentration during the second hour of exercise in L-Arg contributed

to the higher glucose disposal rate in that trial. Very large reductions in plasma

NEFA concentration during exercise, by inhibition of adipose tissue lipolysis with the

nicotinic acid analogue Acipimox, has no (58) or only modest (64) effects on glucose

disposal during exercise in humans. In addition, most (43, 52, 64), but not all (22),

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studies have shown that raising plasma NEFA levels has no affect on glucose disposal

during exercise in humans.

Since insulin is additive on skeletal muscle glucose uptake during contractions (16,

24), it is possible that the small, non-significant, increase in plasma insulin

concentration observed at 90 min of exercise in L-Arg (Figure 1B), mediated the

increase glucose Rd. It is also possible that plasma insulin concentration was

significantly higher within the 75-90 min period of exercise when no measurements

were obtained. Furthermore, it is possible that the eventual “coming together” during

the time-trial of the responses during saline and L-Arg reflect a time dependent

reversal of a potential “insulin surge” at 75-90 min. However, if the plasma insulin

concentration had increased greatly immediately following the start of the L-arg

infusion some inhibition of glucose Ra would have been expected. Further research

is required to examine closely the time course of plasma insulin changes after

infusion of L-arg during exercise.

L-arginine infusion actually significantly increased glucose Ra during exercise

compared with CON (Figure 2B). The mechanism(s) behind this response are

unclear but may relate, in part, to the relative hypoglycemia caused by L-arginine

infusion. Liver glucose output is exquisitely sensitive to small changes in plasma

glucose levels during exercise in humans so the decrease in plasma glucose would be

expected to increase liver glucose output (28). Therefore, it is possible that the

greater glucose Rd in L-arg caused a decrease in the plasma glucose concentration

which then stimulated glucose Ra. It is also possible that L-arg infusion increased the

plasma glucagon concentration which then stimulated liver glucose output since this

has been shown to occur at rest in humans (10). Finally, the L-arg infusion may have

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augmented the exercise-induced increases in glucose Ra by increasing NO, since NO

donors have been shown to potentiate the effect of norepinephrine to increase liver

glucose output in rats and cats (42). It is unlikely that the increased glucose Ra

during exercise in L-arg contributed to the greater glucose Rd in that trial since

plasma glucose levels were lower therefore decreasing the glucose gradient.

Although infusion of L-arginine increases glucose Rd during exercise it does not

appear to effect endurance exercise performance. In both trials the participants were

able to increase the exercise intensity during the performance ride above that of the

first 120 min of exercise at 72% of VO2 peak but there was no difference between the

trials in the time to complete the set amount of work or the average power output

during the performance ride. Accordingly there were similar increases in plasma

glucose concentration, glucose Ra, Rd and CR, increases in plasma lactate and

decreases in plasma NEFA during the performance ride in the two trials. The reason

for including a performance ride in the present study was that we postulated that if L-

arginine increased glucose Rd it may improve exercise performance. We have shown

that carbohydrate ingestion increases glucose Rd and improves exercise performance

in studies utilizing similar methodologies as those used in the present study (39, 40).

However, the increases in glucose Rd with carbohydrate ingestion during prolonged

exercise (39) were substantially greater than the increases observed in the present

study with L-arginine infusion (Figure 2B). Indeed, carbohydrate ingestion increases

the rate of carbohydrate oxidation during prolonged exercise (39-41) but in the

present study the respiratory exchange ratio was identical in the CON and L-Arg

trials at all time points. It has been shown that prior L-arginine infusion has no effect

on VO2 max test exercise time in patients with chronic heart failure (32) and several

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weeks of L-arginine ingestion has no effect on the amount of work completed during

a VO2 max test in healthy humans (1).

It has been shown previously that both L-arginine infusion and oral L-arginine

supplementation improve vascular health (improved endothelial function) (13) and

long-term oral L-arginine supplementation improves insulin sensitivity in people with

type 2 diabetes (44). It is necessary to now determine whether oral L-arginine

supplementation, like L-arginine infusion, increases glucose disposal during exercise.

People with Type 2 diabetes have impaired insulin-stimulated skeletal muscle GLUT-

4 translocation and glucose uptake but their GLUT-4 translocation (33) and glucose

uptake during exercise is normal (23, 34). If we can better understand the

mechanisms regulating skeletal muscle glucose uptake during exercise attempts can

be made to develop pharmacological agents that mimic exercise for people with type

2 diabetes who cannot exercise regularly.

In conclusion, L-arginine infusion during prolonged exercise in humans decreased

plasma glucose concentration. Despite this lower concentration gradient for

facilitated glucose transport in the L-arginine infusion trial, glucose Rd during

exercise was greater than that observed in the control saline infusion trial. As a

result, L-arginine infusion substantially increased the glucose clearance rate during

exercise compared with saline infusion. Given that we and others have previously

shown that L-arginine infusion has no effect on blood flow during exercise, and L-

arginine infusion had no significant effect on plasma insulin concentration, we

suggest that L-arginine infusion increased NO production by skeletal muscle NOS

which then increased muscle glucose uptake. It is also possible that the lower plasma

NEFA concentration during L-arginine infusion played a role in the higher glucose

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disposal although most studies show no affect of alterations in NEFA on muscle

glucose disposal during exercise in humans.

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Acknowledgements

The authors would like to thank the participants for taking part in this study and

acknowledge the technical assistance of Dr Nigel Stepto, Dr Domenic Caredi, Terry

Stephens and Matthew Palmer. This work was supported by grants from the National

Health and Medical Research Council of Australia and Diabetes Australia.

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FIGURE LEGENDS

Figure 1. Plasma glucose concentration (A) and plasma insulin concentration (B) at

rest, during 120 min of steady state exercise at 72 ± 1% VO2 peak and at the end of

the performance ride (249 ± 14 kJ) time-trial (TT) in CON and L-Arg conditions.

Values are mean ± SEM (n=9). * P<0.05 vs. CON.

Figure 2. Rate of glucose appearance (Glucose Ra) (A), rate of glucose

disappearance (Glucose Rd) (B), and glucose clearance rate (Glucose CR) (C) at rest,

during 120 min of steady state exercise at 72 ± 1% VO2 peak and at the end of the

performance ride (249 ± 14 kJ) time-trial (TT) in CON and L-Arg conditions.

Because glucose kinetics are calculated as the change from one time point to the next,

data are plotted midway between sequential time points (eg 7.5 min for the change

from 0 min to 15 min). Values are mean ± SEM (n=9). * P<0.05 vs. CON.

Figure 3. Plasma glycerol (A) and plasma NEFA (B) concentration at rest, during

120 min of steady state exercise at 72 ± 1% VO2 peak and at the end of the

performance ride (249 ± 14 kJ) time-trial (TT) in CON and L-Arg conditions. Values

are mean ± SEM (n=9 for NEFA, n=8 for glycerol). * P<0.05 vs. CON.

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TABLE 1: Mean physiological responses during the last 30 min (90, 105 and 120

min) of 120 min of steady state exercise at 72 ± 1% VO2 peak and time to complete

the performance ride (249 ± 14 kJ) in CON and L-Arg conditions.

CON L-Arg

Oxygen uptake (l.min-1) 3.37 ± 0.17 3.38 ± 0.17

RER 0.90 ± 0.01 0.89 ± 0.01

Heart rate (beats.min-1) 149 ± 4 147 ± 4

Rating of perceived exertion

12.8 ± 0.4 13.0 ± 0.5

Performance time (min) 14.5 ± 0.9 14.1 ± 0.6

Values are mean ± SEM (n=9).

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Figure 1

3.5

4.0

4.5

5.0

5.5 CON

L-Arg

* *

Pla

sma

glu

cose

(mm

ol.l

-1)

0 15 30 45 60 75 90 105 120 135

0

10

20

30

40

50

60

70

CON

L-Arg

Time (min)

Pla

sma

insu

lin(p

mo

l.l-1

)

Saline or L-Arg

Steady state exercise TT

A

B

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Figure 2

0 15 30 45 60 75 90 105 120 135

0

5

10

15

20

CON

L-Arg *

**

Time (min)

Glu

cose

CR

(ml.

kg-1

.min

-1)

Saline or L-Arg

Steady state exercise TT

0

20

40

60

80

100

CON

L-Arg **

*

Glu

cose

Ra

( µm

ol.

kg-1

.min

-1)

A

B

C

0

20

40

60

80

100

CON

L-Arg **

*

Glu

cose

Rd

( µm

ol.

kg-1

.min

-1)

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Figure 3

0 15 30 45 60 75 90 105 120 135

0.00

0.25

0.50

0.75

1.00

CON

L-Arg

**

Time (min)

Pla

sma

NE

FA

(mm

ol.

l-1)

Saline or L-Arg

Steady state exercise TT

0

100

200

300

400

**

CON

L-Arg

*

Pla

sma

Gly

cero

l( µ

mol

.l-1

)A

B