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00005768-200604000-0000800005768_2006_38_660_hickner_citrulline_4article< 93_0_16_6 >Medicine & Science in Sports & Exercise©2006The American College of Sports MedicineVolume 38(4)April 2006pp 660-666L-Citrulline Reduces Time to Exhaustion and Insulin Response to a Graded Exercise Test[BASIC SCIENCES: Original Investigations]HICKNER, ROBERT C.1,2; TANNER, CHARLES J.1; EVANS, CHRIS A.1; CLARK, PAIGE D.1; HADDOCK, AMY1; FORTUNE, CHRIS1; GEDDIS, HEATHER1; WAUGH, WILLIAM2; MCCAMMON, MICHAEL11Human Performance Laboratory and 2Departments of Exercise and Sport Science, and Physiology, East Carolina University, Greenville, NCAddress for correspondence: Robert Hickner, Ph.D., Human Performance Laboratory, East Carolina University, Greenville, NC 27858; E-mail: Hicknerr@mail.ecu.edu.Submitted for publication June 2005.Accepted for publication October 2005.ABSTRACTPurpose: Oral L-arginine supplementation has been shown to improve treadmill time to exhaustion and resting insulin sensitivity in individuals with peripheral vascular disease and type 2 diabetes, respectively. Furthermore, L-citrulline supplementation increases plasma L-arginine concentration to a level higher than that achieved by oral L-arginine supplementation. The purpose of this investigation was therefore to determine whether time to exhaustion during a graded treadmill test, as well as plasma insulin and glucose profiles, could be improved with oral L-citrulline supplementation in healthy individuals.Methods: Seventeen young (18-34 yr), healthy male and female volunteers performed incremental treadmill tests to exhaustion following either placebo or citrulline ingestion (3 g 3 h before test, or 9 g over 24 h prior to testing).Results: Steady-state submaximal respiratory exchange ratio and V̇O2max were not significantly different between placebo and citrulline trials. Treadmill time to exhaustion was lower following citrulline ingestion than during placebo trials (888.2 ± 17.7 vs 895.4 ± 17.9 s; P < 0.05; N = 17), which was accompanied by a higher rating of perceived exertion during exercise in the L-citrulline compared with the placebo condition. There was also an increase in plasma insulin in response to this high-intensity exercise in the placebo, but not in the L-citrulline, condition (P < 0.05).Conclusions: It can be concluded that, contrary to the hypothesized improvement in treadmill time following L-citrulline ingestion, there is a reduction in treadmill time following L-citrulline ingestion over the 24 h prior to testing. The normal response of increased plasma insulin following high-intensity exercise is also not present in the L-citrulline condition, indicating that L-citrulline ingestion may reduce nitric oxide-mediated pancreatic insulin secretion or increased insulin clearance.Nitric oxide (NO) is a gas formed from L-arginine and oxygen via the enzyme NO synthase within the endothelial layer of blood vessels and within many tissues (19,22). NO has been demonstrated to have multiple physiological roles, some of which include increased vasodilatation (9,28), glucose uptake (13,25), and insulin secretion (1,4). In line with these proposed actions of NO, acute L-arginine supplementation has been reported to increase blood flow in hypercholesterolemic individuals (5), increase treadmill time to exhaustion during a graded treadmill stress test in patients with peripheral vascular disease, and enhance aerobic capacity in apolipoprotein E-deficient mice (24). These previous investigations indicate that L-arginine supplementation may be beneficial for individuals with compromised NO production. However, the influence of L-arginine supplementation on maximal aerobic capacity and treadmill time in healthy individuals has not been adequately determined.There is ample evidence to suggest that oxygen delivery is limiting maximal oxygen consumption in healthy humans. Autologous blood transfusion and erythropoietin supplementation result in increased maximal oxygen consumption (7). We have previously reported that NO synthase inhibition decreases local skeletal muscle blood flow during exercise (9), and others have reported reduced maximal oxygen consumption with NO synthase inhibition in healthy young men (11), suggesting a role for NO in increased muscle blood flow during exercise. Although local infusion of L-arginine into muscle did not alter local muscle blood flow during submaximal exercise in healthy young men (9), blood flow response to L-arginine during maximal exercise has not been investigated. L-arginine infusion has also increased glucose clearance during exercise (15) and during insulin infusion (31) in healthy young adults. It is possible that oral L-arginine supplementation will increase oxygen delivery to active muscle during maximal exercise, resulting in increased maximal oxygen consumption and increased treadmill time to exhaustion. This effect would be via NO production from L-arginine. Interestingly, the ingestion of L-citrulline, a byproduct of NO formation from L-arginine, is more effective than L-arginine ingestion in increasing plasma L-arginine concentration. L-arginine, when assimilated over the digestive tract, enters the hepatic circulation where much of the L-arginine is degraded in the liver. L-citrulline is not cleared to a large extent by the liver, instead being taken up by the kidneys and other tissues to be converted to L-arginine (8). It has been demonstrated that acute ingestion of 3-6 g of L-citrulline results in a 60% increase in plasma L-arginine concentration in sickle cell patients, and a doubling of plasma L-arginine concentration in healthy humans (14,32). These values are 30% higher than would be attained following ingestion of a similar dose of L-arginine (32). L-citrulline ingestion may therefore be the preferred means of elevating plasma L-arginine concentrations.We therefore tested the hypothesis that L-citrulline supplementation increases maximal oxygen consumption and treadmill time to exhaustion on a graded treadmill test when compared with placebo in healthy young adults. We also investigated whether L-citrulline ingestion would increase plasma insulin and decrease plasma glucose because of the reported effects of NO to increase insulin secretion and glucose uptake.METHODSSeventeen physically active (>30 min of aerobic exercise > 3× wk−1; V̇O2max 52.1 ± 1.9 mL·kg−1·min−1) healthy nonobese young (18-40 yr) male and female subjects were studied during a graded treadmill exercise test. The percentage of body fat was determined by hydrostatic weighing and calculation of body fat from body density using the equation of Brozek et al. (2). Ten of the subjects underwent the testing described below before and after ingestion of 3 × 3 g (at mealtimes) of L-citrulline or placebo over 24 h (Table 1). A second group of participants (N = 7) ingested only the final 3-g dose (3 h prior to testing) of L-citrulline or placebo to determine whether there was a larger effect with 9 g over 24 h compared with a single 3-g dose ingested at the last meal prior to exercise. Subjects were studied after giving their informed consent to participate according to the university medical center institutional review board of East Carolina University.TABLE 1. Descriptive characteristics.Exclusion CriteriaIndividuals who were suffering from acute or chronic illness, had known cardiovascular or metabolic disease, had known pulmonary disease, were taking medications, smoked, or suffered from chronic injuries that may have influenced maximal exercise performance were excluded. Individuals who had been taking dietary supplements or multivitamins were asked to discontinue such practice 1 wk prior to, and during, any participation in these studies.ProtocolThe participants completed five graded exercise tests to exhaustion on a motorized treadmill. The testing schedule was randomized with respect to L-citrulline or placebo, which were ingested prior to treadmill tests 3 and 5 in a crossover design. The testing data collection outline is provided in Figure 1. Two treadmill tests were conducted prior to the initial supplementation to ensure familiarity with the treadmill and maximal effort for the determination of initial maximal oxygen consumption. A 24-h prescribed diet (55% carbohydrate, 25% fat, 20% protein) was provided for the 24 h prior to testing to eliminate variability based on food intake. An isocaloric diet was provided based on kilocalories per kilogram of lean body mass. Dietary supplements (such as a multivitamin) other than the L-citrulline or placebo provided were not allowed over the course of the study. No exercise was allowed for the 24 h prior to treadmill testing. Subjects were also instructed to avoid caffeine and remain adequately hydrated prior to the testing.FIGURE 1-Study protocol. Min, minimum time between visits; max, maximum time between visits; supplement, randomly assigned to be either placebo or L-citrulline. The subjects and the investigators were blinded to treatment. The boxes below the flow chart describe the duration between visits and whether there was supplementation and diet control or only diet control prior to the subsequent visit.Graded Exercise TestsThe graded exercise test on a motorized treadmill was used to determine maximal oxygen consumption (V̇O2peak), time to exhaustion, and submaximal RER and oxygen consumption. Participants reported to the human performance laboratory 3 h after their most recent supplementation and standardized meal. Each subject ran for approximately 8-20 min on a motorized treadmill. Participants walked at 2.0 mph for 2 min before the treadmill speed was increased to 5.0 mph for the initiation of the treadmill test. Less fit participants exercised at this intensity (5.0 mph: approximately 65% V̇O2peak) for 5 min. This initial steady-state exercise speed allowed determination of fat oxidation by indirect calorimetry during submaximal exercise. The treadmill speed was then increased 1 mph every 2 min until 7.0 mph. The grade of the treadmill was then increased 2% every minute until the subjects could no longer continue. The less fit participants underwent this treadmill protocol for each of their five treadmill tests. Participants with a higher level of fitness and running background (those individuals who reported on questionnaire that they purposefully exercise trained 3× wk−1 or more for a minimum of 30 min per session for the past 6 months) underwent a similar treadmill test for each of their five treadmill tests. However, following the 2.0 mph 2-min warm-up stage, the initial speed for the 5-min exercise stage was 7.0 mph (approximately 65% V̇O2peak), followed by increases of 1.0 mph every 2 min until 9.0 mph, with subsequent 2% increases in treadmill grade every minute. Respiratory gases were continuously collected and analyzed every 20 s by indirect calorimetry (TrueMax 2400, Consentius Technologies, Sandy, UT). V̇O2peak was defined as the maximal oxygen consumption attained during a test where two of the following three criteria were met: 1) an RER ratio > 1.1; 2) attainment of ± 5 bpm of predicted HRmax; or 3) no further increase in oxygen consumption with an increase in exercise intensity. Treadmill time began immediately after 2 min of walking on the treadmill at 2.0 mph and was stopped at the time the subject clutched the treadmill handrail for support to terminate the test.Blood SamplesAfter an initial 10-min resting period in the standing position, blood was drawn before and immediately after each treadmill test by venipuncture of a forearm vein with the participant in the standing position. The blood serum was analyzed for nitrates and nitrites as a marker of NO determined spectrophotometrically using a NO quantitation kit (Active Motif, Carlsbad, CA). Hemoglobin (Sigma diagnostic kit) and hematocrit were determined on resting heparinized whole blood samples for calculation of plasma volume (6). Blood samples were also analyzed for glucose, lactate (YSI 2300 STAT Plus Glucose and Lactate Analyzer, YSI Inc., Yellow Springs, OH), and insulin (Access Immunoassay System, Beckman Coulter, Fullerton, CA). Untreated samples for serum collection were left to clot at room temperature for 15 min before centrifugation. Ethylenediamine tetraacetic acid-coated tubes for plasma collection were kept on ice until centrifugation (3000 × g for 15 min). The supernatant was then pipetted into polyethylene tubes and stored at −80°C until analysis.StatisticsA two-way supplementation (L-citrulline and placebo) by time (pre- and postrun) repeated-measures ANOVA was used to determine the effect of supplementation on blood variables for each dose of L-citrulline (3 or 9 g, or, to increase statistical power, combined 3 and 9 g). One-way ANOVA was used to determine the effect of L-citrulline compared with placebo supplementation on the following variables: maximal aerobic capacity, maximal treadmill time, RER, RPE, and the change in blood variables from pre- to posttreadmill exercise for each dose of L-citrulline. When significance was attained, Newman-Keuls post hoc analysis was used to locate significant differences. Statistics were run on insulin data corrected for changes in plasma volume. Means and SEM are reported. Significance was set at P < 0.05. Data are presented in groups of L-citrulline supplementation of 3 g (N = 10), 9 g (N = 7), and the 9- and 3-g groups combined (N = 17).RESULTSTreadmill time to exhaustion was lower following L-citrulline ingestion (9- and 3-g combined groups) than during placebo trials (888.2 ± 17.7 vs 895.4 ± 17.9 s; P < 0.05, N = 17; Fig. 2A). The lower treadmill time to exhaustion in the L-citrulline compared with placebo condition occurred in 12 of the 17 participants tested. The comparison of V̇O2peak data between L-citrulline and placebo conditions did not reach statistical significance (Fig. 2B); however, there was a positive correlation between the difference in V̇O2peak from L-citrulline to placebo conditions and the difference in treadmill time to exhaustion from L-citrulline to placebo conditions (r2 = 0.40; P < 0.01). The lower treadmill time with L-citrulline supplementation was consistent with a higher RPE with L-citrulline than placebo ingestion (P < 0.05; Fig. 3).FIGURE 2-Total treadmill time (A) and V̇O2peak (B) were determined in 17 healthy young male and female participants who ran on a motorized treadmill during an incremental graded exercise test to exhaustion. Data in the middle of the graph are from seven of the participants who ingested 9 g of L-citrulline (3 × 3 g at meals) in the 24 h prior to the test. Data on the right are from 10 of the participants who ingested only one 3-g dose of L-citrulline 3 h prior to testing. Data on the left are combined data from all 17 individuals. Data are presented as mean ± SEM. * Different from placebo.FIGURE 3-Peak RPE was determined over the last seconds of exercise in 17 healthy young male and female participants who ran on a motorized treadmill during an incremental graded exercise test to exhaustion. Data in the middle of the graph are from seven of the participants who ingested 9 g of L-citrulline (3 × 3 g at meals) in the 24 h prior to the test. Data on the right are from 10 of the participants who ingested only one 3-g dose of L-citrulline 3 h prior to testing. Data on the left are combined data from all 17 individuals. Data are presented as mean ± SEM. * Different from placebo.Blood variables before and at the end of exercise are presented in Table 2. The changes in blood variables in response to exercise are presented in Figure 4. There was no difference in plasma nitrate/nitrite (NOx, a marker of NO) before exercise in the L-citrulline compared with placebo condition, but there was a difference in the change in plasma NOx in response to exercise between L-citrulline (decrease in NOx) and placebo (increase in NOx) ingestion (3-g group and the 9- and 3-g combined group; P < 0.05: Fig. 4A). There was no difference between L-citrulline and placebo conditions in plasma glucose before exercise (Table 2). There were increases in plasma glucose (approximately 1.8 mmol·L−1; Fig. 4B) and blood lactate (approximately 12 mmol·L−1; Fig. 4D) in response to exercise, with no between-group differences in these changes. There was an increase (approximately 4 μIU·mL−1) in plasma insulin in response to exercise following placebo ingestion that was not evident with exercise following L-citrulline ingestion (3-g group and 9- and 3-g combined group; P < 0.05: Fig. 4C). Plasma volume was reduced in response to exercise following both L-citrulline and placebo ingestion (P < 0.05), but the reduction was approximately 30% greater with placebo than L-citrulline ingestion (3-g group and 9- and 3-g combined group; P < 0.05; Fig. 4E). HRmax response to exercise approached a significant difference for the 3-g group data only (L-citrulline: 194 ± 3.6 bpm, placebo: 192 ± 3.2 bpm; P = 0.07).TABLE 2. Blood insulin, glucose, lactate NOx, hematocrit, and hemoglobin before and at the end of a graded maximal exercise test to exhaustion.FIGURE 4-Blood variables were determined before and after exercise in 17 healthy young male and female participants who ran on a motorized treadmill during an incremental graded exercise test to exhaustion. Plasma NOx (A), glucose (B), insulin (C), lactate (D), and plasma volume (E) are presented as change in response to exercise (postexercise/preexercise). Bars in the positive direction indicate increases in response to exercise. Bars in the negative direction indicate decreases in response to exercise. Data in the middle of each graph are from seven of the participants who ingested 9 g of L-citrulline (3 × 3 g at meals) in the 24 h prior to the test. Data on the right are from 10 of the participants who ingested only one 3-g dose of L-citrulline 3 h prior to testing. Data on the left are combined data from all 17 individuals. Data are mean ± SEM. * Different from placebo.There were no differences with respect to submaximal oxygen consumption, RER, or HR between L-citrulline and placebo conditions (data not shown).DISCUSSIONThe primary finding of this placebo-controlled, double-blind, counterbalanced study was that, in contrast to our hypothesis, there was a decrease in performance during this incremental graded treadmill exercise test to exhaustion in 12 of the 17 participants. Of potentially great importance with respect to investigations of insulin secretion and clearance is the additional finding that the previously reported increase in plasma insulin in response to high-intensity exercise (30) is blunted following L-citrulline ingestion. These are the first reports of L-citrulline ingestion resulting in reduced exercise capacity and reduced insulin response in healthy volunteers.L-arginine and exercise performance.The influence of L-arginine supplementation on maximal aerobic capacity and treadmill time to exhaustion in healthy individuals has not been adequately determined (21). We therefore proposed to increase plasma L-arginine concentration, and resultant NO production, via oral L-citrulline supplementation. L-arginine supplementation has been reported to increase treadmill time to exhaustion during an incremental treadmill stress test in patients with peripheral vascular disease and to enhance aerobic capacity in apolipoprotein E-deficient mice (24). There have also been reports that L-arginine supplementation increases treadmill time to exhaustion in rats (16) and reduces the exercise-induced increase in plasma lactate and ammonia (27). L-arginine infusion in humans increases glucose clearance, although with no effect on the amount of work performed during 15 min of cycle ergometry (15). L-arginine supplementation also does not consistently improve endothelial function or local muscle blood flow during exercise in healthy young individuals (3,10). These data indicate that L-arginine supplementation may be more effective in individuals with compromised NO production or availability. Young healthy adults may not suffer from a deficiency in L-arginine or other related factors that would result in impaired blood flow and metabolism in diseased or older adults; furthermore, there may be differences in these populations with respect to the conversion of L-citrulline to L-arginine or subsequent formation of NO from L-arginine. It is therefore not possible to generalize the current findings regarding L-citrulline supplementation and reduced treadmill time to exhaustion in young healthy adults to older adults or individuals with a given disease state.NO production and exercise performance.The original hypothesis that L-citrulline ingestion would result in higher NO production was not supported, in that plasma NOx was not higher in the L-citrulline than the placebo condition prior to exercise. In fact, preexercise plasma NOx was lower in 12 of 17 subjects following L-citrulline compared with placebo ingestion (P = not significant). Furthermore, there was an increase in plasma NOx in response to exercise only following placebo ingestion (Fig. 4A). It is possible that the L-citrulline supplementation resulted in end-product inhibition of the L-arginine/NO reaction and resulted in a lower NO production. Although there is no direct support for this hypothesis in the present investigation, it has been reported that the NO donor sodium nitroprusside, but not L-citrulline, inhibits neuronal NO synthase (29). The inhibitory effect of the NO donor was prevented by an NO scavenger in that study, indicating that NO can reduce NO synthase activity and subsequent NO production (29). There were no direct measures of NO or L-arginine in the present study; however, the plasma NOx data in combination with the small reduction in treadmill time with L-citrulline ingestion suggest that L-citrulline may reduce exercise-induced NO release or increase NO degradation, thereby limiting performance of the graded exercise test to exhaustion.Previous treatments attenuating NO production during intense exercise are those in eNOS knockout mice (18) and a study of L-NAME infusion in humans (11): maximal exercise capacity was reduced in these treatments. In the human study, Jones et al. (11) ascribed the 15-s reduction in incremental cycle ergometry time to exhaustion to an approximately 5% reduction in HRmax and V̇O2max, suggesting a reduced oxygen delivery during maximal exercise. In the present study, the slightly lower HR and V̇O2max with L-citrulline ingestion observed did not reach statistical significance; however, there was a positive correlation between the difference in V̇O2peak from L-citrulline to placebo conditions and the difference in treadmill time to exhaustion. The objective measure of reduced time to exhaustion in the L-citrulline condition was further supported by a higher RPE during exercise in the L-citrulline compared with placebo condition, indicating the participants' subjective opinion of the exercise test was that of a more difficult exercise session.L-citrulline supplementation and insulin response.The data for the blood-related variables suggest that although L-citrulline supplementation did not alter preexercise plasma NOx (nitrate/nitrite: markers of NO production), insulin, glucose, lactate, or plasma volume, there were significant alterations in response to exercise in plasma NOx, insulin, and plasma volume. With respect to the insulin data, it is known that there is increased plasma insulin after high-intensity exercise, as opposed to the progressive decline in plasma insulin with lower-intensity endurance exercise in the fasted state (12,30). There was a lower postexercise plasma insulin concentration with L-citrulline than placebo ingestion in the present study, indicating there may have been a reduced insulin secretion in response to exercise even with a similar increment in blood glucose in response to this high-intensity exercise. NO has been shown to stimulate insulin secretion in both rodent and human pancreas (1,4,20); therefore, it is plausible that a reduced NO production in the pancreas or elsewhere in the participants of the present study resulted in a reduced insulin response to the exercise. There were no measures of insulin flux to determine whether increased insulin clearance rather than, or in addition to, decreased insulin secretion resulted in the lower plasma insulin response to exercise. The lower plasma insulin response to exercise in the context of the similar blood glucose response with L-citrulline supplementation could also be interpreted to be an increase in insulin sensitivity; however, there were no significant differences in plasma insulin or glucose before exercise (Table 2). Furthermore, glucose uptake during exercise is more dependent on contraction-mediated rather than insulin-mediated mechanisms.L-citrulline supplementation and plasma volume.The differential plasma volume response between the L-citrulline and placebo conditions could have been due to an effect of NO on capillary permeability (17,23,26). There may have been less of an exercise-induced increase in capillary permeability in the L-citrulline condition due to reduced NO production, although capillary permeability was not assessed in the present investigation. It should be noted that the difference in plasma volume response to exercise between L-citrulline and placebo conditions was not great enough to alter the above-stated conclusions with respect to differential changes in plasma insulin in response to exercise between the L-citrulline and placebo conditions.CONCLUSIONSIt can be concluded that, in healthy young men and women, contrary to the hypothesized improvement in exercise time, there was a reduction in treadmill time following L-citrulline ingestion over the 24 h prior to performance of a graded treadmill exercise test to exhaustion. There was also a reduction in insulin response to this high-intensity exercise following L-citrulline ingestion that may reflect a reduced NO-mediated insulin secretion or increased insulin clearance. 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