INTRODUCTION

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DURING MODERATE-INTENSITY EXERCISE, in both humans and animals, the increase in liver glucose production can be abolished by an intravenous glucose infusion, demonstrating that liver glucose output is subject to feedback regulation by the blood glucose concentration (3, 9, 24). In contrast, during high-intensity exercise in rats, infusion of glucose reduced, but was unable to totally suppress, liver glucose output (27, 28). These findings suggest that humoral feedback mechanisms cannot prevent the exercise-induced increase in glucose output under such circumstances. During exercise at a high workload, liver glucose output may be regulated by a feed-forward mechanism, whereby motor centers in the brain activate, in parallel, locomotion and neuroendocrine responses that increase glucose production (14, 23). It has previously been shown in trained men that increased blood glucose availability after glucose ingestion inhibits liver glucose output during exercise (19). However, the relative importance of the feedback and feed-forward mechanisms for regulating liver glucose output early in exercise, when feed-forward mechanisms are likely to be dominant, could not be fully elucidated because of the time required for blood glucose to increase after glucose ingestion. The aim of the present study, therefore, was to examine this interaction when blood glucose was increased early in exercise by an intravenous glucose infusion at a rate equal to the average liver glucose output in an initial control trial.

	METHODS

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Subjects. Five trained men [age 20 ± 0.5 (SE) yr, weight 72.6 ± 3.0 kg] volunteered to serve as subjects for the experiment. The experimental procedures and possible risks of the study were explained to each subject verbally and in writing. All subjects gave their informed, written consent, and the experiment was approved by the Human Research Ethics Committee of The University of Melbourne.

Preexperimental protocol. All subjects performed an incremental workload test to exhaustion on an electromagnetically braked cycle ergometer (LODE Instrument, Groningen, The Netherlands) to determine their peak pulmonary oxygen uptake (O_{2 peak}). Mean O_{2 peak} was 4.46 ± 0.27 l/min. For the day preceding each trial, the subjects were provided with a set diet (~14 MJ, 80% carbohydrates) and abstained from strenuous exercise or ingestion of tobacco, caffeine, and alcohol. In addition, they were instructed to consume 5 ml of tap water per kilogram body weight on waking to ensure adequate hydration. The subjects reported to the exercise laboratory in the morning after a 10- to 12-h overnight fast.

Experimental protocol. Each subject was studied during two exercise periods, separated by at least 7 days. In an attempt to examine the effect of increased blood glucose availability on liver glucose output, an exogenous glucose infusion was administered during the second experimental period (Glu). The amount of glucose delivered was equal to the average liver glucose output during exercise measured in an initial control trial (Con). The exercise trials were performed on the same stationary cycle ergometer used in the O_{2 peak} determination. Subjects performed all exercise tests in a laboratory at a temperature of 21 ± 0.5°C, and an electric fan circulated air to minimize thermal stress.

On arrival at the exercise laboratory, all subjects rested quietly on a couch, and indwelling Teflon catheters were inserted into an antecubital vein of one arm for blood sampling and in the contralateral arm for infusion. The catheter for blood sampling was kept patent by flushing with 0.9% saline and every 30 min with 0.5 ml of saline containing 5 units of heparin. After a priming dose of 40 µCi, D-[3-³H]glucose (DuPont, Biotechnology Systems, Wilmington, DE) was infused continuously at a rate of 0.40 µCi/min for the duration of the 2-h rest period and 40 min of exercise. At the completion of the rest period, the subject moved to the cycle ergometer and exercised for 40 min at a workload requiring 77 ± 1% O_{2 peak}. Venous blood samples were obtained at 5-min intervals for the last 15 min of the rest period and throughout exercise for later analysis of plasma glucose and [³H]glucose specific activity. Additional venous samples were obtained immediately before the commencement of exercise, after 10 and 20 min of exercise, and at the completion of the exercise period. These were analyzed for plasma lactate, insulin, glucagon, and catecholamines. Blood for glucose and lactate was placed in fluoride heparin tubes, insulin in lithium heparin tubes, glucagon in lithium heparin tubes containing 200 µl of a protease inhibitor (10% Trasylol), and catecholamines in plain tubes containing EGTA and reduced glutathione. On completion of the exercise, the blood samples were spun, and the plasma was removed and stored at 20°C for later analysis. Plasma for catecholamine analysis was stored at 80°C. In preparation for the lactate assay, 250 µl of plasma were deproteinized in 500 µl of 8% perchloric acid, spun again, and the supernatant was removed and stored at 20°C. Expired gases were collected in Douglas bags at 10-min intervals during exercise for measurement of oxygen uptake and respiratory exchange ratio. Heart rate was measured continuously via telemetry (Polar sports tester, Polar Electro, Finland) and recorded every 10 min during exercise. Subjects were permitted to drink water ad libitum during the trials.

For the Glu trial, all subjects undertook the same protocol as described above. In addition, a 10% glucose solution (Viaflex Baxter Healthcare, NSW, Australia) was delivered via a three-way stop-cock to allow simultaneous infusion of tracer and glucose. The amount of glucose to be delivered was calculated by averaging the liver glucose output measured during the 40-min Con exercise period. In preliminary experiments (n = 3), the intravenous glucose infusion commenced 5 min before exercise so as to avoid delay in administering the glucose. This resulted in a plasma glucose concentration that was significantly higher than in Con at the onset of exercise and throughout the 40-min exercise period. In addition, the increase in hepatic glucose production (HGP) during exercise was completely suppressed in Glu (results not shown). Because the average glucose infusion rate (35.0 ± 2.0 µmol · kg¹ · min¹) in these three subjects was almost identical to the rate of appearance of glucose (Ra) ingested before exercise in a study in which we had observed inhibition of liver glucose output during exercise (18), we were concerned that any effect on liver glucose production may have been due to changes occurring before exercise. Thus, for five subjects (results shown), the infusion commenced at the onset of exercise. The glucose solution was infused for the duration of the exercise at a mean rate of 35.2 ± 2.3 µmol · kg¹ · min¹ by a volumetric infusion pump (Gemini, PCi, v. 7-10. IMED, San Diego, CA).

Analytic techniques. Oxygen and carbon dioxide contents of dried expirate were analyzed by using Applied Electrochemistry S-3A/II and CD-3A analyzers (Ametek, Pittsburgh, PA), whereas volume was measured by using a Parkinson Cowan gas meter. Plasma glucose was measured by using an automated glucose oxidase method (YSI 2300, Yellow Springs, OH), and lactate was determined by using an enzymatic spectrophotometric method (15). Plasma insulin (Incstar, Stillwater, MN) and glucagon were measured by radioimmunoassay (2). The insulin-to-glucagon molar ratio was calculated by dividing the plasma insulin concentration by the plasma glucagon concentration. Plasma catecholamines were determined by using a single-isotope radioenzymatic method (TRK 995, Amersham, UK). For measurement of plasma [³H]glucose specific activity, 500 µl of plasma were mixed with 500 µl 0.3 M Ba(OH)₂ and 500 µl 0.3 M ZnSO₄ and spun. The supernatant (800 µl) was removed and dried overnight. The samples were reconstituted with 0.5 ml of distilled water and 10 ml of scintillant. After 1 h of refrigeration, the samples were counted (LS CA 3801, Beckman Instruments, Irvine, CA). Glucose kinetics at rest and during exercise were calculated by using a modified one-pool non-steady-state model (22), assuming a pool fraction of 0.65 and estimating the apparent glucose space as 25% of body weight. Ra and rate of glucose disappearance (Rd) were determined from the changes in specific activity of [³H]glucose. Although glucose Ra measures total endogenous glucose Ra, the liver is likely to be the predominant, if not sole, source of the increase in glucose production during exercise (25). Thus, in Con, HGP was equal to total Ra, whereas in Glu HGP was calculated as the difference between the measured total Ra and the rate of glucose infusion. The metabolic clearance rate (MCR) of glucose was calculated by dividing glucose Rd by the plasma glucose concentration. The data from the two trials were compared by analysis of variance for repeated measures, with significance at the P < 0.05 level. Specific differences were determined by using the Student-Newman-Keuls post hoc test. All data are reported as means ± SE.

	RESULTS

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There were no differences between trials in oxygen uptake and respiratory exchange ratio during 40 min of exercise (Table 1). During exercise, heart rate was similar between trials, except at 40 min when heart rate was lower (P < 0.05) in Glu compared with Con (Table 1). The plasma glucose concentration was similar at rest and increased during exercise in both trials, but was significantly higher after glucose infusion than during Con (Fig. 1). HGP was similar at rest between trials (Con, 11.4 ± 1.2; Glu, 10.6 ± 0.6 µmol · kg¹ · min¹). After 40 min of exercise, HGP reached a peak of 40.2 ± 5.5 µmol · kg¹ · min¹ in Con (Fig. 1). In Glu, there was complete inhibition of the increase in HGP during exercise, which never rose above the resting level (Fig. 1). There were no differences in glucose Rd at rest between the two trials. During exercise, glucose Rd increased in both trials; however, during the last 15 min of exercise, glucose Rd was significantly greater in Glu than in Con (Fig. 2). No differences were observed between trials for MCR (Fig. 2). Plasma lactate was similar between trials both at rest and during exercise (Table 2). There was a tendency (P = 0.07) for plasma insulin to be higher in Glu (Table 2), whereas glucagon and catecholamines were lower (main effect, P < 0.05) (Table 2). The insulin-to-glucagon molar ratio was significantly higher during exercise in Glu.

View this table: [in this window] [in a new window]   Table 1.   O2, RER, and HR during 40 min of exercise at 77 ± 1% peak O2 with and without intravenous glucose infusion commencing at onset of exercise

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Plasma glucose concentration (top) and hepatic glucose production (HGP; bottom) during 40 min of exercise at 77 ± 1% peak oxygen uptake with (Glu) and without (Con) intravenous glucose infusion commencing at onset of exercise. Values are means ± SE (n = 5 subjects). * Significantly different from Con (top) and Glu (bottom), P < 0.05.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Whole body glucose uptake [glucose rate of disappearance (Rd); top] and metabolic clearance rate (MCR; bottom) during 40 min of exercise at 77 ± 1% peak oxygen uptake with (Glu) and without (Con) intravenous glucose infusion commencing at onset of exercise. Values are means ± SE (n = 5 subjects). * Significantly different from Con, P < 0.05.

View this table: [in this window] [in a new window]   Table 2.   Insulin, glucagon, I/G molar ratio, Epi, NE, and lactate during 40 min of exercise at 77 ± 1% peak O2 with and without intravenous glucose infusion commencing at onset of exercise

	DISCUSSION

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In the present study, evidence for feed-forward activation during exercise at 77 ± 1% O_{2 peak} in trained men was shown in Con by a greater increase in the rate of glucose production than glucose uptake and, subsequently, elevated plasma glucose levels (Figs. 1 and 2). This response was similar to those observed previously (7, 14). However, the increase in liver glucose output was totally suppressed by an intravenous glucose infusion equal to the average glucose output measured during exercise in Con (Fig. 1). This result suggests that an increase in glucose availability provides a humoral feedback signal capable of overriding feed-forward activation. This has also been demonstrated in trained males exercising at 70% O_{2 peak}, where elevation of plasma glucose to a higher level of 10 mM completely suppressed liver glucose production, whereas glucose output was reduced, but not completely abolished, during euglycemia (8). Further evidence for feedback inhibition is provided by animal studies. During high-intensity exercise in running rats, glucose production was regulated primarily by feed-forward activation, but, as humoral feedback mechanisms were elevated late in exercise, glucose production was inhibited (27). Furthermore, early in exercise in rats running at a high workload, feedback inhibition resulted when glucose infusion significantly exceeded the normal increase in HGP (24). In contrast, glucose infusion that elevated blood glucose levels to >10 mM in rats only partly suppressed liver glycogen breakdown (28). Although species variation cannot be excluded (24), this demonstrates the difficulty in determining the glucose concentration at which feedback inhibition can override feed-forward activation during exercise. Given the complexity of the glucoregulatory system, it is likely that regulation of the exercise-induced increase in liver glucose production during strenuous exercise includes both a feed-forward as well as a feedback component. Whether neural feed-forward activation or humoral feedback inhibition dominates seems to be determined by the interaction between motor center activity, as determined by exercise intensity, and the prevailing plasma glucose and insulin levels.

Despite numerous studies that have examined the regulation of liver glucose output during exercise, the stimulus and subsequent regulatory mechanisms are still not completely understood. This is because of the complex and redundant nature of the many neurohormonal mechanisms that regulate liver glucose production (see Ref. 10 for review). In the present study, an intravenous glucose infusion that significantly elevated plasma glucose levels totally suppressed the rise in liver glucose production (Fig. 1). This effect is likely to be mediated directly by the elevated plasma glucose concentration. Increases in plasma glucose have been shown to directly alter liver glycogenolysis and, subsequently, reduce hepatic glucose output by decreasing glycogen phosphorylase and increasing glucokinase activities (20). Although it is possible that inhibition of liver glucose output was mediated by the plasma glucose concentration in the present study, alterations in the plasma levels of glucoregulatory hormones may have also played a role. Glucose infusion results in alterations in the plasma levels of key glucoregulatory hormones (6). In the present study, there was a tendency (P = 0.07) for plasma insulin levels to be higher in Glu (Table 2). The lack of statistical significance is most likely the result of a small sample size and intersubject variability. Plasma glucagon levels were reduced, and the insulin-to-glucagon molar ratio was higher during exercise in Glu (Table 2). Whereas changes in the pancreatic hormones play an important role in regulating hepatic glucose output during low-to-moderate-intensity exercise, especially in dogs (see Ref. 26 for review), this does not seem to be the case in humans exercising at higher intensities (4, 21). However, this does not rule out the possibility that, in the present study, changes in pancreatic hormone levels may have contributed, at least in part, to the reduction in glucose output. Furthermore, small changes in the peripheral levels of insulin and glucagon may not reflect significant alterations in the portal vein concentrations of these hormones. Such changes could account for the reduction in hepatic glucose output during Glu. The role of epinephrine in regulating glucose output is not clearly defined, but reduced liver glucose production may be due to lower plasma epinephrine levels in Glu (Table 2). It is unlikely that direct sympathetic neural innervation regulates liver glucose production during exercise, given that liver-transplant patients have a normal exercise-induced increase in liver glucose production (11); however, reduced plasma norepinephrine levels reflect diminished sympathetic activity that may have contributed to the decrease in liver glucose output. Together, these hormonal changes may partly explain the inhibition of liver glucose output during exercise. A recent finding that the glucose transporter isoform GLUT-4 is expressed in the hypothalamus (15) suggests that this area of the brain may respond directly to elevated blood glucose and/or insulin, resulting in a blunting of the neurohormonal responses to strenuous exercise. In addition, neural feedback from working muscle may play a role in regulating the hormonal and metabolic response to exercise (13). Although it is possible that afferent neural fibers are sensitive to changes in substrate availability, it is unlikely that in the present study neural feedback from contracting muscle can fully account for the alterations in plasma hormones and liver glucose production in Glu (Fig. 1, Table 2), since these changes occurred much earlier than increases in glucose Rd (Fig. 2).

Muscle glucose uptake during exercise is regulated by a number of factors, including membrane glucose transport, intracellular metabolism, and glucose delivery. Muscle glucose uptake is dependent on the prevailing plasma glucose concentration. Limb glucose uptake was elevated in exercising dogs after an increase in blood glucose availability (29). This has also been shown in humans when arm cranking added to cycling increased the arterial glucose concentration and leg glucose uptake (12). Similarly, in cyclists exercising at 73% O_{2 peak}, whole body glucose disposal was increased when plasma glucose was maintained at 10-12 mmol/l (5). Carbohydrate ingestion that elevated plasma glucose has also been found to increase muscle glucose uptake during low-intensity exercise (1) and prolonged strenuous exercise (19) in humans. In contrast, a recent study has suggested that it is the energy demands of the muscle, rather than hyperglycemia, that regulate glucose uptake during intense exercise in humans (17). In the present study, however, glucose uptake tended to increase during the early stages of exercise in Glu and was significantly greater than Con during the last 15 min of exercise (Fig. 2). Because MCR was not significantly different at any time point (Fig. 2), it appears that hyperglycemia increases glucose uptake, even during strenuous exercise in trained men.

In summary, the present study has demonstrated that during strenuous exercise in trained men the rise in HGP was completely suppressed by glucose infusion at a rate equal to the average glucose production observed in the control trial. These results suggest that blood-borne metabolic feedback signals can override feed-forward activation of HGP during strenuous exercise. This effect is most likely to be mediated by the increased plasma glucose level, but alterations in the plasma levels of glucoregulatory hormones may also play a role.