INTRODUCTION

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ONE OF THE MAJOR METABOLIC ADAPTATIONS to endurance exercise training is a decreased utilization of carbohydrate and concomitant increase in fat oxidation during exercise at the same absolute exercise intensity (see Ref. 2 for review). The altered substrate utilization is widely thought to be linked to a greater muscle oxidative potential after training (1, 11, 19). In contrast, shifts in substrate utilization have been detected after short-term endurance training in the absence of an increase in muscle respiratory capacity, suggesting that other factors may play a role (6-8, 16, 17). Short-term endurance training also results in a 6-22% expansion of plasma volume (3). Acute hypervolemia of this magnitude has been shown to attenuate the sympathoadrenal response during submaximal exercise (10), whereas an increase in muscle perfusion is evident after short-term training (18). Such responses may have an impact on intramuscular substrate utilization during exercise. Previous research has demonstrated that an increase in plasma volume attenuates glycogen use and blunts the exercise-induced rise in catecholamines during prolonged submaximal exercise (8). However, the hypervolemia in this study was induced by 3 days of moderate-intensity endurance exercise. Therefore, it is possible that the altered substrate use is a training adaptation and not a result of plasma volume expansion. More recently, acute plasma volume expansion had no effect on total carbohydrate oxidation, glucose kinetics, or estimated glycogen oxidation during prolonged exercise (15). The purpose of the present investigation was to examine the effects of acute plasma volume expansion on skeletal muscle carbohydrate metabolism by measuring muscle glycogen and lactate before and after submaximal exercise.

	METHODS

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Subjects. Seven healthy untrained men [age, 21 ± 1.6 yr; weight, 82.1 ± 5.6 kg (SD)] volunteered as subjects for the experiment. Each subject was informed of the risks associated with the procedures and signed a letter of informed consent before participation. The experiment was approved by the Deakin University Ethics Committee.

Preexperimental protocol. All subjects performed an incremental cycling test to volitional exhaustion on an electromagnetically braked cycle ergometer (LODE Instrument, Groningen, The Netherlands) to determine individual peak pulmonary oxygen uptake (O_{2 peak}). The criteria used to determine O_{2 peak} were a plateau in oxygen uptake (O₂) (<100 ml/min increase) with an increase in work rate and a respiratory exchange ratio (RER) >1.10. Mean O_{2 peak} was 3.36 ± 0.5 l/min. For the day preceding each trial, subjects were provided with a food parcel (~14 MJ, 80% carbohydrate), which they consumed, and were instructed to abstain from exercise and the ingestion of alcohol, caffeine, and tobacco. Additionally, subjects were instructed to consume 5 ml/kg body wt of water on the morning of each trial to ensure adequate hydration and similar pretrial plasma volume levels.

Experimental protocol. Each subject completed two submaximal exercise trials (72 ± 2% O_{2 peak}) for 40 min at an ambient temperature of 21 ± 1°C. On one occasion, exercise was performed without any pretreatment (Con group); on the other, exercise was preceded by plasma volume expansion (PVE group). Plasma volume expansion was achieved by using a plasma substitute, Haemaccel (Behringwerke, Marburg, Germany), which was infused over 15-30 min via a catheter in an antecubital vein. Four subjects performed the PVE trial first and completed the Con trial at least 1 wk later. The order was reversed for the remaining three subjects. On arrival at the laboratory, subjects voided and were weighed. Subjects then rested quietly on a couch, and indwelling catheters were inserted into an antecubital vein for blood sampling and, when required, in the contralateral arm for infusion. The catheter for blood sampling was kept patent by flushing with 0.5 ml of 0.9% saline containing five units of heparin after each sample collection. After the subject lay supine for 20 min, a resting muscle sample was obtained from the vastus lateralis by the percutaneous needle-biopsy technique modified to include suction. Muscle samples were immediately (<15 s) frozen in liquid nitrogen. After biopsy, a resting blood sample was obtained, after which ~7 ml/kg body wt of Haemaccel were infused into the PVE group. A second resting blood sample was obtained at the conclusion of the infusion to determine the magnitude of plasma volume expansion from changes in Hb and hematocrit (Hct) (4).

At the completion of the rest period, subjects exercised for 40 min. Venous blood samples were obtained at 10-min intervals and were analyzed for plasma lactate, glucose, and catecholamines. Blood for glucose and lactate analysis was placed in fluoride-heparin tubes, whereas that for catecholamines was placed in tubes containing EGTA and reduced glutathione. The samples were spun, and the plasma was removed and stored at 20°C and 80°C for glucose and catecholamine analyses, respectively. For lactate, 125 µl of plasma were deproteinized in 250 µl of 8% perchloric acid, spun again, and the extract was stored at 20°C for later analysis. Expired gases were collected in Douglas bags at 10-min intervals during exercise for measurement of O₂ and RER. Heart rate was measured continuously via telemetry (Polar sports tester, Polar Electro, Finland) and recorded every 10 min. Immediately on cessation of exercise, a second muscle sample was obtained from a separate incision on the same leg and immediately (<15 s) frozen. Subjects were not permitted to ingest fluid during the trials.

Analytical 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 volumes were measured by using a Parkinson Cowan gas meter calibrated against a Tissot spirometer. Hb and Hct were measured by using an automated analyzer (Sysmet SE9000, Tao Electronics, Kobe, Japan). 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 (12). Plasma epinephrine and norepinephrine were analyzed by using a single-isotope (³H) radioenzymatic assay (TRK995, Amersham). Muscle samples were weighed and freeze-dried, after which they were reweighed, dissected free of blood and connective tissue, powdered, and placed into two separate aliquots. One was extracted according to the procedures of Harris et al. (9) and analyzed for lactate by using standard enzymatic flurometric techniques (12). Muscle glycogen concentrations were determined on the second aliquot (14). The data from the two trials were compared by a two-way analysis of variance for repeated measures, with significance at the P < 0.05 level. Specific differences were located by Newman-Keuls post hoc test. Where appropriate, paired comparisons were made by t-test. All values are reported as means ± SE.

	RESULTS

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After administration of Haemaccel, Hb and Hct were both reduced (P < 0.05) by 6%, resulting in a 12 ± 2% plasma volume expansion (Table 1). Although we did not measure Hb and Hct (and, therefore, plasma volume changes) during exercise, it has been demonstrated previously (5) that the magnitude of relative plasma volume expansion is maintained during exercise. O₂ and heart rate increased (P < 0.05) during both trials before stabilizing after 20 min. After an increase (P < 0.05) early in exercise, RER was reduced after 20 min (P < 0.05) and remained unaltered for the duration of exercise. However, plasma volume expansion did not affect either heart rate or RER during exercise (Table 2). Total carbohydrate oxidation was similar in the two trials (Con: 108 ± 2 g; PVE: 105 ± 2 g).

View this table: [in this window] [in a new window]   Table 1.   Effect of PVE on Hb, Hct, and changes in BV and PV preceding exercise at 72 ± 2% O2 peak

Neither plasma glucose nor lactate was different when comparing PVE trial with Con during exercise. In both trials, plasma glucose decreased (P < 0.05) and lactate increased (P < 0.01) during the first 10 min of exercise. Thereafter, concentrations of these metabolites were unaltered (Fig. 1). Plasma catecholamines increased (P < 0.05) progressively throughout exercise in both trials but were not different when PVE and Con trials were compared. At 10 min of exercise, plasma epinephrine concentrations were 1.12 ± 0.17 and 0.97 ± 0.15 nmol/l and increased (P < 0.01) to 2.50 ± 0.77 and 1.89 ± 0.52 nmol/l at the conclusion of exercise for the Con and PVE trials, respectively (Fig. 2). Plasma norepinephrine levels were 11 and 28% lower, respectively, in the PVE group compared with Con at 10 and 40 min (Fig. 2, P = 0.06). However, when plasma catecholamine (Cat) values were corrected {[Cat]_corrected = [Cat]_measured × [1 + (PV/100)]} for the increased plasma volume (PV), assuming it was maintained during exercise (5), this tendency for a difference was abolished.

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Plasma lactate concentration (top) and plasma glucose concentration (bottom) during 40 min of exercise at 72 ± 2% peak oxygen uptake (O2 peak) in trials with plasma volume expansion (PVE) and without it (Con). Values are means ± SE (n = 7 men).

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Effects of PVE on plasma epinephrine (top) and plasma norepinephrine concentrations (bottom) during exercise at 72 ± 2% O2 peak. Values are means ± SE (n = 7 men).

Muscle glycogen content was not different when PVE and Con groups were compared either before or after exercise (Fig. 3). Although muscle glycogen use was slightly higher in Con trial (Con: 324 ± 39 vs. PVE 261 ± 54 mmol glucosyl units/kg dry mass), post hoc analysis revealed no significant difference. Muscle lactate content was not different when PVE group was compared with Con either before or after exercise.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Muscle glycogen (top) and muscle lactate (bottom) concentration at rest (0 min; Pre) and at the conclusion of exercise (40 min; Post) at 72 ± 2% O2 peak for PVE and Con groups. Values are means ± SE (n = 7 men).

	DISCUSSION

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The results of the present study suggest that acute plasma volume expansion, similar in magnitude to that seen after short-term endurance training, has no effect on either muscle carbohydrate metabolism or plasma catecholamine levels during 40 min of submaximal exercise in untrained men. This observation suggests that the reduced glycogen use and lactate accumulation, previously observed in response to short-term endurance training (17), are mediated by factors other than vascular hypervolemia.

The 12% increase in plasma volume that we induced in the PVE trial was similar to that seen after short-term training (3). In contrast with the well-accepted changes in carbohydrate metabolism that result from short-term training (6, 7, 19), we observed no change in muscle metabolism. This was reflected in the muscle glycogen, RER, blood glucose, and blood and muscle lactate data. Although we observed a 19% reduction in glycogen use in PVE trial, this was largely due to the results of one subject who, for reasons we cannot explain, had a marked decrease in glycogen use in the PVE trial. In fact, three of seven subjects had an increased net glycogen use when PVE group was compared with Con and, therefore, we are confident that plasma volume expansion had no effect on substrate metabolism. Our data are in agreement with previous investigations that have demonstrated no change in whole body carbohydrate and fat utilization (10, 15) or glucose kinetics and estimated glycogen oxidation (10) in untrained men after plasma volume expansion by dextran infusion. Hence, the changes in substrate metabolism that result from short-term endurance training appear to be mediated by factors other than vascular hypervolemia.

In contrast with previous studies that have employed similar protocols as the present study (5, 10), plasma catecholamines were unaltered by acute hypervolemia. An early adaptation to prolonged exercise training is a reduction in the sympathoadrenal response during exercise (8, 13, 20). Although the exact mechanism remains unclear, the hypervolemia observed at the onset of endurance training appears to significantly reduce the sympathoadrenal response during exercise (5, 7, 10). The reduction in sympathoadrenal activity is reflected by lowered plasma epinephrine and norepinephrine concentrations during the early stages of endurance training (8). Importantly, the reductions observed after 10 days of endurance training are not further manifested after 12 wk (13). The absence of a blunted plasma catecholamine response to exercise in the present study suggests that sympathoadrenal activity during exercise is unaffected by acute vascular hypervolemia of the magnitude we employed, a conclusion different from that in two previous studies (5, 10). Possible explanations for this result include differences in exercise intensity (72 vs. 46 and 60% O_{2 peak}) and duration (40 vs. 120 and 90 min) as well as the agent used to obtain plasma volume expansion (Haemaccel vs. dextran).

In summary, the present study has demonstrated that acute plasma volume expansion, to a level comparable to that obtained with short-term endurance training, failed to alter carbohydrate oxidation, muscle glycogen utilization, or plasma catecholamines during submaximal exercise in untrained subjects. This suggests that plasma volume expansion is unlikely to account for the changes in substrate metabolism observed during the early stages of endurance training.