INTRODUCTION

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GLYCEROL, WHICH CAN BE INGESTED in comparatively large amounts, accumulates in body fluids, except for those of the brain and eyes, increasing osmotic pressure and the total volume of water in the body (16, 30). This hyperhydration could be beneficial for endurance exercise, particularly when performed in the heat (30). Exogenous glycerol, which could be used as an energy substrate in parenteral nutrition (32), could significantly contribute to the energy yield during exercise and modify endogenous energy substrate utilization. In this respect, exogenous glycerol could be converted into glucose by the liver and kidneys or directly used in other tissues that express glycerol kinase (22), such as skeletal muscles (6, 8, 11, 12, 29). Only four studies have described the effect of glycerol administration on the metabolic response to exercise (7, 18, 20, 21). In all these studies, possible changes in fat and glucose oxidation due to glycerol ingestion were tracked by using the respiratory exchange ratio (RER). However, when glycerol is administered and presumably oxidized, changes in RER can no longer be used to directly estimate the respective oxidation of fat and glucose.

In the present experiment, the respective oxidation of exogenous glycerol and glucose (0.36 g/kg each) ingested simultaneously before exercise [120 min at 68% maximal oxygen uptake (O_2 max)] was computed using ¹³C labeling. Changes in fat and endogenous glucose oxidation due to ingestion of the glucose + glycerol mixture were computed using indirect respiratory calorimetry corrected for protein and exogenous glycerol oxidation.

	METHODS

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Subjects. The experiment was conducted on six active and healthy male subjects. Their age, body mass, height, and O_2 max on cycle ergometer were 22 ± 1 yr, 64 ± 2 kg, 175 ± 2 cm, and 4.4 ± 0.2 l/min, respectively (means ± SE). All subjects gave their informed, written consent to participate in the study, which was approved by the Institutional Board on the use of human subjects in research. None of the subjects were smokers, heavy drinkers, under medication, or using recreational drugs.

Experimental protocol. O_2 max and experimental workloads on cycle ergometer (Ergomeca, La Bayette, France) were determined using open-circuit spirometry (1100 medical gas analyzer, Marquette Electronics, Milwaukee, WI) for each subject during a preliminary test session. Subsequently, all subjects performed at 1-wk intervals, between 10:00 AM and midnight, three exercise periods of 120-min duration at a work load corresponding to 68 ± 2% O_2 max (3.02 ± 0.05 l/min). The last evening meal (7:00 PM: 1,300 kcal, 55% carbohydrates, 30% fat, and 15% proteins) and the morning breakfast (7:30 AM: 800 kcal, 60% carbohydrates, 30% fat, and 10% proteins) were provided to the subjects. To keep a low-background ¹³C enrichment of expired CO₂, ingestion of carbohydrates from plants with the C₄ photosynthetic cycle, which are naturally enriched in ¹³C (14), was avoided during the period of experiments. Subjects also refrained from exercising and drinking coffee and alcohol for 2 days before each experiment.

¹³C labeling. To measure the respective oxidation of glycerol and glucose in the mixture, glycerol artificially enriched in ¹³C was ingested along with unlabeled glucose in trial 2, whereas unlabeled glycerol was ingested along with artificially enriched [¹³C]glucose in trial 3. For this purpose, glycerol and glucose naturally poor in ¹³C (¹³C/¹²C = 29.6, and 25.2 [-¹³C]PDB-1, respectively) were ingested with or without addition of artificially enriched [U-¹³C]glycerol or [U-¹³C]glucose (¹³C/C ratio > 99%; Isotec, Miamisburg, OH) to achieve a final ¹³C/¹²C close to +245 [-¹³C]PDB-1 (actual values measured by mass spectrometry were +244.5 and +243.8 [-¹³C]PDB-1 for glycerol and glucose, respectively). This high ¹³C enrichment of labeled glucose and glycerol provided a very strong signal in expired CO₂ vs. the comparatively small changes in background enrichment of expired CO₂ observed from rest to exercise (Ref. 28 and Fig. 1). In addition, ¹³C/¹²C in expired CO₂, in trials 2 and 3, was corrected for the shift observed when the placebo was ingested (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   13C-to-12C ratio in expired CO2 observed during exercise when the placebo, [13C]glucose + unlabeled glycerol, or unlabeled glucose + [13C]glycerol was ingested. Values are means ± SE. a Significantly different from glucose + glycerol trials; b significantly different from [13C]glucose + unlabeled glycerol trial; c significantly different from resting values from this point to the end of exercise (P < 0.05).

Measurements. Measurements and blood sampling were made at rest before ingestion of exogenous substrates and every 20 min during the exercise period. Carbohydrate and fat oxidation were computed from carbon dioxide production (CO₂) and oxygen consumption (O₂), corrected for protein oxidation, based on urea excretion over the 120-min observation periods. For this purpose, the amount of urine produced during the observation period was measured, and the volume of sweat produced during exercise was estimated from change in body mass, taking into account fluid and substrate intake, loss of mass through CO₂ production, and water loss in the lungs (15). Urea concentration was measured in urine and in a sample of sweat taken with the aid of absorbent gauze placed in a small plastic bag taped on the back of the subjects. For the measurement of ¹³C/¹²C in expired CO₂, 80-ml samples of expired gases were collected and stored in vacutainers (Becton Dickinson, Franklin Lakes, NJ) until analysis. Blood samples (6 ml) were drawn through a catheter (Baxter Health Care, Valencia, CA) inserted into an antecubital vein at the beginning of experiment for measurements of plasma glucose, free fatty-acid, glycerol, and insulin concentrations. Plasma, urine, and sweat samples were stored at 80°C until analysis.

Computation. The oxidation rate of exogenous glucose (trial 2) and glycerol (trial 3) (M_exo, g/min) was computed as follows (28)

(1)

where CO₂ is in liters (STDP) per minute, R_exp is the ¹³C/¹²C observed in expired CO₂, R_ref is the ¹³C/¹²C in expired CO₂ at rest before exercise, R_exo is the ¹³C/¹²C in the artificially labeled exogenous glucose or glycerol ingested, and k is the volume of CO₂ provided by the complete oxidation of glucose and glycerol (0.7426 and 0.7304 l/g for glucose and glycerol, respectively). The computation of the amounts of exogenous substrate oxidized is made assuming that, in response to exercise, ¹³C is not irreversibly lost in pools of tricarboxylic acid cycle intermediates and/or bicarbonates, and ¹³CO₂ recovery in expired gases is complete or almost complete (4, 13). However, the ¹³C/¹²C in expired CO₂ only slowly equilibrates with ¹³C/¹²C in the CO₂ produced in the tissues (24). To take into account this delay between ¹³CO₂ production in the tissues and at the mouth, the computations were only made during the last 80 min of the observation period, thus allowing for a 40-min equilibration period at the beginning of exercise.

(2)

(3)

Statistics. Data are presented as means ± SE. The main effects of time and exogenous substrate ingested and time-substrate interactions were tested by repeated-measures analysis of variance (Statistica package). Newman-Keuls post hoc tests were used to identify the location of significant differences (P  0.05) when the analysis of variance yielded a significant F ratio.

	RESULTS

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No significant difference was observed for O₂, CO₂, and urea excretion between experimental trials (Table 1). Figure 1 shows the ¹³C enrichment of expired CO₂ at rest before ingestion of the placebo or the ¹³C-labeled mixtures of glucose and glycerol and during the 120-min exercise period. ¹³C/¹²C in expired CO₂ at rest was similar in all experimental trials (22.7 ± 0.2 [-¹³C]PDB-1, pooled data; n = 18). When the placebo was ingested, a slight and progressive increase in ¹³C/¹²C in expired CO₂ was observed throughout the exercise period. At all time points, these values were much lower than those observed when the mixture of glucose and glycerol was ingested. After ingestion of these substrates, ¹³C/¹²C in expired CO₂ increased progressively, peaked ~60 min into the exercise, and then declined. However, ¹³C/¹²C in expired CO₂ rose slower and reached lower peak values after [¹³C]glycerol than after [¹³C]glucose ingestion.

The amounts of glucose and glycerol oxidized are shown in Table 1. Over the last 80 min of exercise, 12.1 ± 0.7 g of exogenous glucose were oxidized. The amount of exogenous glycerol oxidized was significantly (21%) lower (10.0 ± 0.8 g). Compared with the placebo trial, no significant changes in fat or total glucose oxidation were observed when the glucose + glycerol mixture was ingested. In contrast, endogenous glucose oxidation was slightly (14%) but significantly reduced (123.7 ± 11.7 vs. 144.4 ± 13.0 g).

Changes in plasma metabolite and insulin concentrations are shown in Fig. 2. In the placebo trial, plasma glycerol concentration increased slightly, reaching a peak value of 0.24 ± 0.03 mmol/l at the end of exercise. In contrast, a large increase in plasma glycerol concentration was observed in response to the ingestion of the glucose + glycerol mixture, with peak values of 2.2 ± 0.2 mmol/l reached 40 min into the exercise period. The small decrease in plasma glucose concentration during the exercise did not reach statistical significance, and no significant difference was observed between experimental conditions. In response to exercise, plasma insulin significantly decreased in a similar fashion with ingestion of the placebo and the glucose + glycerol mixture. Plasma free fatty acid concentration rose progressively in response to exercise in both experimental trials. However, compared with the placebo trial, the values observed were lower when the glucose + glycerol mixture was ingested, the difference reaching statistical significance during the last 20 min of exercise.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Plasma glycerol, glucose, and free fatty acid (FFA) and insulin concentrations (indicated by brackets) observed in response to exercise in the placebo and glucose + glycerol trial. In the glucose + glycerol trial, blood sampling was randomly performed in only 1 of the 2 trials. a Significantly different from glucose + glycerol trial; c significantly different from resting values from that point to the end of exercise (P < 0.05).

	DISCUSSION

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In the present experiment, the amount of glucose ingested along with exogenous glycerol was small (0.36 g/kg or 23.3 ± 0.6 g) compared with that ingested in other reports on the oxidation of exogenous glucose during exercise (see Ref. 9 for a review). Thus the amount of exogenous glucose oxidized over the last 80 min of exercise was comparatively low (12.1 ± 0.7 g or 52% of the load), accounting for only 4.1% of the total energy yield. These data are in line with those from the only study in which such a low amount of glucose was ingested. Indeed, Péronnet et al. (26) reported that, when 30 g of glucose were ingested immediately before the beginning of a 90-min period of exercise at 68% O_2 max, the amount of exogenous glucose oxidized was 13.0 ± 4.2 g. This observation suggests that adding comparatively large amounts of glycerol to a glucose drink does not interfere with glucose absorption and oxidation. This probably stems from the fact that intestinal absorption of glucose, as well as the entry of glucose into the cell, depends on selective transport mechanisms, which are different from those involved in the transport of glycerol (16).

There are no data on the metabolic fate of exogenous glycerol administered before prolonged exercise or on the possible changes in fuel oxidation. Several authors have attempted to describe possible changes in glucose vs. fat oxidation when 0.38-1.0 g/kg of glycerol were ingested before exercise of 80- to 150-min duration at 50 to 73% O_2 max (7, 18, 20, 21) using O₂ and/or the RER. Compared with exercise with ingestion of water, no significant change in RER was observed when glycerol was administered. However, the RER can be used for computing the respective contributions of glucose vs. fat oxidation to the energy yield only when no other substrate is actually oxidized. To correctly estimate the respective contribution of fat and glucose oxidation to the energy yield, when another substrate such as glycerol is ingested and presumably oxidized, O₂ and CO₂ should be corrected for the volume of O₂ and CO₂ used and produced in glycerol oxidation (0.730 and 0.852 l/g, respectively). For this purpose, the oxidation of exogenous glycerol should be measured first, for example, by using ¹³C labeling as in the present experiment.

Over the last 80 min of exercise, a large percentage of exogenous glycerol ingested was oxidized: 43% of the load or an average of 10.0 ± 0.8 g or 1.34 ± 0.01 mmol/min. This rate of oxidation is ~40% lower than the indirect estimation made by Gleeson et al. (7) (2.2 mmol/min) from the reduction of plasma glycerol concentration over 86 min of exercise, assuming that the glycerol space is 65% body mass. This could be because, in the present experiment, the amount of glycerol administered was ~60% lower than in the study by Gleeson et al.: 0.36 vs. 1 g/kg, which could indicate that exogenous glycerol oxidation, like exogenous glucose oxidation, increases with the amount ingested. In addition, in the Gleeson et al. study, because of the large amount of glycerol administered, plasma glycerol concentration (16.7 mmol/l at the beginning of exercise) was well above the renal threshold for glycerol reabsorption (~1.6 mmol/l) (30). Consequently, a significant percentage of glycerol was probably lost in urine (31). In the present experiment, a much lower dose of glycerol was administered, and the peak plasma glycerol concentration observed was only 2.2 mmol/l, which compares well with data from Murray et al. (21) (2.8 mmol/l ~60 min after ingestion of 0.38 g/kg of glycerol). Urinary glycerol excretion was not measured. However, plasma glycerol concentrations in the 2-3 mmol/l range are not associated with large glycerol loss in urine (23).

In the present experiment, compared with the oxidation of exogenous glucose in the mixture, the oxidation of exogenous glycerol was ~20% lower. However, because the energy potential of glycerol is 18% higher than that of glucose (4.57 vs. 3.87 kcal/g), the contribution of exogenous glycerol oxidation to the energy yield was similar to that of exogenous glucose oxidation (4.0%). The fraction of the exogenous glycerol load that was actually oxidized during the last 80 min of exercise (43%) was slightly higher than that of exogenous lactate (~35%) for similar amounts ingested (25). In addition, in this previous study on the metabolic fate of exogenous lactate (25), in agreement with what was reported by others, we observed that ~25 g of lactate, in the form of various salts, was the maximal amount that can be ingested without gastrointestinal discomfort. In contrast, large amounts of glycerol can be ingested before exercise with no or minimal gastrointestinal discomfort (up to 1 g/kg every 6 h), although this could be associated with headache due to cerebral dehydration (30). Accordingly, exogenous glycerol oxidation could significantly contribute to the energy yield, although the possible beneficial effect of glycerol ingestion in endurance performance remains a matter of debate (7, 18, 20, 30).

Oxidation of exogenous glycerol, and of exogenous glucose provided in the same mixture, did not modify fat oxidation during the exercise period, although plasma fatty acid concentration was lower when exogenous substrates were ingested, compared with ingestion of the placebo. A lower concentration of plasma free fatty acids is a consistent observation in response to exercise when glycerol is administered (7, 18, 20, 21). Gleeson et al. (7) have suggested that this does not reflect a reduction in lipolysis and free fatty release from adipose tissue but a higher rate of disappearance of blood-borne free fatty acids through reesterification in the liver because of the large availability of glycerol. Recent evidence from Guo and Jensen (8) indicates that reesterification of free fatty acids using blood-borne glycerol could also occur in the skeletal muscles.

Ingestion and oxidation of exogenous glycerol and glucose significantly reduced endogenous glucose oxidation. This phenomenon has been regularly reported when glucose is administered (9) and appears to be due mainly to a reduction in glucose released from the liver (3, 19) with no consistent reduction in muscle-glycogen utilization (9). Miller et al. (20) also failed to observed any muscle-glycogen sparing effect during prolonged, sustained exercise when large amounts of glycerol were ingested.

It is generally accepted that the liver and, in a lesser manner, kidneys are the main organs involved in the removal of plasma glycerol (see references in Ref. 10) because of their substantial glycerol kinase activity (16). It also is believed that the main fate of glycerol in these tissues is conversion into glucose (10). However, a number of studies in dogs (Ref. 29 and references in Ref. 10) and humans (2, 12) in the postabsorptive state at rest have shown that the percentage of endogenous glycerol turnover (R_t) converted into glucose only ranges from 20 to 56%. In addition, Previs et al. (29), in dogs infused with epinephrine, and Landau et al. (12), in humans submitted to a 60-h fast, have shown that the net uptake of glycerol by the liver and kidneys accounts for only 17-30% and 4-17% of endogenous glycerol R_t, respectively. Landau et al. also reported a significant uptake of glycerol by the forearm and estimated that the total muscle mass could account for ~13% of glycerol R_t. Uptake of glycerol by the muscle was also reported by Elia et al. (6) and Kurpad et al. (11), and, recently, Guo and Jensen (8) have shown that blood-borne glycerol was incorporated into muscle triacylglycerols. These data are consistent with the observation that glycerol kinase activity is present in a variety of tissues (16), including skeletal muscles (22). Taken together, these observations suggest that exogenous glycerol could be converted into glucose in the liver before being oxidized but could also be taken up directly and oxidized in peripheral tissues. However, results from the present experiment do not allow the respective contributions of these two routes in the oxidative disposal of the glycerol load ingested before exercise to be determined.