INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

CARBOHYDRATES, IN THE FORM of muscle glycogen and blood-borne glucose, are important substrates for contracting skeletal muscle. Among the numerous factors that regulate carbohydrate metabolism in muscle, there is evidence that adrenergic stimulation can affect both uptake of glucose by muscle and the rate of intramuscular glycogen utilization. In humans at rest, both basal and insulin-stimulated glucose clearance are impaired during epinephrine infusion (3). Furthermore, studies in the rat have shown that skeletal muscle is the main tissue in which glucose uptake is decreased by epinephrine (6). The effects of epinephrine on utilization of blood glucose during exercise are less well studied. However, epinephrine infusion reduces leg glucose uptake in humans during cycling exercise (30). Similarly, epinephrine infusion in running dogs inhibits glucose clearance but does not affect hepatic glucose production (28). Moreover, administration of propranolol inhibits the effects of epinephrine on glucose clearance in exercising dogs (28), implicating -adrenergic mechanisms in this restraint of glucose uptake.

In contrast to the inhibitory effects of -adrenergic stimulation on glucose uptake by muscle, studies in animals (28, 53) and humans (9, 30) have demonstrated that epinephrine infusion increases muscle glycogen utilization during submaximal exercise. Furthermore, adrenomedullation in rats (52) or -adrenergic blockade (27) in dogs reduces muscle glycogen use during exercise. In dogs, -adrenergic blockade also reverses the effect of epinephrine infusion on muscle glycogenolysis (28). It is apparent, therefore, that -adrenergic mechanisms can exert opposing effects on muscle glycogenolysis and the uptake of glucose by muscle.

It is well known that exercise is a potent stimulus for epinephrine secretion. However, the magnitude of the plasma epinephrine response is affected by carbohydrate availability. In humans during exercise, plasma epinephrine concentrations are higher in fasted than in fed subjects (15, 49). Conversely, short-term (3-4 days) consumption of a high-carbohydrate diet (29) or ingestion of glucose during exercise (45) attenuates the exercise-associated increase in plasma epinephrine. Given the aforementioned effects of -adrenergic stimulation on carbohydrate metabolism in muscle, it is possible that such alterations in the plasma epinephrine response can modify the relative contributions of blood-borne glucose and intramuscular glycogen to total carbohydrate oxidation (CHO_ox) during exercise.

Few studies have examined the role of adrenergic mechanisms in regulation of carbohydrate metabolism in horses during exercise. However, similar to other species, -adrenergic blockade accelerates glucose utilization in horses during low- and moderate-intensity exercise. Carbohydrate availability also modifies the sympathoadrenal response to sustained exertion. Gabbard et al. (13) have reported that, compared with a high-carbohydrate meal (corn), preexercise consumption of feed low in soluble carbohydrate (alfalfa hay) exacerbated the plasma catecholamine response to light exercise. Furthermore, the increase in glucose supply associated with an intravenous infusion of glucose attenuates the increase in plasma epinephrine in horses during exercise at 35% of maximum oxygen uptake (O_{2 max}) (16). Thus there is evidence that -adrenergic stimulation constrains glucose uptake in horses during exercise. Moreover, on the basis of plasma epinephrine concentrations, an increase in carbohydrate availability reduces -adrenergic stimulation during exercise. Taken together, these findings suggest that, under conditions of increased carbohydrate availability, attenuation of the sympathoadrenal response may contribute to an increase in whole body glucose disposal. To date, however, this hypothesis has not been tested.

The present studies were, therefore, undertaken to determine the effects of glucose supply (preexercise oral glucose administration vs. water placebo) and adrenergic mechanisms (preexercise glucose administration with and without an intravenous infusion of epinephrine during exercise) on tracer-determined whole body glucose uptake in horses during moderate-intensity exercise. It was hypothesized that preexercise glucose administration would attenuate the sympathoadrenal response to exercise and increase utilization of blood glucose, evidenced by an increase in glucose rate of disappearance (R_d). Conversely, the increase in -adrenergic stimulation associated with epinephrine infusion would reverse the effects of preexercise glucose administration such that whole body glucose disposal would be similar to that measured in control (a 24-h fast before exercise) trials. Because -adrenergic stimulation can also affect muscle glycogenolysis, a further objective was to determine the effects of these interventions on net muscle glycogen utilization.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

All animal experiments were conducted after approval by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University and were performed in compliance with their guidelines and recommendations.

Horses. The subjects were six horses (2 Standardbred and 4 Thoroughbred; all geldings), 3-9 yr old, body mass 408-527 kg (471 ± 25 kg, mean ± SD). The horses were conditioned and undertaking regular treadmill exercise (5 days/wk) for 2 mo before the study. Four days per week, horses exercised for 30-45 min at 55% of O_2max, and on the fifth day a protocol of moderate- (20 min at 55% O_2max) and higher (10 min at 75% O_2max) intensity exercise was completed. During the conditioning and experimental periods, the horses were housed indoors and fed a diet of timothy grass and alfalfa hay and a pelleted concentrate ration, and they had access to a salt and mineral block. Between experimental trials, horses received 3 days of light treadmill exercise (20 min of trotting at 4-4.5 m/s with the treadmill set at a 4° incline).

Preliminary testing. For each horse, O_{2 max} and the relationship between oxygen consumption (O₂) and speed were determined during an incremental exercise test 1 wk before the first experiment. The incremental exercise test consisted of the horse running on a high-speed treadmill (Sato) inclined at 4° for 90 s at 4 m/s, after which the treadmill speed was increased by 1 m/s every 90 s until the horse was no longer able to maintain its position on the treadmill. O₂ was measured every 10 s during the exercise test. O_2max was defined as the value at which O₂ reached a plateau, despite further increases in speed. A plateau was defined as a change in O₂ of <4 ml · kg¹ · min¹ with an increase in speed. From linear regression analysis (speeds below O_2max), the running speed that elicited 55% of O_2max was calculated for each horse.

Experimental design. The effects of preexercise glucose administration and of epinephrine infusion on carbohydrate metabolism during moderate-intensity exercise were examined in a three-way crossover study. All horses undertook 60 min of treadmill exercise at a workload equivalent to 55% O_2max in each of three experimental conditions: 1) 1 h after administration of a 20% glucose solution (2 g/kg body wt; G trial); 2) 1 h after administration of a 20% glucose solution and with an intravenous infusion of epinephrine (0.2 µmol · kg¹ · min¹), commencing at the onset of exercise (GE trial); and 3) 1 h after administration of water only (F trial). For each horse, the administered fluid volumes (glucose solution or water) were the same (~8-9 liters). Trials were separated by 7 days, and the order of the trials was randomized but balanced among treatments.

Experimental protocol. The horses were fasted for 24 h before each experiment and were confined to their stalls during this time period. Body weight was measured on entry to the laboratory. After aseptic preparation and local anesthesia of the overlying skin, catheters (14 gauge, 51/4 in.; Angiocath, Becton Dickinson) were inserted into the right and left jugular veins for isotope infusion and blood collection, respectively. Thereafter, a blood sample was obtained for subsequent determination of background isotopic enrichment. For determination of glucose kinetics, a primed (18.0 µmol/kg), continuous (0.248 ± 0.007 µmol · kg¹ · min¹) infusion of [6,6-²H]glucose (99% enriched; Cambridge Isotopes, Cambridge, MA) in 0.9% saline was then initiated with the use of a calibrated infusion pump (PHD 2000; Harvard Apparatus, South Natick, MA). After a 90-min equilibration period, during which the horses stood in stocks, the glucose or water treatments were administered by nasogastric gavage. Five minutes before exercise, a sample of middle gluteal muscle was obtained by percutaneous biopsy (see Sampling procedures). Thereafter, the horses were positioned on the treadmill (4° incline), and a loose-fitting face mask for measurement of respiratory gas exchange was applied. A thermocouple (model T-180; Physitemp Instruments, Clifton, NJ) attached to a thermometer (BAT-10, Physitemp Instruments) was inserted 20-25 cm beyond the anal sphincter for measurement of temperature within the rectum during exercise. The horses completed a 5-min warm-up (3 m/s treadmill belt speed) followed by 60 min of running at a speed calculated to elicit 55% O_2max. The rate of [6,6-²H]glucose infusion was tripled (0.747 ± 0.02 µmol · kg¹ · min¹) during the warm-up. In the GE trial, an epinephrine solution (12.5 mg of epinephrine diluted in 500 ml of 0.9% saline for a concentration of 25 µg/ml) was infused (0.2 µmol · kg¹ · min¹) by a second pump (volumetric infusion pump, model Vet/IV 2.2, Heska), whereas an equivalent volume of 0.9% saline was administered during the F and G trials. The epinephrine or saline solutions were infused via a three-way stopcock to allow simultaneous administration of the tracer and epinephrine or saline. The epinephrine and saline infusions were commenced at the onset of exercise (after completion of the warm-up). During the exercise test, fans mounted 0.5 m in front and to the sides of the treadmill were used to maintain an air velocity of 3.5-4 m/s over the horse. Ambient conditions were similar for all trials; values for room temperature and relative humidity during the experiments were 22.4 ± 0.9°C and 58 ± 5% (means ± SD), respectively. The horses were reweighed after completion of the exercise protocol.

Respiratory gas exchange measurements. O₂, carbon dioxide production (CO₂), and respiratory exchange ratio (RER) were measured with an open-circuit calorimeter (Oxymax-XL, Columbus Instruments, Columbus, OH) as previously described (24). Flow through the system was ~1,500 l/min STP when the horse was stationary and 9,000 l/min during running. Data for expired O₂ (electrochemical cell, Columbus Instruments) and CO₂ (single-beam nondispersive infrared sensor, Columbus Instruments) concentrations were measured continuously and were reported at 10-s intervals. The gas-analysis system was calibrated before the start of each exercise test by using gas mixtures with O₂ and CO₂ concentrations that spanned the measurement range. The overall accuracy of the system was verified repeatedly by the nitrogen dilution method (10). Discrepancy between simulated O₂ produced by nitrogen dilution and the value measured by the system was ±3% at nitrogen flow rates equivalent to a O₂ of 54 l/min (~140 ml · kg¹ · min¹ for a 385-kg horse). Standard equations were used to calculate O₂ and CO₂ and R values were calculated by dividing CO₂ by O₂.

Rectal temperature. Temperature within the rectum (T_re) was measured at rest before the start of exercise and at 10-min intervals during the exercise trial. The thermocouple had a response time of ~1°C/s and was calibrated in a heated water bath with a precision thermometer (Fisher Scientific, Mississauga, Ontario).

Sampling procedures. Blood samples were obtained at 75, 60, 45, 30, and 15 min before exercise and at 0, 5, 10, 20, 30, 40, 50, and 60 min of exercise (where the "75" sample was collected 75 min after the start of isotope infusion and the water or glucose solutions were administered immediately after collection of the "60" sample). Blood samples were divided (6-ml aliquots) into four different tubes for subsequent analysis. Two aliquots of each sample were placed in evacuated tubes containing EDTA. These samples were later analyzed for plasma isotopic enrichment, hematocrit, plasma total protein, nonesterified fatty acid (NEFA), and glycerol concentrations. A 5-ml aliquot was placed in a tube containing sodium fluoride-potassium oxalate for subsequent determination of plasma glucose and lactate concentrations. The final aliquot was placed in a tube containing no additive and was later analyzed for serum insulin concentration. Additional blood samples for subsequent measurement of epinephrine and norepinephrine concentrations were obtained at 0, 10, 20, 40, and 60 min of exercise and were placed in test tubes that held 120 µl of a solution containing 0.24 M EGTA-reduced glutathione. Plasma or serum was obtained by centrifugation (3,000 rpm for 20 min at 4°C) within 30 min of collection and frozen at 20°C (80°C for hormone and tracer samples) until analysis.

Plasma isotopic enrichment. Plasma samples (0.7 ml) were deproteinized by adding 1.2 ml of 0.3 N Zn(SO)₄ and 1.2 ml of 0.3 N Ba(OH)₃. Each tube was then vortexed and incubated in the ice bath for 20 min. After centrifugation (3,000 rpm for 20 min at 4°C), the supernatant was harvested, and the water was removed from the tubes by vacuum centrifugation (Savant Instruments, Farmingham, NY). The penta-acetate derivative of [6,6-²H]glucose was prepared by adding 100 µl of a 2:1 acetic anhydride and pyridine mixture to the dried sample. After a 60-min incubation at 60°C, the reaction mixture was partitioned by sequential addition of 1.5 ml double-distilled water and 0.4 ml of methylene chloride. After gentle shaking, the tubes were centrifuged at 2,000 rpm for 10 min. The upper water phase was discarded, and the remaining methylene chloride phase was evaporated under N₂. Before injection into the gas chromatograph-mass spectrometer, the samples were dissolved in 50 µl of ethyl acetate. Standards of known isotopic enrichment were prepared in an identical fashion and were analyzed with each batch of samples. Isotopic enrichment was determined by gas chromatography-mass spectrometry on a Hewlett-Packard 5989A mass spectrometer equipped with a 30-m × 0.25-mm B-5 capillary column (J & W Scientific, Folsom, CA) and with the use of 1-µl injections. The initial oven temperature was set at 110°C and was gradually increased by 35°C/min until it reached a final temperature of 255°C. The transfer line was set at 250°C, the source temperature was set at 200°C, and the quadrupole temperature was set at 115°C. Ions were formed by electron impact ionization (70 eV), and molecular ions of mass-to-charge ratios (m/z) of 200 (m + 0), 201 (m + 1), and 202 (m + 2) were selectively monitored, i.e., m/z 200 and m/z 202 correspond to the unlabeled and labeled ions, respectively. The tracer-to-tracee ratio was calculated directly from measured ion abundance ratios and was equal to R  R_o, where R and R_o represent the measured tracer and tracee ion abundance ratios for enriched and unenriched (background or preinfusion) samples, respectively. Correction was made for the contribution of singly labeled molecules (m/z 201) to the apparent enrichment at m/z 202 (61). The intra-assay coefficient of variation was 1.5 ± 0.5%, and the interassay coefficient of variation was 5.6 ± 2.1%. To control for between-day variability, all samples for a given horse (F, G, and GE) were analyzed during the same analytic session.

Plasma biochemical analyses. Plasma glucose and lactate concentrations were measured by the glucose oxidase and lactate oxidase methods, respectively, with an automated analyzer (Yellow Springs Instruments, Yellow Springs, OH). Urine glucose concentration was also measured by use of the glucose oxidase autoanalyzer. Plasma NEFA concentration was determined by using a commercial kit that employs an enzymatic colorimetric method (NEFA test kit; Wako Chemicals USA, Dallas, TX). Plasma glycerol concentration was measured by an enzymatic spectrophotometric method [triglycerides kit 337A (without the triglyceride hydrolysis step); Sigma Chemical]. Intra- and interassay coefficients of variation for these biochemical methods were <1.0% and 2.5%, respectively. Hematocrit was measured by the microhematocrit technique. Plasma total protein was measured by refractometry (Cambridge Instruments, Buffalo, NY). All samples were analyzed in duplicate.

Plasma hormone analyses. Plasma epinephrine and norepinephrine concentrations were determined by HPLC using electrochemical detection (42). Serum immunoreactive insulin (IRI) was determined in duplicate by use of a commercially available RIA (insulin kit, Coat-a-Count Diagnostics, Los Angeles, CA) that has been validated for horse blood (51). Intra- and interassay coefficients of variation were 6.0 ± 1.5% and 11.5 ± 2.1%, respectively.

Muscle glycogen and lactate. Muscle samples were weighed and subsequently freeze-dried. The freeze-dried samples were reweighed and dissected free of any blood and connective tissue. For each sample, duplicate pieces of muscle were analyzed. One portion was extracted according to the general procedures of Harris et al. (23), and duplicate extracts were analyzed for lactate content by enzymatic fluorometric methods (41). Muscle glycogen concentrations (as glucosyl units) were determined on a second freeze-dried aliquot, which was extracted and analyzed according to the procedure of Passoneau and Lauderdale (48). Intra-assay (12 replicates of a single sample) and interassay (6 replicates) coefficients of variation for the muscle glycogen assay were 2.5% and 4.1%, respectively.

Calculations of glucose kinetics. Glucose rate of appearance (R_a) and R_d (=tissue uptake) at rest were calculated by using the steady-state tracer dilution equation (61)
where F is the infusion rate of the isotope (in µmol · kg¹ · min¹); IE_i and IE_p are the stable isotopic enrichment of the infusate and plasma, respectively; and 1 accounts for the tracer's contribution to the turnover rate of the substrate (61). The rate of infusion was calculated by multiplying the infusion pump rate by the concentration of glucose in the infusate. During exercise, R_a and R_d were calculated according to the non-steady-state equation developed by Steele (57). This equation is modified for use with stable isotopes as the amount of tracer infused is no longer negligible
and
where V_d is the effective volume of distribution, E is the plasma isotopic enrichment, C_m is the measured plasma concentration of the tracee, and dE/dt and dC_m/dt are maximum rates of change in enrichment and glucose concentration, respectively, as a function of time. By using this fixed, one-compartment model of Steele, it is assumed that 1) the apparent glucose space is 25% of body weight, and 2) 65% of this space represents the rapidly mixing portion of the glucose pool. Therefore, the effective V_d for glucose was assumed to be 162 ml/kg. However, our interpretation of the data presented herein is not affected by the assumed value for V_d because the difference in R_a did not exceed 10% when values were calculated using values for V_d ranging from 40 to 210 ml/kg.

Rates of energy expenditure and whole body substrate oxidation. Total energy expenditure (TEE) and absolute rates of CHO_ox and lipid oxidation were calculated by using the following equations (12)

(1)

(2)

(3)

where O₂ and CO₂ are in liters per minute and it was assumed that protein oxidation made negligible contribution to O₂ and CO₂. The calculated values were based on respiratory gas exchange values averaged over 5-min intervals for the first 10 min of exercise and averaged over 10-min intervals thereafter. CHO_ox in grams per minute was converted to micromoles per kilogram per minute by dividing the molecular weight of glucose (180) and the horse's body weight. Similarly, rates of fat oxidation were converted to micromoles per kilogram per minute by dividing by the molecular weight of palmitate (259) and the horse's body weight.

Statistical analyses. Unless otherwise stated, data are presented as means ± SE. The data were analyzed as a three-way crossover design by use of a two-way repeated-measures ANOVA [repeated measures on treatment (i.e., F, G, or GE) and time factors]. As the data for muscle glycogen did not exhibit homogeneous variances, these data were subject to logarithmic transformation before ANOVA. Data for net muscle glycogen utilization and the absolute and relative contributions by different substrates to TEE during the 30-60 min period of exercise were analyzed by a one-way repeated-measures ANOVA (repeated measures on the treatment factor). Percent data (relative contributions by different substrates) were subject to arcsine transformation before ANOVA. The null hypothesis was rejected at  = 0.05 for the main effects (treatment and time) and  = 0.10 for the interaction. Significant differences identified by ANOVA were isolated using the Student-Newman-Keuls post hoc test. The Sigmastat 2.0 software package (Jandel Scientific, San Rafael, CA) was used for statistical computations.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Individual values for O_2max ranged from 127 to 168 ml · kg¹ · min¹ (mean 141 ± 6.8 ml · kg¹ · min¹). Mean running speed during the exercise protocol was 5.5 ± 0.2 m/s (range 5.2-6.0 m/s). The mean O₂ measured during the 60 min of exercise corresponded to a relative workload of 55 ± 1.0% of O_2max (range 53-57%). T_re was not different throughout exercise in F compared with G. However, T_re was significantly lower in GE than in G and in F between 30 and 60 min of exercise (Table 1). Preexercise body weight was similar among the three trials (F: 493 ± 15; G: 495 ± 17; GE: 496 ± 15 kg). Mean values for loss of body weight during exercise were higher in GE (18.8 ± 1.2 kg) than in F (15.5 ± 1.2 kg) and G (15.3 ± 1.3 kg). However, this difference did not reach statistical significance (P = 0.15; power at  = 0.05:0.21).

View this table: [in this window] [in a new window]   Table 1.   Hematocrit, plasma total protein and lactate concentrations, and rectal temperature before and during 60 min of exercise at 55 ± 1% of O2max

Plasma glucose concentration. At rest and before administration of glucose or water, plasma glucose concentrations were similar among the three trials (Fig. 1). During the 60-min period after administration of the oral glucose load, plasma glucose concentration increased during both G and GE (8.2 ± 0.4 and 8.3 ± 0.3 mM, respectively; P < 0.05 vs. F) but remained unchanged in F (5.1 ± 0.1 mM). During exercise in F, plasma glucose concentration increased steadily to reach a peak of 10.3 ± 0.5 mM. In G, plasma glucose decreased from preexercise values during the first 20 min of exercise but subsequently increased, and, between 20 and 60 min of exercise, plasma glucose concentration was similar in F and G. During GE, epinephrine infusion resulted in marked hyperglycemia during exercise, with peak values of 17.2 ± 0.3 mM. Plasma glucose concentrations were higher (P < 0.05) throughout exercise in GE than in F. Similarly, plasma glucose was higher (P < 0.05) in GE than in G between 20 and 60 min of exercise (Fig. 1).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Plasma glucose concentration during rest and 60 min of exercise at 55% of maximum oxygen uptake (O2 max) when fasted (F), administered glucose 60 min before exercise (G), or administered glucose 60 min before exercise and infused with epinephrine during exercise (GE). Values are means ± SE; n = 6. * Mean glucose concentration during G and GE significantly greater than during F trial, P < 0.05. # Mean plasma glucose concentration during GE significantly greater than during F and G, P < 0.05.

Serum insulin. As expected, oral glucose administration in G and in GE resulted in a substantial increase (time × treatment, P < 0.0001) in serum IRI (Fig. 2). During exercise, serum IRI decreased in all trials but remained elevated (P < 0.05) in the G trial compared with F and GE for the first 30 min of exercise. No differences in serum IRI concentration existed among trials between 40 and 60 min of exercise.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Serum immunoreactive insulin (IRI) concentration during rest and 60 min of exercise at 55% of O2 max. Values are means ± SE; n = 6. * Mean IRI concentration during G and GE significantly greater than during F trial, P < 0.05. # Mean IRI concentration during G significantly greater than during F and GE, P < 0.05.

Urine glucose. Five horses in the G trial urinated during the early postexercise period, whereas urine was obtained from all six horses in the GE trial. On average, urine was collected 15-20 min after exercise. The mean volume of urine collected in G and GE was 1.9 ± 0.1 and 2.1 ± 0.2 liters, respectively. Urine glucose concentration was significantly higher in GE (47.5 ± 14.5 mM; range 9.5-121.4 mM) than in G (mean 5.7 ± 2.6 mM; range 2.1-8.5 mM).

Plasma catecholamines. Preexercise epinephrine and norepinephrine concentrations did not differ among trials (Fig. 3, A and B). Epinephrine infusion (GE) resulted in plasma concentrations three- to fourfold higher (P < 0.0001) than those in F and G (Fig. 3A). On the other hand, plasma norepinephrine concentrations were lower (P < 0.001) in GE than in F and G throughout exercise (Fig. 3B). After 60 min of exercise, plasma epinephrine was significantly lower in G than in F (Fig. 3A).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Plasma epinephrine (A) and norepinephrine (B) concentrations before and during 60 min of exercise at 55% of O2 max. Values are means ± SE; n = 6. * Values for GE significantly different from F and G, P < 0.001. # G significantly lower than F, P < 0.05.

Hematocrit, plasma total protein, NEFA, glycerol, and lactate. Administration of glucose or water was not associated with changes in hematocrit and plasma total protein concentration (t = 75 min vs. t = 0 min in Table 1). In all trials, hematocrit and plasma total protein increased significantly early during exercise, but no differences were detected among trials.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Plasma glycerol (A) and nonesterified fatty acid (NEFA; B) concentrations during rest and 60 min of exercise at 55% of O2 max. Values are mean ± SE; n = 6. a Values for F and GE significantly greater than those for G, P < 0.05. b Values for GE significantly greater than those for F, P < 0.05. c Values for F significantly greater than those for G and GE, P < 0.05.

Gas exchange and whole body CHO_ox and fat oxidation. There was a small but significant (P < 0.001) increase in O₂ during exercise, but O₂ was similar among the three trials (Table 2). There was a significant (P < 0.0001) treatment × time interaction for RER. In GE, RER was greater than 1.0 for the first 15 min of exercise and was significantly higher compared with G and F during this time period. RER was higher (P < 0.05) in GE than in F for the remainder of exercise. RER was also higher in G than in F at most time points during the exercise trial. However, RER was not different between 20 and 60 min of exercise in GE compared with G (Table 2).

View this table: [in this window] [in a new window]   Table 2.   Oxygen uptake, respiratory exchange ratio, and calculated rates of carbohydrate and fat oxidation during 60 min of exercise at 55 ± 1% of O2max

1

1

Glucose kinetics. Before oral administration of glucose, plasma isotopic enrichment (Fig. 5), glucose R_a (Fig. 6A), and glucose R_d (Fig. 6B) were similar among the three trials. In F, an isotopic steady state was maintained throughout the preexercise period, but isotopic enrichment decreased during exercise despite the threefold increase in tracer infusion rate. During both G and GE, isotopic enrichment decreased after oral glucose administration and was lower (P < 0.05) throughout exercise compared with that in F (Fig. 5).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Plasma [6,6-2H]glucose enrichment during rest and 60 min of exercise at 55% of O2 max. Values are means ± SE; n = 6. * Values for F significantly greater than those for G and GE, P < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Glucose rate of appearance (Ra; A) and disappearance (Rd; B) during rest and 60 min of exercise at 55% of O2 max. Values are means ± SE; n = 6. a Values for G significantly greater than those for F, P < 0.05. b Values for G significantly greater than those for GE, P < 0.05. c Values for GE significantly greater than those for F, P < 0.05.

1

1

1

1

1

1

1

1

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Glucose metabolic clearance rate (MCR) during 60 min of exercise at 55% of O2 max. Values are means ± SE; n = 6. * Values for G significantly greater than those for F and GE, P < 0.05.

Muscle glycogen and lactate. Preexercise muscle glycogen concentration was similar among the three trials. However, postexercise muscle glycogen concentration was lower (P < 0.04) in GE than in F and G (Fig. 8A). Therefore, net muscle glycogen utilization was higher (P < 0.03) in GE (349 ± 44 mmol/kg dry muscle) compared with F (218 ± 28 mmol/kg dry muscle) and G (201 ± 35 mmol/kg dry muscle). There was no difference in net muscle glycogen utilization in F compared with G. Similarly, the exercise-associated increase in muscle lactate concentration was similar in the F and G trials (Fig. 8B). However, end-exercise muscle lactate was higher (P < 0.05) in GE than in F and G. 

View larger version (20K): [in this window] [in a new window]   Fig. 8.   Muscle glycogen (A) and lactate (B) concentrations before (Pre-Ex) and after (Post-Ex) 60 min of exercise at 55% of O2 max. Values are means ± SE; n = 6. dw, Dry weight. * Significant difference compared with Pre-Ex values, P < 0.05. # Post-Ex values significantly different in GE compared with F and G, P < 0.05.

View this table: [in this window] [in a new window]   Table 3.   Total carbohydrate oxidation, rate of glucose disappearance, and muscle glycogen (and lactate) oxidation during exercise at 55 ± 1% of O2max

Overall pattern of substrate utilization. Figure 9 depicts the estimated relative caloric contribution from fat, muscle glycogen (plus lactate), and blood glucose during the final 30-min period of exercise. The total rate of energy expenditure was similar among the three trials (342 ± 18, 346 ± 21, and 350 ± 22 cal · kg¹ · min¹ for F, G, and GE, respectively). In F, the relative energy expenditure from fat, blood glucose, and muscle glycogen was 32 ± 2, 5.7 ± 0.5, and 62.3 ± 2%, respectively (Fig. 9). The contribution by fat to total energy was ~33% lower in G (22.5 ± 2%) and GE (20 ± 3%) compared with F. Thus the contribution by all sources of carbohydrate (blood glucose, muscle glycogen, lactate) to energy use was significantly higher in G (77 ± 3%) and GE (80 ± 2%) compared with F (68 ± 2%). In GE there was a shift to greater use of muscle glycogen (74.6 ± 3%), whereas the contribution by blood glucose (5.5 ± 1%) was similar to that during F. In contrast, the relative contribution of blood glucose during G (12.5 ± 2%) was significantly greater than in F and GE, whereas the contribution from muscle glycogen (65 ± 2%) was lower (P < 0.05) compared with GE but not different from F (Fig. 9).

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Relative caloric contributions from other carbohydrates (CHO) (muscle glycogen plus lactate), lipid, and blood glucose in 6 horses during the 30- to 60-min period of exercise. * Values significantly different between G and GE vs. F, P < 0.05. ** Contribution from blood glucose greater in G than in F and GE, P < 0.05. # Contribution from other carbohydrates greater in GE than in F and G, P < 0.05.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The present study examined the interactive effects of increased blood glucose availability (preexercise glucose administration) and increased circulating epinephrine (preexercise glucose administration and infusion of epinephrine during exercise) on carbohydrate metabolism in horses during moderate-intensity exercise. The main findings were 1) a more than twofold increase in whole body glucose uptake during exercise after oral glucose administration compared with exercise after a 24-h fast; 2) almost complete inhibition of the increase in glucose R_d associated with preexercise glucose administration produced by an infusion of epinephrine; 3) an increase in net intramuscular glycogen use and lactate accumulation when epinephrine was infused during exercise; and 4) augmentation of CHO_ox in both trials preceded by oral administration of glucose.

Glucose kinetics. As expected, preexercise administration of glucose resulted in marked hyperglycemia (Fig. 1) and hyperinsulinemia (Fig. 2). The increase in plasma glucose concentration was accompanied by increments in glucose R_a and R_d. Similarly, during exercise, glucose R_a was significantly higher in the trials preceded by glucose administration compared with the fasting condition. In humans, ingestion of glucose 30-50 min before exercise results in an increase in glucose R_a during exercise compared with placebo trials (1, 44). However, preexercise glucose ingestion markedly suppresses hepatic glucose production during exercise, and the increase in glucose R_a reflects ongoing intestinal uptake of glucose (44). In the present study, we did not measure the contribution of gut-derived R_a (intestinal uptake) to the total glucose R_a. Nonetheless, it is probable that continued uptake of glucose from the intestinal tract contributed to the higher glucose R_a in the trials preceded by oral glucose administration. However, systemic glucose availability during exercise was lower in GE than in G (Fig. 6A). The reason for this difference cannot be determined from our data.

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Muscle glycogen utilization. A second objective of this study was to determine the effect of preexercise glucose administration, with and without epinephrine infusion during exercise, on intramuscular glycogen utilization. In humans, the hyperglycemia and hyperinsulinemia resulting from glucose ingestion 30 to 60 min before exercise has been associated with an increase in muscle glycogen utilization in some (7, 21), but not all (11, 20), studies. The increase in muscle glycogen usage under these circumstances has been attributed to a reduction in the supply of free fatty acids in contracting skeletal muscle, thereby increasing the reliance on muscle glycogen as a fuel source (4). There also is conflicting evidence regarding the effect of preexercise carbohydrate ingestion on muscle glycogenolysis in horses during exercise. Lawrence and colleagues (37) reported that, compared with exercise after an overnight fast, glycogen utilization was higher when horses consumed 1 kg of corn grain (a high-carbohydrate meal) 1, 3, or 5 h before exercise. However, other studies from the same laboratory utilizing similar preexercise feeding regimens did not find an increase in intramuscular glycogenolysis when a carbohydrate meal was ingested before exercise (36, 38). Similarly, in the present study, we did not detect an effect of preexercise glucose administration (G trial) on net muscle glycogen degradation during exercise (Fig. 8A).

Overall pattern of substrate utilization. The results of the present study indicate that preexercise glucose administration in horses results in increased CHO_ox during exercise (Table 2 and Fig. 9). During the final 30 min of exercise, CHO_ox was ~15-17% higher in G than in F (Table 3), and, overall, the contribution by carbohydrate to TEE was ~10% higher in G. During the same time period, rates of fat oxidation were ~30-35% lower in G than in F (Table 2). Previous studies in humans during moderate-intensity exercise also have demonstrated increased CHO_ox, with a concomitant reduction in fat oxidation, when carbohydrate (glucose or fructose) is ingested before exercise (8, 25). In humans, the reduction in fat oxidation is due to inhibition of lipolysis (25) and an increase in CHO_ox in muscle (8). Horowitz et al. (25) reported that a 10-30 µU/ml increase in plasma insulin concentration before exercise completely abolished the exercise-associated increase in lipolysis. Furthermore, this low rate of lipolysis limited the availability of free fatty acids during exercise (25). Similarly, in the present study it is possible that the higher insulin concentrations after glucose administration reduced whole body lipolysis, evidenced by the lower plasma NEFA and glycerol concentrations during exercise in G (Fig. 4).