INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

ALTHOUGH COMPLEX MECHANISMS regulate endogenous glucose production [rate of appearance (R_a)] and utilization [rate of disappearance (R_d)] during exercise, sympathoadrenergic mechanisms are thought to play an important role, particularly during heavy exertion (8, 32, 45, 53). However, the results of several recent investigations have suggested that sympathoadrenergic mechanisms do not play an important role in the glucose R_a response, at least in humans and dogs. Attenuation of sympathetic nerve activity to the liver and adrenal medulla, by means of anesthesia of the celiac ganglion, did not affect glucose R_a in humans during exercise at ~75% of maximum O₂ uptake (O_2 max) (30). Similarly, selective - and -adrenergic blockade of the liver did not alter the glucose R_a response during heavy exercise in dogs (11). Furthermore, physiological increases in plasma epinephrine (Epi) do not appear to play a major role in mediating the exercise-induced increase in glucose R_a (25, 26). These findings indicate that neither sympathetic liver nerve activity nor circulating Epi is a major stimulus for glucose R_a during exercise. On the other hand, adrenergic mechanisms may be important in the regulation of the exercise-induced increase in muscle glucose uptake. In humans, physiological increases in plasma Epi inhibit glucose clearance during exercise (26, 35). Furthermore, -blockade (propranolol administration) augments glucose R_d during submaximal and maximal exercise (39, 46). Taken together, these findings support the hypothesis that -adrenergic mechanisms regulate glucose uptake during exercise.

Horses have an extremely high capacity for aerobic metabolism, as reflected by mass-specific rates of O_2 max that are two- to threefold higher than those of human athletes (13). Therefore, for exercise at a given percentage of O_2 max, metabolic rate and absolute energy requirements are two to three times higher in horses than in humans. Despite these observations, few studies have examined mechanisms for mobilization and utilization of fuel substrates in the horse during exercise. Similar to other species, hyperglycemia is a feature of moderate- and high-intensity exercise (42). In addition, -blockade has been shown to abolish the increase in plasma glucose concentration associated with sprint exercise (48). However, inasmuch as there have been no reports of the effects of -blockade on glucose turnover in the horse during exercise, it is not known whether the lower plasma glucose during exercise under -blockade reflects reduced glucose R_a or augmented glucose R_d.

In the present study, we used a graded exercise protocol to examine the effects of workload and -adrenergic mechanisms on the kinetics of glucose R_a and R_d in horses. We hypothesized that, compared with low-intensity exercise, moderate-intensity work would result in a mismatch between glucose R_a and R_d (R_a > R_d), manifested as an increase in plasma glucose concentration. We further hypothesized that -adrenergic mechanisms would underlie this restraint of glucose R_d, such that nonselective -adrenergic blockade would mitigate the mismatch between glucose R_a and R_d and increase glucose R_d during higher-intensity exercise. Therefore, the specific objective of this study was to examine the effects of exercise intensity and -adrenergic blockade on endogenous production and whole body uptake of glucose during consecutive 30-min bouts of exercise at ~30 and ~60% O_2 max with and without prior administration of the -blocker propranolol.

	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.

Experimental design. The effects of nonselective -adrenergic blockade on glucose kinetics and whole body substrate utilization during graded exercise were examined in a balanced, randomized crossover study. Each of six horses was studied on two occasions during 60 min of graded exercise: the first 30-min exercise period was undertaken at ~30% O_2 max and then the workload was increased to ~60% O_2 max. One trial was conducted under control conditions (C trial); in the other trial the exercise protocol was performed 15 min after administration of the _1 + 2-adrenoceptor blocking agent propranolol (0.22 mg/kg iv, P trial). For all horses, there was a 1-wk interval between trials.

Horses. Six horses (3 Standardbred and 3 Thoroughbred, 4 geldings and 2 mares), 4-7 yr of age and 409-490 kg [452 ± 28 (SD) kg] body mass, were studied. All horses were housed indoors during the experimental period, fed a diet of timothy grass-alfalfa hay and mixed grain, and had access to a salt-mineral block. All horses were conditioned and undertaking regular treadmill exercise for 3 mo before the study. 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 O₂ uptake (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 2° for 90 s at 4 m/s, and then 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_2 max 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 (with data from speeds below O_2 max), the running speed that elicited 30 and 60% O_2 max was calculated for each horse.

Experimental protocol. All experiments began between 0730 and 0800; food was withheld for 12 h before each experiment, and the horses had been confined to their stalls for the preceding 24 h. After aseptic preparation and local anesthesia of the overlying skin, catheters (14 gauge, 5.25 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 [0.22 ± 0.02 (SD) µmol · kg¹ · min¹] infusion of [6,6-²H]glucose (99% enriched; Cambridge Isotopes, Cambridge, MA) in 0.9% saline was then initiated using a calibrated infusion pump (model PHD 2000, Harvard Apparatus, South Natick, MA). During a 2-h equilibration period, horses stood in stocks. Fifteen minutes before commencement of the exercise protocol, propranolol (0.22 mg/kg) or an equivalent volume of 0.9% saline was administered intravenously. After collection of blood for final baseline hormone, substrate, and glucose kinetic determinations (see Blood sample collection and analysis), the horses were positioned on the treadmill (2° incline), and a loose-fitting facemask for measurement of respiratory gas exchange was applied. A thermocouple (model T-180, Physitemp Instruments, Clifton, NJ), attached to a thermometer (model BAT-10, Physitemp Instruments), was inserted 20-25 cm beyond the anal sphincter for measurement of temperature within the rectum (T_re) during exercise. The horses then began running at a speed calculated to elicit 30% O_2 max. The rate of [6,6-²H]glucose infusion was doubled at the onset of exercise (0.44 ± 0.03 µmol · kg¹ · min¹). After 30 min of exercise, the treadmill speed was increased to achieve a workload of 60% O_2 max. Exercise was continued for a further 30 min or until development of fatigue, as evidenced by an inability to keep pace with the treadmill, despite verbal encouragement. Isoprenaline (1 µg/kg body wt iv bolus) was administered 10 min after completion of exercise to verify that -adrenergic blockade had been maintained during the experiment. HR was recorded at 1-min intervals for 10 min after isoprenaline administration. 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; room temperature and relative humidity during the experiments were 17.2 ± 0.6°C and 35 ± 4% (means ± SE), respectively.

Respiratory gas exchange measurements. O₂, CO₂ production (CO₂), and respiratory exchange ratio (RER) were measured with an open-circuit calorimeter (Oxymax-XL, Columbus Instruments, Columbus, OH), as previously described (22). Flow through the system was ~1,500 l/min STP with the horse stationary and 9,000 l/min during running. The gas analyzers were calibrated before the start of each exercise test with 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 (14). 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 RER values were calculated by dividing CO₂ by O₂.

Rectal temperature. T_re was measured at rest before the start of exercise and at 5-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, ON, Canada).

Blood sample collection and analysis. Blood samples for determination of plasma isotopic enrichment and glucose concentrations were obtained at 30, 15, 0, 5, 10, 20, 30, 35, 40, 50, and 60 min of exercise (where 30, 15, and 0 refer to samples taken 75, 90, and 105 min, respectively, after the start of tracer infusion and 0 min is the sample collected just before the onset of exercise) and placed in tubes containing EDTA and sodium fluoride-potassium oxalate. When the exercise trial was terminated because of fatigue, the final blood sample was obtained at the point of fatigue. Additional blood samples were obtained at 30, 15, 0, 5, 15, 30, 45, and 60 min (or the point of fatigue) for subsequent measurement of hematocrit, plasma total protein, lactate, nonesterified fatty acid (NEFA), glycerol, glucagon, insulin, Epi, and norepinephrine (NE) concentrations. Blood samples (6 ml) were placed in tubes containing sodium fluoride-potassium oxalate (plasma lactate), EDTA (hematocrit, plasma total protein, NEFA, glycerol), EDTA-aprotinin [10,000 kallikrein inhibitor units/ml; Trasylol, FBA Pharmaceuticals, New York, NY (glucagon)], 120 µl of a solution containing 0.24 M EGTA-reduced glutathione (Epi, NE), or no additive (serum insulin). 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 [6,6-²H]glucose enrichment was determined as previously described (18). Briefly, the penta-acetate derivative of glucose was formed and then analyzed using electron impact gas chromatography-mass spectrometry (model 5889A, Hewlett-Packard, Palo Alto, CA). 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 (TTR) was calculated directly from measured ion abundance ratios as follows: TTR = R  R₀, where R and R₀ represent the measured tracer-to-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 (59). The intra- and interassay coefficients of variation were 1.5 ± 0.5 and 5.6 ± 2.1%, respectively.

Plasma biochemical analyses. Plasma glucose concentration was measured spectrophotometrically using the hexokinase reaction with a commercial kit (Glucose-HK kit; Sigma Chemical), and plasma lactate concentration was measured using an automated lactate oxidase method (Sport 1500 lactate analyzer, Yellow Springs Instruments, Yellow Springs, OH). Plasma NEFA concentration was determined using a commercial kit that employs an enzymatic colorimetric method (NEFA test kit, Wako Chemicals, Dallas, TX). Plasma glycerol concentration was measured by using an enzymatic spectrophotometric method [triglycerides kit 337A (without trigylceride 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 Epi and NE concentrations were determined by HPLC by use of electrochemical detection (33). 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 (37). Intra- and interassay coefficients of variation were 6.0 ± 1.5 and 11.5 ± 2.1%, respectively. Plasma immunoreactive glucagon (IRG) was determined in duplicate by use of a commercially available RIA (glucagon kit, Coat-a-Count Diagnostics). Pooled equine plasma was used to partially validate the assay for horse plasma. Specificity for equine glucagon was demonstrated by dilutional parallelism between standard solutions and serial dilutions of endogenous glucagon in equine plasma (r = 0.987). Accuracy was demonstrated by addition of porcine glucagon to equine plasma at 20-285 pmol/l. Linear regression of the recovery curve showed a correlation coefficient of 0.9929. The intra-assay precision for 12 replicates (6 duplicates) of equine plasma with a mean concentration of 31 and 75 pmol/l was 7.8 and 5.2%, respectively. The interassay coefficient of variation for the same samples was 13.1 and 14.8%, respectively. For the insulin and glucagon RIA, analysis of experimental samples was completed in a single analytic session.

Calculations of glucose kinetics. Glucose R_a and R_d at rest were calculated using the steady-state tracer dilution equation (59)
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 (59). The rate of infusion was calculated by multiplying the infusion pump rate by the concentration of glucose in the infusate. During exercise, glucose R_a and R_d were calculated using the non-steady-state equation developed by Steele and modified for use with stable isotopes (50)
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. With use of 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. Glucose metabolic clearance rate (MCR) was calculated by dividing glucose R_d by the plasma glucose concentration. Glucose R_a was assumed to represent hepatic glucose production (HGP), although a small contribution from renal glycogenolysis and gluconeogenesis is possible.

Rates of energy expenditure and whole body substrate oxidation. Total energy expenditure (TEE) and absolute rates of carbohydrate (CHO) and lipid oxidation were calculated as follows (15)

(1)

(2)

(3)

where O₂ is in liters per minute and it was assumed that protein oxidation made a negligible contribution to O₂ and CO₂ (i.e., nonprotein RER). The calculated values were based on respiratory gas exchange values averaged over 5-min intervals. CHO oxidation (CHO_ox) in grams per minute was converted to micromoles per kilogram per minute by dividing the molecular weight of glucose (mol wt 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 (mol wt 259) and the horse's body weight. Muscle glycogen (plus lactate) oxidation was calculated as the difference between total CHO_ox and glucose R_d. Coggan et al. (9) reported that, in human subjects, ~90% of glucose R_d is oxidized during submaximal exercise. Therefore, glucose R_d provides a reasonable estimate of plasma glucose oxidation during exercise. Finally, the absolute and relative contributions by plasma glucose, other CHO sources (muscle glycogen and lactate), and lipid to total energy expenditure during the 20- to 30- and 35- to 45-min periods of exercise were estimated using standard caloric equivalents (4.2 kcal/g CHO, 9.0 kcal/g lipid).

Statistical analyses. Values are means ± SE. The data for all dependent measures were analyzed using a two-way ANOVA for repeated measures, with treatment (control vs. propranolol) and time as independent factors. Inasmuch as the data for Epi and NE did not exhibit homogeneous variances, these data were subject to logarithmic transformation before ANOVA. Percent data 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_2 max ranged from 123 to 159 ml · kg¹ · min¹ (mean 137.2 ± 4.9 ml · kg¹ · min¹). Mean running speeds during the graded exercise protocol were 4.2 ± 0.1 and 7.3 ± 0.2 m/s, which corresponded to relative workloads of 32.8 ± 1.0 and 58.8 ± 1.5% O_2 max. The relative workloads in the C and P trials were similar; i.e., -blockade did not affect the O₂-speed relationship. Duration of exercise differed between treatments. Whereas all horses completed the 60-min protocol in the C trial, none of the horses finished the P trial. Mean duration of exercise in the P trial was 49.9 ± 1.2 min (range 47-56 min). Despite the shorter duration of exercise in the P trial, end-exercise T_re was significantly greater in the P trial than in the C trial (Table 1). Postexercise isoprenaline administration resulted in a rapid and sustained cardioacceleration in all horses in the C trial, whereas there was little change in HR after isoprenaline challenge in the P trial (Table 2). Subjectively, the horses appeared quieter and less motivated to run after propranolol administration.

Plasma glucose concentration and kinetics. Data for plasma glucose concentration, isotopic enrichment, and glucose R_a (HGP) and R_d are presented in Figs. 1 and 2. At rest, plasma glucose concentration was similar in the two experiments. In the C trial, plasma glucose concentration was not significantly changed from rest during the lower workload but increased progressively during exercise at the higher workload, peaking at the end of exercise (Fig. 1A). In contrast, plasma glucose rose rapidly in the P trial and was significantly greater than in the C trial after 20 min of exercise. Despite the doubling of tracer infusion rate at the start of exercise, in both trials there was a progressive decrease in plasma isotopic enrichment (Fig. 1B). HGP and glucose R_d were similar at rest between trials (Fig. 2). In the C trial, HGP increased progressively during exercise at ~30% O_2 max; at 30 min HGP was almost fourfold higher than preexercise values (30.5 ± 3.6 µmol · kg¹ · min¹). The doubling of work intensity was accompanied by a proportional increase in HGP with peak values of 54.4 ± 4.0 µmol · kg¹ · min¹ at 50 min (Fig. 2A). Although the pattern of change in HGP was similar between trials, mean HGP was 40-50% higher in the P trial than in the C trial between 10 min and the end of exercise (Fig. 2A).

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Plasma glucose concentration (A) and isotopic enrichment (B) at rest and during exercise at 30 and 60% of maximum O2 uptake (O2 max) in the control trial and after administration of propranolol (0.22 mg/kg, 15 min before exercise). Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from control, P < 0.05.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Rate of plasma glucose appearance (Ra, A) and disappearance (Rd, B) at rest and during exercise at 30 and 60% O2 max in the control trial and after administration of propranolol (0.22 mg/kg, 15 min before exercise). Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from control, P < 0.05.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Metabolic clearance rate of glucose (MCR) during exercise at 30 and 60% O2 max in the control trial and after administration of propranolol (0.22 mg/kg, 15 min before exercise). Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from control, P < 0.05.

Plasma hormone concentrations. Basal serum IRI and plasma IRG were very similar in the C and P trials (Fig. 4). During exercise in the C trial, serum IRI did not change significantly, whereas there was a small but significant decrease in IRI during exercise in the P trial (Fig. 4A). Serum IRI was significantly higher in the C trial than in the P trial throughout exercise. Plasma IRG did not change significantly during exercise in the P trial. In contrast, in the C trial there was a progressive increase in IRG during exercise at 30% O_2 max, with a more substantial increase during exercise at the higher workload (Fig. 4B). Consequently, plasma IRG was significantly higher in the C trial than in the P trial at several time points during exercise. The glucagon-to-insulin molar ratio (G/I) increased during exercise in both trials (Fig. 5). However, as a consequence of the decline in serum IRI, G/I was higher (P < 0.05) in the P trial than in the C trial during exercise at 30% O_2 max.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Serum immunoreactive insulin (A) and plasma immunoreactive glucagon (B) at rest and during exercise at 30 and 60% O2 max in the control trial and after administration of propranolol (0.22 mg/kg, 15 min before exercise). Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from propranolol, P < 0.05.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Glucagon-to-insulin ratio during graded exercise in the control trial and after administration of propranolol. Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from control, P < 0.05.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Plasma epinephrine (A) and norepinephrine (B) at rest and during exercise at 30 and 60% O2 max in the control trial and after administration of propranolol (0.22 mg/kg, 15 min before exercise). Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from control, P < 0.05.

Hematocrit, plasma total protein, lactate, glycerol, and NEFA. Hematocrit was significantly increased by 5 min of exercise and increased further coincident with the increment in workload. Plasma total protein followed a pattern similar to that for hematocrit (Table 1). Hematocrit and plasma total protein were similar between the two trials. Plasma lactate concentrations were also similar in the two trials. Plasma lactate decreased slightly during exercise at 30% O_2 max and then increased to reach 5.04 ± 0.7 and 4.45 ± 0.65 mmol/l in the C and P trials, respectively (Table 1). Plasma glycerol and NEFA concentrations during exercise were significantly lower in the P trial than in the C trial. Although there were progressive increases in plasma glycerol and NEFA throughout exercise in the C trial, glycerol concentration was unchanged in the P trial, whereas plasma NEFA was decreased relative to preexercise values (Fig. 7).

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Plasma nonesterified fatty acid concentration ([NEFA], A) and glycerol (B) at rest and during exercise at 30 and 60% O2 max in the control trial and after administration of propranolol (0.22 mg/kg, 15 min before exercise). Values are means ± SE for 6 horses. Horizontal error bar indicates SE for exercise duration in propranolol condition. * Significantly different from propranolol, P < 0.05.

Respiratory gas exchange and whole body substrate oxidation. Tables 3 and 4 show the steady-state gas exchange data and the total calculated CHO_ox and fat oxidation rates during the two trials. Whereas O₂ was similar in both trials, RER was significantly greater in the P trial than in the C trial between 20 and 50 min of exercise (Table 3). The total rate of energy expenditure was similar between trials during exercise at ~30% O_2 max (0.21 ± 0.03 kcal · kg¹ · min¹) and ~60% O_2 max (0.40 ± 0.04 kcal · kg¹ · min¹; Fig. 8). During the lower workload in the C trial, there was a progressive decrease in CHO_ox that was matched by a similar increase in the rate of fat oxidation (Table 4). The subsequent increase in workload was associated with an almost threefold increase in CHO_ox compared with exercise at 30% O_2 max. However, absolute rates of fat oxidation remained unchanged during exercise at the higher workload. Compared with the control condition, propranolol treatment resulted in significant suppression of fat oxidation between 20 and 50 min of exercise. In contrast, rates of CHO_ox were significantly higher in the P trial than in the C trial between 30 and 50 min (Table 4). Muscle glycogen (and lactate) oxidation, calculated as the difference between total CHO_ox and glucose R_d, was not different between trials (Table 5). Therefore, the higher CHO_ox in the P trial than in the C trial can be attributed to greater use of plasma glucose for energy production.

View this table: [in this window] [in a new window]   Table 3.   Steady-state gas exchange data during exercise at 30% and 60% O2max under control conditions or after administration of propranolol

View larger version (32K): [in this window] [in a new window]   Fig. 8.   Contribution of energy from different substrate sources during the 20- to 30-min and 35- to 45-min periods of exercise under control conditions and after administration of propranolol. Values are means ± SE for 6 horses. CHO, carbohydrate. * Significantly different from control, P < 0.05.

View larger version (39K): [in this window] [in a new window]   Fig. 9.   Percent energy expenditure derived from plasma glucose, other CHO (muscle glycogen, lactate), and lipid during the 20- to 30-min and 35- to 45-min periods of exercise under control conditions and after administration of propranolol. Values are means ± SE for 6 horses. * Significantly different from control, P < 0.05

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

This report represents one of the first descriptions of glucose turnover in the horse during sustained exertion. Furthermore, this study is the first to evaluate the role of -adrenergic mechanisms in the regulation of glucose kinetics and whole body substrate utilization in this species during exercise. The most significant findings were 1) a >40% increase in glucose R_a and R_d during exercise after induction of -adrenergic blockade, 2) increased reliance on plasma glucose for energy transduction during exercise under -blockade, 3) a -blockade-associated reduction in endurance capacity, as reflected by the decrease in exercise duration in the P trial, and 4) no change in the rate of fat oxidation after the transition from exercise at ~30% O_2 max to exercise at ~60% O_2 max, such that the additional energy expenditure at the higher workload was met solely by an increase in CHO_ox.

Critique of methods. A graded exercise protocol was chosen to examine the effects of two work intensities on our measures of glucose kinetics and substrate utilization. Inasmuch as the two workloads were completed in a single exercise protocol, it is possible that some of the observed alterations in dependent measures were due to time-related, rather than exercise intensity-related, effects. Nonetheless, the large increments in glucose R_a and R_d observed after the step change in workload suggest that exercise intensity was an important determinant of the response.

Plasma hormones. Catecholamine (Epi and NE) concentrations were higher in the P trial than in the C trial, whereas -blockade attenuated the exercise-associated rise in IRG concentrations and lowered IRI levels. Previous studies in humans at rest and during exercise (1, 17, 46) have demonstrated increased catecholamine concentrations during -blockade. Our data do not allow for definition of the mechanism for this increase in Epi and NE. However, in humans during moderate-intensity exercise, -blockade decreases hepatic (6) and splanchnic (1) blood flow. Inasmuch as the gut and liver are the primary sites of catecholamine clearance at rest and during exercise (10), it is possible that the increased plasma Epi and NE result from decreased clearance. It is likely that similar mechanisms accounted for the increased Epi and NE observed in the P trial.

Glucose kinetics. In accord with findings in humans and other animals (53), during low-intensity exercise in the C trial, the increase in HGP was closely matched to the increment in glucose R_d, and plasma glucose concentration was largely unchanged. Conversely, heavier exercise (~60% O_2 max) resulted in a mismatch between glucose R_a and R_d (Fig. 2), and plasma glucose increased progressively (Fig. 1A). We hypothesized that -blockade would alleviate this mismatch by enhancement of glucose uptake; indeed, glucose R_d and MCR were ~40 and ~50% higher, respectively, in the P trial than in the C trial, and the difference in glucose R_d was greatest during exercise at ~60% O_2 max (Fig. 2B). However, throughout exercise in the P trial, HGP exceeded glucose R_d by ~20%, and plasma glucose concentrations were ~1.2-2.0 mM higher in the P trial than in the C trial between 20 and 50 min of exercise. Thus the increment in HGP under -blockade cannot be due solely to increased demand for hepatic glucose supply, and it is likely that -blockade enhanced some component of a feedforward mechanism for regulation of HGP.

Substrate utilization. -Blockade augmented CHO_ox during exercise at both workloads (Table 4, Figs. 8 and 9). Furthermore, given the increase in blood glucose uptake in the P trial and the similar calculated rates of muscle glycogen oxidation in the P and C trials, our data indicate that the increase in total CHO_ox was due to increased utilization of plasma glucose. Previous studies in humans (39, 46) and in dogs (27) also have demonstrated an increased reliance on plasma glucose for energy production during exercise after -blockade. In the present study, the lower rates of fat oxidation during exercise in the P trial may be explained by a decrease in fatty acid availability. The lower plasma NEFA and glycerol concentrations in the P trial (Fig. 7) are consistent with a -blockade-mediated decrease in lipolysis (58). Furthermore, in human subjects, -blockade decreases intramuscular triglyceride breakdown in skeletal muscle during submaximal exercise (52). Taken together, these mechanisms would limit fatty acid availability in active skeletal muscle during -blockade. Alternatively, the increase in glucose uptake during exercise in the P trial may have inhibited fatty acid oxidation by more direct mechanisms. Recent studies in humans have demonstrated reduced long-chain fatty acid transport and oxidation within muscle mitochondria under conditions of increased plasma glucose uptake and accelerated glycolytic flux (12).

Exercise duration. In the present study, -blockade resulted in an ~17% reduction in exercise duration. Previous studies in humans and horses (48) and in ponies (44) have firmly established that -adrenoceptor blocking agents impair exercise performance, although the mechanism(s) responsible for this decrement in performance is not well understood. In the present study, a likely explanation for the reduction in exercise duration in the P trial is an impairment in thermoregulation. Despite the reduction in exercise time, end-exercise T_re was ~0.8°C higher in the P trial than in the C trial. Similarly, in a study by Sexton and Erickson (44), end-exercise pulmonary artery blood temperature was ~1.2°C higher in ponies that had received propranolol. Hyperthermia has been implicated in the development of fatigue during submaximal exercise (24). In horses, fatigue during exercise at ~65% O_2 max coincides with a pulmonary artery blood temperature of ~42.5°C (24). Inasmuch as T_re is ~0.5 to 1.0°C lower than central blood temperature in horses during submaximal exercise (23), it is likely that central blood temperature was approaching 42.5-43°C at the end of exercise in the P trial. Given that sweating in the horse is mediated via ₂-adrenoceptors (23), this exacerbation of exercise hyperthermia can, in part, be explained by a decrease in evaporative heat loss during exercise under -blockade.