Muscle glycogen is an important energy substrate used during high-intensity1–3 and prolonged low-intensity4,5 exercise in horses. Standardbred trotters can reduce their glycogen concentration by 40% after an intense training session.6 Therefore, the time needed for replenishment of muscle glycogen after exercise is an important factor in determining the recovery time needed for horses between training sessions or races.
In horses, the synthesis of muscle glycogen after intense exercise is a slow process and requires between 48 and 72 hours, even if horses are fed a diet high in starch and simple sugars.6–9 In comparison, complete restoration of muscle glycogen after prolonged intense exercise in humans can occur within 24 hours, provided sufficient carbohydrates are ingested.10,11 A study12 involving human athletes revealed that the rate of glycogen synthesis in muscle can be substantially increased by ingestion of soluble carbohydrates immediately after exercise. In contrast, oral administration of glucose or a glucose polymer early after completion of glycogen-depleting exercise in horses has not been reported to enhance glycogen replenishment, compared with glycogen replenishment after administration of a placebo.13,14
Insulin stimulates uptake of glucose by muscle cells and activates glycogen synthase, the rate-limiting enzyme for glycogen synthesis.15 Several studies involving humans have attempted to increase the serum concentration of insulin after exercise to optimize the rate of muscle glycogen synthesis. The stimulus for insulin secretion from the pancreas is increased concentrations of blood glucose as well as certain amino acids and proteins that exert a synergistic effect on insulin release.16,17 Studies18,19 involving humans have shown that pancreatic insulin secretion and glycogen replenishment after exercise are enhanced when protein or certain amino acids are ingested in addition to carbohydrates following exercise, compared with insulin secretion and glycogen replenishment achieved when carbohydrates are ingested alone. However, the results of another study20 involving humans indicated that the addition of a protein-amino acid mixture does not increase the rate of muscle glycogen synthesis when the intake of carbohydrates is high (1.2 g/kg).
In horses, only a limited amount of carbohydrates can be provided orally without causing adverse effects; therefore, the addition of amino acids to supplemental carbohydrates may be a potential strategy to increase glycogen synthesis after exercise. To our knowledge, only 1 study21 has been conducted to investigate the effect of the addition of amino acids to orally administered carbohydrates after exercise on glycogen synthesis in horses. In that study,21 the addition of leucine to a single low dose of glucose increased plasma insulin concentration, but glycogen synthesis was not enhanced during the 22.5 hours immediately after exercise. Given that the availability of carbohydrates after exercise has an important role in glycogen synthesis, it is possible that oral administration of high doses of glucose after exercise would enhance the replenishment of muscle glycogen in horses. The purpose of the study reported here was to investigate whether the addition of leucine to a moderate amount of glucose (0.5 g/kg/h) administered orally at 2-hour intervals during the period immediately after glycogen-depleting exercise would increase the secretion of insulin and thereby enhance glycogen synthesis in horses.
Materials and Methods
Animals—Twelve Standardbred horses (7 geldings and 5 mares; body weight, 406 to 536 kg; age, 4 to 9 years) owned by the National Center for Education in Trotting at Wången, Sweden, were used in the present study. All horses had been in training and were in racing condition. The horses were housed individually in box stalls and were released into a paddock daily for 5 hours. Three days per week, the horses were trained outdoors in 60-minute sessions. Each week, the first day of training consisted of trotting (11 m/s) on a track for 2,140 m. The second day of training consisted of walking and trotting (< 6 m/s) for approximately 15 km. The third day of training consisted of intermittent exercise on an uphill slope. Exercise during the 2 experimental periods was performed in winter between 8:30 am and 11:00 am and with good weather and track conditions.
Each horse's daily intake of feed on a dry-matter basis was 6.5 to 8.0 kg of haylage (10.3% crude protein and 9 MJ of metabolizable energy/kg) and 3.3 to 4.8 kg of a commercial pelleted concentratea (11.9% crude protein and 11 MJ of metabolizable energy/kg). Metabolizable energy was estimated as previously described.22,23,b Water and salt were provided ad libitum. The horses were fed at 6:00 am, noon, 5:00 pm, and 9:00 pm. Horses were fed both haylage and the commercial pelleted feed except for the feeding at noon, at which time only haylage was fed. Horses were acclimated to the diet for 3 weeks before the study was initiated. The study was approved by the Ethical Committee for Animal Experiments, Uppsala, Sweden.
Experimental design—The study was performed as a crossover study design with 2 oral treatments, water (control treatment) or glucose and leucine, administered during the period immediately following intense intermittent exercise known to decrease muscle glycogen content by approximately 40%.6 Concentrations of serum glucose, plasma insulin, and plasma leucine were monitored during the 6-hour period immediately after exercise. Muscle glycogen concentrations were determined before and for 24 hours after exercise. There was a 6-week interval between exercise trials, and the order of treatments was randomized for each horse. For 2 days after performing each exercise trial, horses were exercised in an automatic horse walker for 30 minutes twice daily. After that period, the horses returned to their standard training regimens and were exercised regularly between the exercise trials.
Experimental protocol—Feed was withheld for 9 hours prior to each exercise trial, but the horses were allowed free access to water until the start of exercise. On the morning of each exercise trial, a 2% lidocaine solutionc was injected into the skin over a jugular vein and a catheterd was inserted into a jugular vein and secured with sutures. A 25-cm IV extension line with a 3-way stopcocke was then attached to the catheter. After collection of blood samples to determine resting (baseline) concentrations of insulin, glucose, and leucine, the horses were exercised. Each exercise trial involved a warm-up period during which horses moved at a slow trot (7 m/s) on a straight track for a distance of 4,000 m. Following the warm-up period, horses moved at a trot on an uphill slope (elevation increased 24 m) for 500 m at a speed of approximately 9 m/s and then walked back to the start of the slope. The horses moved at a trot up the same slope 7 times. The speed was adjusted slightly in accordance with each horse's conditioning such that each horse achieved a heart rate of approximately 210 beats/min at the end of each sprint up the slope. After completion of the trial, the horses moved at a slow trot (7 m/s) on a track for approximately 2,000 m back to the stable. The horses performed each exercise trial in pairs, with the horses matched by age and training status.
Heart rate was recorded continuously before, during, and immediately after completion of the exercise trial with a pulse meter.f Blood samples were obtained immediately after completion of the exercise trial. The horses were then allocated by a random draw to receive the control (water) or glucose and leucine treatment; for each exercise pair, 1 horse was assigned to each treatment. At 15 minutes after completion of exercise, horses received the first bolus of the assigned treatment (time 0). The glucose plus leucine treatment consisted of 3 boluses of glucose (1 g/kg as a 10% solution) administered via a nasogastric tube at 0, 2, and 4 hours after exercise; leucineg (0.1 g/kg) was added to the boluses of glucose administered at 0 and 4 hours. For the control treatment, tap water (10 mL/kg) was administered via a nasogastric tube at 0, 2, and 4 hours after exercise. After each exercise trial, blood samples were collected at 0 hours, at 15-minute intervals during the first hour, and then at 30-minute intervals for the following 5 hours. Each horse was kept individually in a box stall with no access to water or feed for the 6-hour period during which blood samples were collected, but was fed immediately after the final blood sample had been obtained.
Muscle biopsy specimens were obtained from a gluteus medius muscle before each exercise trial, within 10 minutes after completion of each exercise trial, and 3, 6, and 24 hours after exercise. The area around the biopsy site was anesthetized by the injection of a 2% lidocaine solution. Biopsy specimens were obtained at a depth of 6 cm with an aseptic needle biopsy technique.24 The gluteus medius muscle (right or left) from which each biopsy specimen was obtained was alternated between subsequent collections. Immediately after collection, muscle biopsy specimens were frozen in liquid nitrogen and stored at −80°C until analysis.
Sample analysis—Blood samples were collected in evacuated tubesh that contained sodium heparin for determination of plasma insulin and leucine concentrations and in evacuated tubes that contained no additives for determination of serum glucose concentration. All tubes were centrifuged for 15 minutes at 2,700 × g, and then plasma or serum was harvested. Plasma and serum samples were frozen and stored for 3 days in a freezer at −20°C and then transferred to a freezer at −80°C, where the samples were stored until analysis.
Serum glucose concentrations were measured with an automated analyzer.i Plasma insulin concentrations were measured in duplicate with a commercial ELISA,j which had been validated for use in horses.k The intra-assay coefficient of variation for the insulin assay ranged between 1.9% and 4.8% for individual batches, and the interassay coefficient of variation was 8.0%.
Plasma leucine concentration was measured via high-performance liquid chromatography separation; all samples were assayed as single samples. For each sample, protein was precipitated with 5% trichloroacetic acid in a 1:10 dilution of plasma followed by centrifugation at 2,700 × g. The supernatant was then collected and stored at −80°C until analysis. Leucine concentration was measured by means of reverse-phase high-performance liquid chromatography in accordance with a previously reported method25 with some modifications. Briefly, a 5-μm, 150 × 3.9-mm C18 columnl was used at a fixed temperature of 45°C. Prior to injection, precolumn derivatization of samples was performed with 0.04M O-phthalaldehyde and separation was performed with a previously described gradient.26
Frozen muscle biopsy specimens were freezedried, and visible blood, connective tissue, and fat were removed via dissection before analysis. Acid hydrolysis of the freeze-dried muscle samples was performed, and then total glycogen concentration was measured in duplicate via a fluorometric hexokinase method.27
Calculations and statistical analysis—The net synthesis rate for total glycogen was determined by dividing the difference in glycogen concentration between samples obtained at 2 time points by the interval (in hours) between those time points. The AUC for glucose and insulin was calculated with a computer software programm by means of the trapezoidal approximation.
Data were analyzed with statistical software.n Treatment differences at each time point were analyzed with least squares means. Logarithmic transformations were used to normalize data for serum glucose, plasma insulin, and plasma leucine concentrations. Treatment differences in the mean response for serum glucose and plasma insulin concentrations during the treatment period were determined via a paired t test. All results were expressed as mean ± SE, except for logarithmically transformed data, which were expressed as the geometric mean ± 95% CI on the original scale after back transformation. For all analyses, values of P < 0.05 were considered significant.
Results
Animals—One horse became lame during an exercise trial and was excluded from the present study. Therefore, results were reported for only the 11 horses that completed both exercise trials. The overall mean ± SE heart rate at the end of the 2 exercise trials was 210 ± 2 beats/min for the first trial and 211 ± 1 beats/min for the second trial.
Glucose and insulin concentrations—Serum glucose concentration increased immediately after exercise for both treatments, and there was no significant difference in glucose concentration between treatments (Figure 1). When horses received the control treatment, serum glucose concentration decreased to the baseline glucose concentration by 30 minutes after exercise and remained at that concentration for the rest of the observation period. Administration of the first dose of glucose and leucine at 0 hours resulted in an increase in serum glucose concentrations during the 90 minutes immediately after exercise. The glucose concentration remained elevated until 150 minutes after exercise and then gradually decreased to the baseline concentration by 300 minutes after exercise, despite oral administration of additional glucose at 2 hours and glucose and leucine at 4 hours after exercise. The mean ± SE serum glucose concentration during the 6-hour period immediately after exercise was significantly higher for the glucose and leucine treatment (9.1 ± 0.2 mmol/L), compared with the mean ± SE serum glucose concentration for the control treatment (5.9 ± 0.15 mmol/L). The AUC for serum glucose concentration during the 6 hours immediately after exercise was greater when horses received the glucose and leucine treatment (Figure 2), compared with the AUC for serum glucose concentration when horses received the control treatment. At the intervals of 0 to 2 hours and > 2 to 4 hours, the AUC for the serum glucose concentration was significantly higher for the glucose and leucine treatment than that for the control treatment; however the AUC for the serum glucose concentration did not differ between the treatment groups during the interval from > 4 to 6 hours.
Geometric mean ± 95% CI serum glucose concentration for 11 healthy Standardbred horses before (baseline; −1 hour) and after intermittent glycogen-depleting exercise and during the 6-hour period immediately after exercise. Fifteen minutes after completion of exercise was designated as time 0. Horses received a control treatment (water, 10 mL/kg, PO, at 0, 2, and 4 hours after exercise; white circles) or a combination of glucose (1 g/kg as a 10% solution, PO, at 0, 2, and 4 hours after exercise) and leucine (0.1 g/kg, PO, at 0 and 4 hours after exercise; black circles). Each horse received each treatment once, and there was a 6-week interval between treatments. *Within a time point, values for treatments differ significantly (P < 0.05). †Within a treatment, value differs significantly (P < 0.05) from the baseline value.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Geometric mean ± 95% CI serum glucose concentration for 11 healthy Standardbred horses before (baseline; −1 hour) and after intermittent glycogen-depleting exercise and during the 6-hour period immediately after exercise. Fifteen minutes after completion of exercise was designated as time 0. Horses received a control treatment (water, 10 mL/kg, PO, at 0, 2, and 4 hours after exercise; white circles) or a combination of glucose (1 g/kg as a 10% solution, PO, at 0, 2, and 4 hours after exercise) and leucine (0.1 g/kg, PO, at 0 and 4 hours after exercise; black circles). Each horse received each treatment once, and there was a 6-week interval between treatments. *Within a time point, values for treatments differ significantly (P < 0.05). †Within a treatment, value differs significantly (P < 0.05) from the baseline value.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Geometric mean ± 95% CI serum glucose concentration for 11 healthy Standardbred horses before (baseline; −1 hour) and after intermittent glycogen-depleting exercise and during the 6-hour period immediately after exercise. Fifteen minutes after completion of exercise was designated as time 0. Horses received a control treatment (water, 10 mL/kg, PO, at 0, 2, and 4 hours after exercise; white circles) or a combination of glucose (1 g/kg as a 10% solution, PO, at 0, 2, and 4 hours after exercise) and leucine (0.1 g/kg, PO, at 0 and 4 hours after exercise; black circles). Each horse received each treatment once, and there was a 6-week interval between treatments. *Within a time point, values for treatments differ significantly (P < 0.05). †Within a treatment, value differs significantly (P < 0.05) from the baseline value.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE AUC for serum glucose concentration at 2-hour intervals and for 6 hours after intermittent glycogen-depleting exercise in 11 healthy Standardbred horses that received a control treatment (black bars) or combination of glucose and leucine (gray bars). *Within a time interval, values for treatments differ significantly (P < 0.05). a,bWithin a treatment, values with different letters differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE AUC for serum glucose concentration at 2-hour intervals and for 6 hours after intermittent glycogen-depleting exercise in 11 healthy Standardbred horses that received a control treatment (black bars) or combination of glucose and leucine (gray bars). *Within a time interval, values for treatments differ significantly (P < 0.05). a,bWithin a treatment, values with different letters differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE AUC for serum glucose concentration at 2-hour intervals and for 6 hours after intermittent glycogen-depleting exercise in 11 healthy Standardbred horses that received a control treatment (black bars) or combination of glucose and leucine (gray bars). *Within a time interval, values for treatments differ significantly (P < 0.05). a,bWithin a treatment, values with different letters differ significantly (P < 0.05). See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
For the glucose and leucine treatment, plasma insulin concentration increased rapidly during the first 60 minutes after the initial administration, reached a peak concentration at 150 minutes, and then plateaued between 180 and 270 minutes (Figure 3). During the final hour of the 6-hour observation period, plasma insulin concentration decreased despite the administration of glucose and leucine at 4 hours after exercise For the control treatment, exercise induced a slight increase in plasma insulin concentration, but it rapidly returned to the baseline concentration and remained at that concentration for the remainder of the 6-hour observation period after exercise. The mean ± SE plasma insulin concentration during the 6-hour period after exercise was significantly higher for the glucose and leucine treatment (701 ± 39 ng/L), compared with that for the control treatment (112 ± 7 ng/L). The AUC for the mean insulin response during the 6-hour observation period after exercise was approximately 7 times as high as the AUC for the glucose and leucine treatment, compared with that for the control treatment (Figure 4). The AUCs calculated during each time interval (0 to 2 hours, > 2 to 4 hours, and > 4 to 6 hours) were significantly different between the treatments. For the glucose and leucine treatment, insulin response was significantly higher from > 2 to 4 hours, compared with that during the other intervals (0 to 2 hours and > 4 to 6 hours).
Geometric mean ± 95% CI plasma insulin concentration before and after intermittent glycogen-depleting exercise and during the 6-hour period after exercise in 11 healthy Standardbred horses. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Geometric mean ± 95% CI plasma insulin concentration before and after intermittent glycogen-depleting exercise and during the 6-hour period after exercise in 11 healthy Standardbred horses. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Geometric mean ± 95% CI plasma insulin concentration before and after intermittent glycogen-depleting exercise and during the 6-hour period after exercise in 11 healthy Standardbred horses. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE AUC for plasma insulin concentration at 2-hour intervals and for 6 hours after intermittent glycogen-depleting exercise in 11 healthy Standardbred horses. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE AUC for plasma insulin concentration at 2-hour intervals and for 6 hours after intermittent glycogen-depleting exercise in 11 healthy Standardbred horses. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE AUC for plasma insulin concentration at 2-hour intervals and for 6 hours after intermittent glycogen-depleting exercise in 11 healthy Standardbred horses. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Leucine concentrations—Plasma leucine concentration increased from the baseline measurement to 0 hours for both treatments, and there was no significant difference in leucine concentration between treatments at baseline or 0 hours (Figure 5). For the control treatment, plasma leucine concentration decreased to the baseline concentration by 60 minutes after exercise and remained at that concentration for the remainder of the 6-hour observation period. Administration of the first dose of glucose and leucine at 0 hours resulted in a substantial increase in plasma leucine concentration by 60 minutes after exercise. Thereafter, plasma leucine concentration decreased, plateaued between 120 and 210 minutes, and then continued to decrease until the second administration of leucine at 4 hours after exercise. Following the second dose of leucine, the plasma leucine concentration continued to increase until the end of the 6-hour observation period.
Geometric mean ± 95% CI plasma leucine concentration before and after intermittent glycogen-depleting exercise and during the 6-hour period after exercise in 11 healthy Standardbred horses. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Geometric mean ± 95% CI plasma leucine concentration before and after intermittent glycogen-depleting exercise and during the 6-hour period after exercise in 11 healthy Standardbred horses. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Geometric mean ± 95% CI plasma leucine concentration before and after intermittent glycogen-depleting exercise and during the 6-hour period after exercise in 11 healthy Standardbred horses. See Figure 1 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Muscle glycogen concentration—Exercise resulted in a significant reduction in glycogen concentration in the gluteus medius muscle for both treatments (Figure 6). Muscle glycogen concentration decreased by 41.0% and 38.7% from the baseline concentrations for the glucose and leucine treatment and control treatment, respectively. The mean ± SE rate of glycogen synthesis during the 6-hour observation period after exercise did not differ between the glucose and leucine treatment (1.8 ± 4.7 mmol of glucosyl/kg of dry weight/h) and control treatment (0.6 ± 1.5 mmol of glucosyl/kg of dry weight/h). Twenty-four hours after exercise, muscle glycogen concentrations were 72.4% and 70.0% of the baseline concentration for the glucose and leucine treatment and control treatment, respectively.
Least squares mean ± SE muscle glycogen concentration before and after intermittent glycogen-depleting exercise and during the 24-hour period after exercise in 11 healthy Standardbred horses. a–cValues with different letters differ significantly (P < 0.05). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE muscle glycogen concentration before and after intermittent glycogen-depleting exercise and during the 24-hour period after exercise in 11 healthy Standardbred horses. a–cValues with different letters differ significantly (P < 0.05). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Least squares mean ± SE muscle glycogen concentration before and after intermittent glycogen-depleting exercise and during the 24-hour period after exercise in 11 healthy Standardbred horses. a–cValues with different letters differ significantly (P < 0.05). See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 73, 6; 10.2460/ajvr.73.6.867
Discussion
In the present study, the addition of leucine to an orally administered glucose solution provided at 2-hour intervals immediately following intense exercise in horses did not affect the rate of muscle glycogen synthesis even though plasma insulin concentration was substantially increased. Exercise resulted in a moderate decrease in muscle glycogen concentration that was consistent for both treatments. Therefore, the stimulus for glycogen synthesis immediately after exercise was similar for both treatments. An exercise trial that consisted of repeated sprints up a slope was chosen because it is a common training method for Standardbred horses in Sweden and causes considerable glycogen depletion.6 The 6-hour treatment and observation period has been used by other investigators14 to compare the effect of route of glucose administration on glycogen synthesis in horses. The present study used the same dosage schedule (1 g/kg at 0, 2, and 4 hours after exercise) for orally administered glucose as used in the other study.14 This administration schedule provided glucose during a time of enhanced insulin sensitivity in muscle11 and has not been associated with adverse effects in horses.14 In humans, the period of increased insulin sensitivity immediately after exercise is characterized by a substantial increase in glucose uptake and glycogen synthesis in muscle.28 The administration of leucine at 0 and 4 hours after exercise was chosen because results of another study21 indicated that oral administration of leucine stimulated an insulin response that lasted for 4 hours in horses.
Horses have a slow rate of muscle glycogen replenishment following intense exercise; it can take up to 72 hours to restore muscle glycogen concentration following glycogen-depleting exercise.7–9 In horses, it is difficult to increase the rate of glycogen synthesis after exercise by oral administration of glucose8,9,29; however, IV administration of glucose increases the rate of replenishment of muscle glycogen after exercise.13,14,30 Following high-intensity exercise in horses, the concentration of insulin after IV administration of glucose is approximately 3 times as high as that after oral administration of glucose.14 This suggests that plasma insulin concentration is an important determinant for the rate of glycogen synthesis after exercise in horses as well as in humans. However, horses are not as sensitive to insulin following exercise as are humans.31 Thus, in horses, a substantial increase in insulin concentration is likely necessary to stimulate muscle glycogen synthesis after exercise.32
In the present study, the primary objective was to determine the effect of oral administration of glucose and leucine after exercise on plasma insulin concentration in horses. Insulin has been reported as an activity unit (mU/L) in horses. That unit was determined on the basis of the activity of the human insulin molecule, but it is unknown whether human and horse insulin molecules have the same activity. Therefore, it is preferable to use a quantitative unit for equine insulin. An equine-specific ELISAj for insulin analysis has been developed that reports insulin concentration in nanograms per liter. The equine-specific ELISA has been validatedk and compared with a commonly used radioimmunoassay methodo for the analysis of equine insulin concentrations. The calculated conversion factor for equine insulin from milliunits per liter, determined with the radioimmunoassay method, to the quantitative unit nanograms per liter, determined with the equine-specific ELISA,j is 10 (unpublished data). Because most studies that have been conducted to evaluate the effect of exercise on insulin response in horses have used the radioimmunoassay method, the calculated conversion factor enables the comparison of the results of the present study with those of previous studies.
Investigators of another study14 that involved horses found that the rate of muscle glycogen synthesis was higher when glucose (3 g/kg) was administered IV than when an equivalent dose of glucose was administered orally during the 6 hours immediately after exercise. However, the rate of glycogen synthesis following oral administration of glucose did not differ from that following administration of a placebo treatment.14 For the present study, the amount of glucose administered and the times at which the glucose was administered after exercise were identical to those in the previous study.14 In the study reported here, mean plasma insulin concentration following oral administration of glucose and leucine (701 ng/L, corresponding to 70 mU/L) was similar to the mean serum insulin concentration (75 mU/L) after IV infusion of glucose and higher than the mean serum insulin concentration (35 mU/L) after oral administration of glucose in the other study.14 Thus, oral administration of glucose and leucine increases plasma insulin concentration after exercise in horses and can be an used as an alternative to IV administration of glucose.
The findings of the study reported here are similar to those of another study32 conducted to evaluate serum insulin concentration after oral administration of a single dose of glucose and leucine in horses following exercise. However, in the present study, instead of a single dose of glucose (1 g/kg) and leucine (0.3 g/kg) administered in combination, which was used in the other study,32 glucose alone (1 g/kg) was administered at 3 time points (0, 2, and 4 hours) and glucose with the addition of leucine (0.1 g/kg) was administered at 2 time points (0 and 4 hours) to horses after exercise. In the present study, despite a substantial increase in plasma insulin concentration during the period after exercise, there was no effect on the rate of glycogen synthesis, which is similar to findings of another study.21 In that study,21 the addition of leucine to orally administered glucose increased plasma insulin concentration only 3 to 4 times, compared with plasma insulin concentration before exercise. In the present study, plasma insulin concentration after oral administration of glucose and leucine following exercise increased > 10 times, compared with the plasma insulin concentration before exercise. A possible reason for the discrepancy in the magnitude of plasma insulin responses between that study21 and the present study is that we used much higher doses of glucose and leucine. Although the increased concentration of plasma insulin during the 6 hours immediately after exercise did not affect the rate of muscle glycogen synthesis in the present study, it is possible that exercise that depletes muscle glycogen to a greater extent or administration of glucose and leucine for an extended time after exercise, at higher doses, or more frequently might have affected glycogen synthesis.
Results of the present study and those of another study14 indicate that insulin was not the rate-limiting factor for synthesis of muscle glycogen when glucose was administered orally at 2-hour intervals. In the present study, serum glucose concentration began to decrease at 150 minutes after exercise despite the additional oral administration of glucose at 2 hours after exercise and glucose and leucine at 4 hours after exercise. The reason serum glucose concentration continued to decrease despite additional oral administration of glucose is unknown, but it is possible that some of the glucose was extracted by the liver during this time. Entrapment of glucose in the gastrointestinal tract is less probable because it is evident that the administered leucine was absorbed from the gastrointestinal tract following the last administration of glucose and leucine 4 hours after exercise. Therefore, administration of oral glucose at 2-hour intervals may not increase and maintain the blood glucose concentration sufficiently to enhance synthesis of muscle glycogen in horses. It is also possible that the rate of glucose uptake by muscle was greater than the rate of glucose absorption from the gastrointestinal tract, but the glucose must have then been oxidized in the mitochondria of skeletal muscle because there was no enhancement of muscle glycogen synthesis. In 1 study,14 IV administration of glucose after exercise increased the mean glucose concentration by 11 mmol/L, compared with the mean glucose concentration achieved after administration of a placebo, whereas oral administration of an equivalent dose of glucose increased the mean glucose concentration by only 2.1 mmol/L. These results suggest that the availability of glucose, rather than insulin, is the key determinant of muscle glycogen replenishment after exercise in horses.
The absence of insulin sensitivity after exercise in horses31 may be the reason glycogen synthesis was not enhanced in the horses of the present study even though plasma insulin concentration increased significantly after oral administration of glucose following exercise. In horses, the number and activity of glucose transporter type 4, the protein responsible for insulin-regulated translocation of glucose into muscle cells, and the activity of glycogen synthase in skeletal muscle after exercise are lower, compared with those in humans and rodents, which results in a slower rate of glucose transport and glycogen synthesis despite glucose availability.8,14,31,33 Additionally, horses have decreased availability of triglycerides, glycerol, and nonesterified fatty acids during recovery from exercise, compared with that for humans.7,21 It is also possible that the availability of glucose for glycogen synthesis was decreased because a proportion of the administered glucose was used for energy production within the muscle cell. Investigators34 have found that oral administration of acetate as an alternative energy source can enhance muscle glycogen synthesis in horses immediately after exercise. In that study,34 muscle concentrations of acetyl-coenzyme A and acetylcarnitine were significantly higher when horses were administered acetate, compared with those concentrations when horses were not administered acetate. Those investigators34 suggested that the administered acetate was oxidized in the tricarboxylic acid cycle into ATP, which allowed glucose to be preferentially used for glycogen synthesis in skeletal muscle.
In the present study, oral administration of glucose and leucine to horses after exercise resulted in a significant increase in plasma insulin concentrations, and the insulin response was similar to that achieved by an equivalent dose of glucose that was administered IV in another study.14 However, the increased insulin concentration had no effect on the rate of glycogen synthesis in horses during the 6-hour period immediately after exercise.
ABBREVIATIONS
| AUC | Area under the curve |
| CI | Confidence interval |
Krafft Grund, Lantmännen Krafft AB, Falkenberg, Sweden.
Lindberg JE, Ragnarsson S. Prediction of energy content in forage for horses (abstr), in Proceedings. 1st Nordic Feed Sci Conf 2010;127–128.
Xylocaine, lidocaine 2%, AstraZeneca AB, Södertälje, Sweden.
Intranule, 2.0 × 105 mm, Vygon, Ecouen, France.
Discofix, B. Braun Melsungen AG, Melsungen, Germany.
Polar Electron OY, Kempele, Finland.
l-Leucine, Sigma-Aldrich Sweden AB, Stockholm, Sweden.
BD Vacutainer, Becton-Dickinson, Plymouth, Devon, England.
Architect ci8200, Abbott Scandinavia AB Diagnostics, Solna, Sweden.
Insulin equine ELISA, Mercodia AB, Uppsala, Sweden.
öberg J, Lilliehöök I, Wattle O, et al. Validation of a species specific enzyme-linked immunosorbent assay for measurement of serum insulin in horses (abstr) J Vet Intern Med 2009;23:778–779.
Resolve C18 90å, Waters Corp, Mass.
SigmaPlot software, version 11, SPSS Inc, Chicago, Ill.
PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC.
Coat-A-Count RIA, DPC, Siemens Medical Solutions Diagnostics, Los Angeles, Calif.
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4 Essén-Gustavsson B, Karlström K, Lindholm A. Fibre types, enzyme activities and substrate utilization in skeletal muscle of horses competing in endurance rides. Equine Vet J 1984; 16:197–202.
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