Single-injection glucose kinetics with compartmental modelling during rest and low-intensity exercise in horses


  • K. H. TREIBER,

    Corresponding author
    1. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, USA; Department of Clinical Sciences, New Bolton Center, Kennett Square, Pennsylvania, USA; and The Equine Studies Group, WALTHAM Centre for Pet Nutrition, Melton Mowbray, UK.
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  • R. C. BOSTON,

    1. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, USA; Department of Clinical Sciences, New Bolton Center, Kennett Square, Pennsylvania, USA; and The Equine Studies Group, WALTHAM Centre for Pet Nutrition, Melton Mowbray, UK.
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  • R. J. GEOR,

    1. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, USA; Department of Clinical Sciences, New Bolton Center, Kennett Square, Pennsylvania, USA; and The Equine Studies Group, WALTHAM Centre for Pet Nutrition, Melton Mowbray, UK.
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  • T. M. HESS,

    1. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, USA; Department of Clinical Sciences, New Bolton Center, Kennett Square, Pennsylvania, USA; and The Equine Studies Group, WALTHAM Centre for Pet Nutrition, Melton Mowbray, UK.
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  • P. A. HARRIS,

    1. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, USA; Department of Clinical Sciences, New Bolton Center, Kennett Square, Pennsylvania, USA; and The Equine Studies Group, WALTHAM Centre for Pet Nutrition, Melton Mowbray, UK.
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    1. Department of Animal and Poultry Sciences, Virginia Polytechnic Institute and State University, Blacksburg, USA; Department of Clinical Sciences, New Bolton Center, Kennett Square, Pennsylvania, USA; and The Equine Studies Group, WALTHAM Centre for Pet Nutrition, Melton Mowbray, UK.
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Present address: Nutrition Department, Fort Worth Zoo, 3358 Creston Avenue, Fort Worth, Texas 76133, USA. Email: Tanja Hess's present address is: Department Equine Sciences, Colorado State University, Fort Collins, Colorado, USA.


Reasons for performing study: Tools and criteria to evaluate and understand glucose metabolism are essential to optimise equine energy utilisation for exercise performance and reduced metabolic health risks.

Objectives: To re-evaluate models of glucose kinetics in the horse at rest and during endurance type exercise using a single injection technique and compartmental modelling.

Methods: Twelve exercise trained Arabian geldings received a bolus of 100 µmol/kg bwt [6,6-2H]glucose i.v. while at rest and while running at ∼4 m/s on a treadmill. Tracer and tracee glucose curves from 4–150 min after the bolus dose (while the subject maintained its resting or exercising state) were described by a 2 term exponential decay curve. Compartmental modelling was performed simultaneously for each horse's resting and exercise curves using an ‘exercise effect’ parameter for each compartmental exchange rate during exercise.

Results: Exercise increased all rate constants and transport flows for glucose between compartments by 110–145% (P≤0.004). Total glucose transport through the system increased from 8.9 ± 0.6 µmol/min/kg/bwt at rest to 25.0 ± 1.1 µmol/min/kg bwt during exercise (P<0.001). Exercise decreased the volume of the primary glucose compartment by 8% (P = 0.006) and increased plasma glucose clearance rate by almost 200% (P<0.001). Turnover times and mean residence times were decreased approximately 60% by exercise (P<0.001), whilst turnover rates were increased 125% (P<0.001).

Conclusions: Single-injection tracer kinetics and compartmental modelling represent a valuable tool to quantify tracee availability to and use by tissue.

Potential relevance: This technique could represent a beneficial tool for future studies exploring the role of glucose metabolism in equine exercise performance and metabolic disease.


Tracee glucose concentration, mmol/l


Volume of compartment i, ml/kg bwt


Tracee mass of compartment i, µmol/kg bwt


Fractional turnover rate constant for total output from compartment i, /min


Fractional transfer rate of glucose to compartment i from compartment j, /min


Rate of tracee transport to compartment i from compartment j, µmol/min/kg bwt


Total glucose leaving and entering the system, µmol/min/kg bwt

Clearance rate

Glucose disposal from plasma, ml/min/kg bwt


Mean residence time of glucose in compartment i, min

Turnover timei

Time to transport Qi from compartment i, min

Turnover rate

Total glucose leaving (and entering) compartment i, µmol/min/kg bwt


Glucose homeostasis in the horse is of increasing interest as associations between glucose metabolism and diseases such as pars pituitary intermedia dysfunction and laminitis are elucidated (Treiber et al. 2006a). Mathematical models including the euglycaemic/hyperinsulinaemic clamp and the minimal model are now used regularly in the horse to evaluate glucose and insulin dynamics and their link with potential health risks such as obesity, reproductive complications and laminitis (Hoffman et al. 2003; Vick et al. 2007). Exercise is commonly recommended to normalise glucose metabolism and reduce such health risks. Circulating glucose concentrations as well as muscle glycogen metabolism may present limiting factors in exercise performance as glucose in its stored and circulating form is essential to fuel muscle function, particularly during high intensity exercise. Primed glucose tracer infusion studies have previously been applied to thehorse to evaluate glucose kinetics under various exercise conditions (Geor et al. 2000a,b,c), using a one compartment model. Such a model, while providing an empirically adequate simplification, does not accurately describe the physiological distribution of glucose in most species.

Single injection tracer technique allows for a compartmental analysis of the glucose system based on a physiological representation of glucose distribution and exchange more comprehensive than previously achieved through infusion techniques. Single-injection glucose kinetic studies were last performed in the horse over 30 years ago (Evans 1971; Argenzio and Hintz 1972; Anwer et al. 1976); however, compartmental modelling of glucose kinetics has only ever been applied to 4 resting ponies (Argenzio and Hintz 1972). Since then, technological advancement in stable-isotopes and mathematical modelling have made single-injection kinetic studies and compartmental analysis more accessible. The results from the single injection technique will support or challenge assumptions of glucose primed-infusion studies in the horse, such as the glucose distribution space and the relevance of the one compartmental model - and may reveal new insights into adaptations of glucose kinetics during exercise. As far as the authors are aware, compartmental modelling has never been applied to tracer curves during exercise in any species.

The present study details the methodology behind single-injection stable isotope tracer glucose kinetics with compartmental modelling in horses during rest and constant low-intensity exercise. The results were expected to describe the most comprehensive multi-compartmental glucose system in horses to date and quantify the upregulations of corresponding parameters of glucose transport during exercise to meet increased energy demand.

Materials and methods

Twelve Arabian geldings were selected to be representative of horses that compete in low-intensity endurance exercise competitions. Horses were matched by body condition score (BC) (Henneke et al. 1983), weight and age. Horses were maintained in 2 matched pastures of mixed grass/legume and supplemented with 1 kg twice daily of concentrate, either a fat and fibre rich feed (n = 6) or a starch and sugar rich sweet feed (SS, n = 6) (Treiber et al. 2006b). The effect of diet adaptations within this trial is described in a companion paper (Treiber et al. 2008). For the current paper, each horse served as its own control and both diets were typical for pasture kept horses supplemented with a commercial horse feed so the results of the current analysis (reported across all 12 horses, independent of diet) should be representative of a general population of exercise-trained Arabians.

All horses had been conditioned for 10 months to running on a treadmill before the present tests were undertaken. Each horse was exercised for approximately 1 h, 3 times/week. Exercise consisted primarily of treadmill work (at least once per week) and work in an automatic walker (walk, trot). Horses were also exercised an equivalent amount by riding or lungeing when the treadmill and walker were not available.

Each horse underwent 2 similar tracer tests, one at rest and one during exercise. Horses were randomly assigned to test days with 6 horses exercising first and 6 horses resting first. All horses were maintained on pasture until the morning of the test and given no concentrate for at least 15 h prior to the test. A catheter (MILACATH, 14 gauge, 13 cm)1 was inserted into the jugular vein the morning of each test and horses allowed at least 30 min to rest following the procedure. Basal samples were then taken, followed by initiation of the test. For practical and ethical reasons a single catheter was used for dosing and sampling. Samples were taken and the catheter flushed prior to the initial 4 min point and discarded to account for mixing time and possible residual tracer. Horses had at least 8 days to recuperate between tests and missed one training session before and one training session after their exercise test. All tests were initiated between 09.00 and 10.00 h. The study was approved by the Institution's Animal Care and Use Committee and was part of a series of studies on glucose metabolism in this group of horses (Treiber 2006).


Horses had access to grass hay and water ad libitum throughout this test. Following baseline samples, an i.v. glucose tracer dose (100 µmol/kg bwt of [6,6-2H] glucose, 98% enriched)2 in 0.9% saline solution was administered rapidly through the catheter. Blood samples were collected at 4, 5, 6, 7, 8, 10, 12, 14, 16, 18, 20, 23, 26, 30, 35, 40, 50, 60, 70, 90, 110, 130 and 150 min following the glucose injection.


Each horse was placed on the treadmill and warmed-up by walking for 10 min at 1.8 m/s at a 0% slope followed by trotting at a 2% slope and 60% of its lactate threshold (∼4 m/s) as determined by a lactate breakpoint test performed previously (Treiber et al. 2006b). Trotting at this speed was continued for the duration of the test (170 min). A sample was taken after 15 min of trotting at 60% of lactate threshold, constituting the 0 min sample for the exercise test. The tracer dose was then applied and sampling continued identical to that at rest. Heart rate was measured throughout the exercise test by a commercial digital heart rate monitor (Polar Pacer)3.

Blood sample handling

Blood was withdrawn into tubes containing EDTA anticoagulant (Vacutainer evacuated blood collection tubes)4 and placed in ice water until centrifuged at 3000 g for 10 min. Plasma was removed within 30 min of collection and frozen at -20°C until analysis. Plasma glucose was analysed by enzymatic assay (Beckman Instruments, Glucose Procedure #16-UV)5. The intra-assay coefficient of variation (CV) of duplicate samples was <1%.

Plasma [6,6-2H]glucose enrichment was determined by gas chromotography mass spectrometric analysis (GCMS) with electron impact for penta-acetate derivatives of glucose. Protein was precipitated from 0.2 ml of sample plasma by adding 2 ml acetone. The mixture was centrifuged at 3000 g for 10 min at 4°C and the removed supernatant vacuum-dried. The dried remnant was dissolved in 0.5 ml of a 2:1 v/v acetate anhydride/pyridine and heated on a heating block at 60°C for 10 min. After cooling to room temperature (5–10 min), 1.0 µl of the solution was injected on the GCMS system (Hewlett-Packard 6890 GC with 5973 N Mass Selective Detector)6. Percent isotope enrichment was determined from the ratio of peak areas for ions 100 (labelled glucose) and 98 (unlabelled glucose) using the selective ion-monitoring mode (Shipley and Clark 1972). The intra-assay CV of duplicate samples was <1%.


Tracer as fraction of dose/l (Tr) was calculated as


where t is the time in min, E the plasma isotopic enrichment (%), G(t) the plasma glucose concentration (mmol/l) at t min and D the tracer dose (mmol). Curves of Tr(t) were fit using WINSAAM software ( for 2 and 3 term exponential decay functions of the form


Likelihoods for the 2 and 3 term exponential functions were compared by Akaike's Information Criterion (AICc), autocorrelation of the residuals (Durbin-Watson Statistic) and resolution of the parameter estimates. Parameters were considered unresolved by a fractional standard deviation >0.50.

Once a 2 compartment model was selected, the curves were fitted using WINSAAM software (for additional detail on the WINSAAM model, see Treiber 2006). Tracer curves from rest and exercise were fitted simultaneously with resting curves providing a baseline for rate constants (kij, /min) describing the proportion of glucose moving to compartment i from compartment j. An ‘exercise effect’ parameter (pij) was added to each rate constant for fitting of the exercise curve such that:


This approach assumes a similar structure to the glucose system during rest and exercise (as evidenced by the curves) and takes into account the fact that both tests together represent one individual.

From the determined curves and rate constants, the remaining model parameters (compartment volumes, masses, rate constants and transport flow rates) could be determined (DiStefano 1983; Landaw et al. 1984; Chen et al. 1985). Results are reported for only one possible 2 compartment model (with loss from the second compartment only); however, parameter bounds for all possible 2 compartment models can be determined from the results reported.

Noncompartmental parameters that are consistent and identifiable for every possible 2 compartment model were also determined, including clearance rate (ml/min/kg bwt) (Jeukendrup et al. 1999; Geor et al. 2000c), mean residence time (MRT, min) (Hoffman et al. 2003; Vick et al. 2007), turnover time (min) and turnover rate (µmol/min) (Shipley and Clark 1972).


Statistics were performed using Intercooled Stata Version 97. Differences between exercise and rest for plasma glucose, heart rate and model parameters were determined using a 2-way ANOVA with horses nested in diet and, where appropriate, time was entered as a repeated measure. Diet effect was included in the model to ensure that the exercise effects reported here exist independent of diet effects. However, values and variability reported are pooled across all 12 horses and, therefore, represent the range of values expected in a population of horses consuming a variety of diets. Interaction effects at specific time points were evaluated by regression with interaction expansion and clustering by horse. Two sample comparisons were performed using the Kruskal-Wallis statistic. Significance was assessed at P<0.05 and a trend at P<0.10. Results are reported as mean ± s.e. unless otherwise indicated.


Horses weighed 456 ± 13 kg, had BC scores of 5.3 ± 0.3 (range 4–7), and were 11 ± 1 years old (range 5–16). There was no difference in bwt between the exercise and resting test (P = 0.14). Mean temperature during the test was 7 ± 1°C.

Horses ran at 3.9 ± 0.1 m/s (range 3.3–4.3 m/s). Mean heart rate during exercise ranged from 90–130 beats/min for individual horses. Heart rate tended to decline (P = 0.086) from 113 ± 4 beats/min at 40 min to 108 ± 3 beats/min at 150 min (Fig 1).

Figure 1.

Heart rates of 12 horses during the exercise test. Resting baseline values were taken (0 min) followed by a 25 min warm-up after which horses ran at a constant speed (∼4 m/s). *Significantly different from 40 min (P≤0.046).

Plasma glucose

Plasma glucose concentrations during the trials are shown in Figure 2. Glucose was not different overall between rest and exercise (P = 0.32) but showed different patterns over the course of the tests (P = 0.021, time x test interaction). The tracer glucose bolus at rest resulted in increased (P≤0.016) plasma glucose concentration compared to baseline from 4–40 min with the greatest increase (4 min post glucose bolus) being 15%. For the remainder of the trial, plasma glucose concentration were not different (P≥0.093) from baseline. During exercise there was a 12% increase (P<0.001) from baseline after the glucose bolus, but basal glucose concentrations were re-established by 10 min (P≥0.063). After 90 min of exercise, glucose values were lower (P = 0.031) than basal and continued to decline for the remainder of exercise, reaching concentrations 13% lower than basal at 150 min.

Figure 2.

Plasma glucose concentrations at rest (solid circles) and exercise (open circles) in trained Arabians. At 0 min, an i.v. bolus of 100 µmol/kg bwt of [6,6-2H]glucose was administered. For the exercise test, a 25 min warm-up occurred before the 0 min dose. Significant difference (P≤0.031) from respective baseline (0 min value) is indicated by the asterisk over a broken bar for resting values and an asterisk over a solid bar for exercise.

Glucose tracer curves

Plasma enrichment curves are shown in Figure 3. An example of fits for one, 2 and 3 phase exponential equations during rest and exercise is shown for one horse in Figure 4. The one phase exponential equation was discarded based on clear systematic deviation. At rest, the AICc recommended a 3 phase curve for 8 of the 12 horses. Of these only 6 demonstrated improved residuals and subsequently parameters could be satisfactorily resolved for only 3 horses. None of the criteria supported a 3 phase exponential curve during exercise. Accordingly, a conservative 2 compartment model was selected for compartmental analysis (Fig 5).

Figure 3.

Plasma glucose tracer enrichment curves for 12 trained Arabian geldings following an i.v. bolus of 100 µmol/kg bwt of [6,6-2H]glucose at rest or during low intensity exercise. Curves are fitted by 2 term exponential decay functions.

Figure 4.

One, 2 and 3 term exponential decay functions fitted to glucose tracer curves during rest and exercise for one trained Arabian gelding. Systematic deviations were apparent for the one term function (solid line) and improved by adding the second exponential term (dashed line). Addition of a third exponential term (dotted line) did not considerably improve the fit.

Figure 5.

Two compartment models of glucose kinetics. (a) Complete 2 compartment model with rate constants (kij, /min), volumes (Vi) and masses (Qi). (b, c) Identifiable models with all loss from primary compartment (b) or secondary compartment (c) representing minimum (min) and maximum (max, heavy arrow) bounds for parameters.

Model analysis

One possible 2 compartment model and parameters during exercise and rest are shown in Figure 6. Exercise increased all kij and Rij by 110–145% (P≤0.004 for test effect). Total glucose transport through the system (EGP) increased from 8.9 ± 0.6 µmol/min/kg bwt at rest to 25.0 ± 1.1 µmol/min/kg bwt during exercise (P<0.001).

Figure 6.

Two compartment model of the glucose space for 12 Arabian horses. Values are reported as median (interquartile range) at rest (R) and as effected by exercise (EE). Rate constants (kij) are expressed as percent (i.e. ×100). NS indicates a nonsignificant difference between rest and exercise tests. Units are /kg bwt.

Exercise decreased V1 by 8% (P = 0.006) and tended to decrease Q1 by 5% (P = 0.054). Exercise did not impact the volume or mass of the secondary glucose compartment.

Noncompartmental parameters are shown in Figure 7. Exercise increased plasma glucose clearance rate by almost 200% (P<0.001) and mean residence times by approximately 60% (P<0.001). Turnover times were also decreased approximately 60% by exercise, and turnover rate increased 125% by exercise (P<0.001).

Figure 7.

Noncompartmental parameters of glucose kinetics for a 2 compartment model at rest or during exercise in trained Arabian geldings. Values are reported as median (interquartile range) at rest (R) and as effected by exercise (EE). Turnover rate constants (kii) are expressed as percent (i.e. ×100). ‡Indicates parameters which are model dependant (k01 = 0). Units are per kg bwt.


This study demonstrated that single injection tracer kinetics can be applied to horses at rest and during constant low-intensity exercise to provide a detailed compartmental description of the glucose system. A model for glucose kinetics during rest and exercise in the horse was determined, demonstrating how glucose kinetics are upregulated during exercise to meet increased energy demands. The technique demonstrated here could represent a beneficial tool for future studies exploring how glucose metabolism can impact exercise performance and metabolic health risks.

Tracer kinetic studies rely on the assumption that the tracee remains at steady state, ensuring constancy of tracer kinetics so parameters can be solved based on steady state relationships. At rest, horses had access to hay and water throughout the test. The availability of feed and water ensured the animals remained calm. Consumption of forage during similar tests has not been shown to significantly impact circulating glucose concentrations (Jose-Cunilleras et al. 2002; Kronfeld et al. 2004) due to the low soluble carbohydrate content and slow consumption of the forage. Glucose arriving in the circulation would be predominantly from gluconeogenesis following hindgut fermentation of forage, which would have been consumed in all horses prior to the test. Feed deprivation, which is typical for glucose studies in other species, was deemed inappropriate as horses are continuous feeders. Feed deprivation potentially alters glucose kinetics and dynamics as the subject switches to stored energy sources and energy conservation, a condition that should be carefully considered when interpreting the results of metabolic studies particularly in animals accustomed to continuous intake.

Glucose steady-state during exercise has been considered difficult to achieve and single-injection tracer models have rarely been applied (Finegood et al. 1992). However, by providing a period of acclimation (approximately 30 min) constant low or moderate intensity exercise results in relatively constant plasma glucose concentrations, as demonstrated in this study. The changes in total glucose concentration both at rest and during exercise were small (<1 mmol/l or 15% deviation from baseline) and at all times concentrations were within the reference range for horses. A concomitant small insulin response at rest (an increase of <50 pmol/l disappearing by 30 min, data not shown) provided further confidence that these data represent a near steady state with minimal perturbations. For future studies, a smaller bolus may be warranted to further reduce perturbations. For the present study, the assumption that the observed changes in total glucose concentration did not affect model parameters was tested by modelling tracer and tracee curves simultaneously. We found that accounting for the change in total glucose concentration resulted in <1% change in the estimates for EGP. Furthermore, the observed change in glucose concentration during the first part of the test is largely attributable to tracer glucose, rather than the concentration of tracee. These finding support the contention that deviation from steady-state assumptions was insufficient to influence our conclusions. This finding is consistent with simulations of nonsteady states in rabbits (Atkins 1980a).

Selection of a conservative 2 compartment model is consistent with most glucose kinetics models in animals (Anwer et al. 1976; Radziuk et al. 1978; Atkins 1980b). A third glucose compartment may also be elucidated under certain conditions and assumptions, particularly when the sample period is several hours long (Cobelli et al. 1984; Jacquez 1992; Gastaldelli et al. 1997). This 3 compartment model is considered to represent the plasma compartment, a rapidly exchanging interstitial compartment and a slowly exchanging interstitial compartment. For this study, the third compartment could not be satisfactorily characterised in 150 min, with the variability in parameter estimates too large to provide useful information. During exercise the rapid clearance of plasma tracer probably further contributed to the poor resolution of a third compartment. The 2 compartment model selected does not differentiate the rapidly exchanging compartment from the central compartment, and therefore is theorised to represent a primary compartment of plasma and rapidly exchanging interstitial fluid, and a secondary compartment of slowly exchanging interstitial fluid.

The volumes estimated for the compartments in this study and others support their physiological interpretation (Insel et al. 1975). In the present study the primary compartment represented 13.5% of bwt at rest and 11% of bwt during exercise. Approximately 4% of bwt is estimated for plasma volume, 4% for red blood cells, with the rest of the compartment representing the rapidly exchanging interstitial fluid. The secondary compartment in this study made up the remainder of the glucose space to estimate a total glucose distribution of approximately 24% bwt at rest and 22% bwt during exercise. These values for rest are similar those reported across species for glucose space and extracellular fluid (Steele et al. 1956; Baker et al. 1959; Kronfeld 1977). To our knowledge this is the first study to estimate the volume of glucose spaces in a 2 compartment model during exercise. The tendency for V1 and V2 to be lower during exercise translated into a 9% lower volume of the total glucose space during exercise. The loss of plasma and interstitial volume is probably attributable to fluid shifts redistributing vascular volume from nonworking to working tissue and moving fluid into active muscle (Harrison 1985). Slightly higher estimates for loss of plasma volume during exercise (13–15%) have been reported with exercise in the horse and man independent of sweat loss (Fortney et al. 1981; Sejersted et al. 1986; Nyman et al. 2002). The difference may be due to the nature of the exercise or the cooler ambient temperature in the present study.

A 2 compartment representation of glucose kinetics has been used extensively in the past to describe single-injection tracer experiments. Originally single-injection curves were analysed according to a simple one compartment model (identical to the ‘model independent’ analysis of infusion studies) to calculate irreversible loss (glucose leaving not to return, R01, µmol/min) and total entry rate (all glucose leaving/entering the sampled compartment, R11, µmol/min) (White et al. 1969) (see Appendix, Equation 12). However a discrepancy was observed between total entry rate and irreversible loss indicating that some glucose was exchanging with another compartment (R12 = R21, µmol/min), thus a new term was applied, attributing this difference to ‘recycling’. These factors essentially describe a 2 compartment model with the sampled compartment exchanging with a peripheral compartment.

A 2 compartment model with loss from both compartments is a priori unidentifiable, meaning that unique solutions cannot be determined for all rate constants describing exchange amongst compartments (Shipley and Clark 1972). No firm physiological basis exists for the assumption that glucose disposal occurs from only one compartment; both compartments are considered to contain interstitial fluid from which glucose could be expected to be irreversibly cleared into tissue. However, minimal and maximal values for all parameters can be determined by selectively setting the irreversible loss from each compartment to its minimal value (i.e. 0); the minimal and maximal values represent the parameter estimates identified by the 2 extreme models assuming loss from only one compartment (Figs 5b, c). For clarity, we chose to report the results from the 2 compartment model with loss from the secondary compartment only (Fig 5c).

The rate of glucose entering the system (EGP) in resting trained Arabian geldings (∼9 µmol/min/kg bwt) was similar to that previously determined for trained, overnight fasted Thoroughbred, Arabian and Standardbred horses using glucose tracer primed-infusion methods (Geor et al. 2000a,c; Pagan et al. 2002). As in this study, exercise increased glucose entering and leaving the sampled pool in horses under glucose-tracer infusion, with the percent increase proportional to the exercise intensity (∼200% at 35% VO2max and ∼400% at 50–55% VO2max) (Geor et al. 2000a,c; Jose-Cunilleras et al. 2002; Pagan et al. 2002). The intensity of exercise in the present study was based on individual lactate threshold (Treiber et al. 2006b), i.e. metabolic signalling occurring in skeletal muscle rather than mechanical speed (Coggan et al. 1992; Coyle 1995). Sixty percent of lactate threshold was selected to represent an intensity that each horse could maintain for the duration of the test and resulted in speeds comparable to those performed in recreational endurance rides. The mean absolute speed run in the present study (3.9 ± 0.1 m/s) was similar to that run at 35% VO2max (4.4 ± 0.2 m/s) and glucose entering and leaving the glucose system in the present study increased by 200%. Therefore kinetics studies in exercising horses demonstrate consistency despite differences in breed, training, modelling techniques and the fasting vs. grazing state.

The effect of exercise on additional compartmental and noncompartmental parameters determined by kinetics modelling is not surprising as the augmented exchange of glucose facilitates the increase in glucose utilisation. This augmentation may be attributable to redistribution of vascular volume increasing glucose availability to muscle tissue. Catecholamines may also increase EGP directly or by suppressing insulin while increasing glucagon (Wasserman et al. 1989; Gustavson et al. 2003; Sumida et al. 2003).

During exercise glucose transport through the system increased ∼16 µmol/min/kg bwt, probably representing greater utilisation of plasma glucose for energy during exercise as glucose leaving circulation is expected to be entering working tissue for metabolism (Jeukendrup et al. 1999). Plasma glucose utilisation might be expected to spare muscle glycogen, but studies in man and horses have not demonstrated this effect (Bosch et al. 1996; Geor et al. 2000c; Hess et al. 2004). It is more likely that increased uptake of glucose could be attributable to upregulation of the glycolytic pathway, promoting both glucose and glycogen use.

The volume of distribution for the sampled compartment of primed-infusion experiments has been assumed to be 162 ml/kg bwt (or 65% of the extracellular space estimated as 25% bwt) (Geor et al. 2000a,b,c; Pagan et al. 2001) according to Steele (Steele et al. 1956). Results from the present study, however, indicate that 162 ml/kg bwt is an overestimation of the sampled compartment volume, attributable to a one compartment assumption, limited observation period and a failure to account for fluid shifts, which are expected to occur in the exercise state. Applying the volume for the sampled compartment determined from the present single-injection study (110 ml/kg bwt) to data from primed infusion studies indicates an overestimation of glucose rate of apperance (Ra) and glucose rate of disappearance (Rd) of approximately 10% due to the volume assumption (Geor et al. 2000a,b,c; Pagan et al. 2001; Jose-Cunilleras et al. 2002). Although noteworthy, this overestimation would not be expected to influence the physiological interpretation of data from primed-infusion studies as the exercise-induced increase observed in Ra and Rd is much greater than the potential overestimation (Geor et al. 2000a,b,c; Pagan et al. 2001). Another assumption that contributes to misestimation of Ra and consequently Rd under nonsteady state conditions is the inability to account for intercompartmental exchange (i.e. R12 or ‘recycling’). As with glucose distribution, the deviation from steady state determines the degree of error resulting from inaccurate assumption.

The results of the present single-injection kinetics study with compartmental modelling suggest that the assumptions made in ‘model independent’ analyses do not grossly impact the conclusions of those analyses. At the same time, new insights provided by the compartmental modelling of the present study, including characteristics of the glucose distribution space, intercompartmental exchange, and the impact of exercise, provide a stronger foundation for future kinetics studies.

This study introduced a new tool for evaluating the glucose system in horses. This technique and its characterisation of the glucose system offer a unique window into characterising glucose metabolism and its impact on equine exercise performance. Quantifying the impact of exercise on glucose metabolism offers important insight into the potential benefits of exercise as a strategy to reduce the risk of obesity and metabolic disease.


This study was funded by the John Lee Pratt Fellowship, the WALTHAM Centre for Pet Nutrition and Equine Guelph. Thanks also to Leah Larsen and Louisa Gay for technical assistance.

Conflicts of interest

None declared.

Manufacturers' addresses

1 MilaInternational, Inc., Erlanger, Kentucky, USA

2 Sigma-Aldrich, St Louis, Missouri, USA.

3 Polar CIC Inc., Port Washington, New York, USA.

4 Fisher Health Care, Chicago, Illinois, USA.

5 Sigma Diagnostics, St Louis, Missouri, USA.

6 Hewlett-Packard, Palo Alto, California, USA.

7 StataCorp, College Station, Texas, USA


Appendix 1: Determination of kinetic parameters

The equations used to solve for parameters of the 2 compartment model are as follows: Rest and exercise curves for tracer fraction of dose (Tr(t), /l) were fitted for each horse using WINSAAM, providing estimates for the y-intercept, the kij (k02, k12, k21) at rest, and the exercise effect on each kij (pij) (p02, p12, p21, respectively). Initially, pij were permitted to be negative and 3 horses demonstrated a p12<0. However, these negative pij were not resolved (fsd>0.5) and therefore not significantly different from 0. Thus pij were limited to values ≥0, resulting in resolved fits where p12 = 0 for these 3 horses. From the resulting parameter estimates, the remaining compartmental and noncompartmental parameters were determined algebraically according to the following equations:


By definition, in the steady-state, the sum of glucose mass arriving in any compartment equals the sum of the mass leaving irreversibly and to all of the n other compartments in the system:


where Rj0 (often termed Uj) is glucose nonexchanging input/production into compartment j. As the only glucose mass entering compartment 2 in this model is R21, the mass of compartment 2 (Q2) can be determined:


From the above equations, parameters bounds for all possible 2C models can be determined. For any tracer curve fit with a 2 phase exponential, kii and kij·kji are a priori identifiable and independent of model assumptions (Landaw et al. 1984).


uniquely identifiable parameter bounds for the remaining model parameters of the a priori unidentifiable model (Fig 5a) are determined by sequentially setting the loss from each compartment to 0 such that some kji =−kii.