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Perfused rat hindlimb is suitable for skeletal muscle glucose transport measurements

Jørgen F. P. Wojtaszewski1, Allan B. Jakobsen1, Thorkil Ploug2, and Erik A. Richter1

1 Copenhagen Muscle Research Centre, August Krogh Institute and 2 Department of Medical Physiology, The Panum Institute, Copenhagen University, DK-2100 Copenhagen, Denmark

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

It has been postulated that the perfused rat hindlimb is unsuitable for measurements of muscle glucose transport [P. Hansen, E. Gulve, J. Gao, J. Schluter, M. Mueckler, and J. Holloszy. Am. J. Physiol. 268 (Cell Physiol. 37): C30-C35, 1995]. The aim of the present study was therefore to critically evaluate the suitability of this preparation for glucose transport measurements using the extracellular marker mannitol and the glucose analogs 3-O-methyl-D-glucose or 2-deoxy-D-glucose. In all three muscle fiber types studied, the rate of 2-deoxy-D-glucose uptake during perfusion was linear from 1 to 40 min during maximal insulin stimulation and from 1 to 15 min during maximal electrical stimulation. Uptake of 2-deoxy-D-glucose was not increased by an increase in perfusate flow. Combined stimulation with a maximal insulin concentration and electrical stimulation elicited additive effects on 2-deoxy-D-glucose uptake in slow- and fast-twitch oxidative but not in fast-twitch glycolytic muscle fibers. Furthermore, in muscles having high glucose transport capacities 3-O-methyl-D-glucose is less suitable than 2-deoxy-D-glucose because of rapidly developing nonlinearity of accumulation. Our findings clearly demonstrate that the perfused hindlimb is suitable for measurements of muscle glucose transport and that the most feasible glucose analog for this purpose is 2-deoxy-D-glucose.

3-O-methyl-D-glucose; 2-deoxy-D-glucose; insulin; contractions

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

SKELETAL MUSCLE constitutes almost 40% of the total human body mass and displays a high responsiveness to various stimuli of glucose uptake, e.g., insulin and contractions. Thus skeletal muscle plays a central role in the control of glucose homeostasis, and the regulatory mechanisms involved in skeletal muscle glucose uptake are therefore studied extensively. It is generally accepted that the transport process through the muscle surface membrane, i.e., the plasma and the T tubule membrane, is rate limiting for muscle glucose uptake under most physiological conditions. It is therefore important to be able to measure the rate of muscle glucose transport under various physiological conditions.

Measurements of intracellular accumulated 3-O-methyl-D-glucose, a nonmetabolizable D-glucose analog, is a widely used method to determine muscle glucose transport rate. However, during high rates of glucose uptake, intracellular accumulation of 3-O-methyl-D-glucose may result in a significant cellular efflux of the analog causing underestimation of the glucose transport capacity. By use of the analog 2-deoxy-D-glucose, the risk of a significant cellular efflux, even under very high rates of glucose transport, is diminished due to intracellular trapping by phosphorylation catalyzed by hexokinase. However, product inhibition of hexokinase by accumulated glucose 6-phosphate may, under certain conditions, e.g., muscle contractions, limit the phosphorylation capacity so that the rate-limiting step is shifted from the glucose transport process. This would cause an increasing intracellular concentration of nonphosphorylated 2-deoxy-D-glucose that will lead to cellular efflux of the analog and subsequent underestimation of the glucose transport capacity.

In vitro, incubated muscles and the perfused rat hindquarter are the most widely used models for the evaluation of muscle glucose metabolism, including glucose transport, under various physiological conditions (for review, see Ref. 2). The maintained in situ position of the muscles, the possibility of electrical stimulation through an intact nervous innervation, and especially the use of the muscle's own capillary bed for nutritive supply make the perfused hindquarter physiologically superior to the isolated incubated muscle preparation. Yet criticisms have been raised against the usefulness of the perfused rat muscle in the measurements of muscle glucose transport (4, 9). The main argument against the model is that the equilibrium process of substrate between perfusate and extracellular space is slower (5-10 min) in the perfused hindlimb (4) than in incubated thin and split muscles (4-5 min) (6). This slower rate of equilibrium in the hindlimb was postulated to interfere with measurements of muscle glucose transport, although no data supporting this argument were presented (4).

The purpose of the present study was therefore to evaluate the usefulness of the perfused rat hindquarter model for muscle glucose transport measurements under various conditions known to stimulate glucose transport.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Animals. All experiments were approved by the Danish Animal Experiments Inspectorate and complied with the "European Convention for the Protection of Vertebrate Animals Used for Experiments and Other Scientific Purposes" (Council of Europe no.123, Strasbourg, France, 1985). Male Wistar rats weighing 200-250 g were used in the study and were maintained on a 12:12-h light-dark cycle and received standard rat diet and water ad libitum.

Surgical procedure. The rats were anesthetized by an intraperitoneal injection of pentobarbital sodium (5 mg/100 g body wt). Surgery was performed as described by Ruderman et al. (18). In some instances, perfusion of only one hindlimb was carried out and therefore ligation of the contralateral common iliac artery and vein was performed.

Perfusion medium. All perfusions were carried out using a cell-free perfusate consisting of Krebs-Ringer bicarbonate buffer solution, 5% bovine serum albumin (fraction V, Sigma Chemical) dialyzed twice for 24 h against 25 vol of Krebs-Ringer bicarbonate buffer (pore size 10-15 kDa), 0.15 mM pyruvate, and 4.2 IU/ml heparin. Media having this simple composition were used initially during all perfusions. For measurement of glucose transport, either 8 mM 3-O-methyl-D-glucose or 8 mM 2-deoxy-D-glucose and 1 mM mannitol together with radioactive labeled tracers as specified below were added to the perfusate. When 3-O-methyl-D-glucose uptake was measured 3-O-[methyl-14C(U)]-D-glucose (sp act 315 mCi/mmol) and D-[1-3H(N)]mannitol (sp act 22.5 mCi/mmol) were added to the perfusate yielding an activity of 0.050 and 0.075 µCi/ml, respectively. For 2-deoxy-D-glucose uptake measurements 2-[2,6-3H]deoxy-D-glucose (sp act 51 Ci/mmol) and D-[1-14C]mannitol (sp act 57 mCi/mmol) yielding an activity of 0.075 and 0.050 µCi/ml, respectively, were used.

Perfusion procedure. The perfusion apparatus included an artificial lung by means of which the arterial perfusate was continuously gassed with a mixture of 95% oxygen-5% carbon dioxide. The oxygen pressure and pH of the arterial perfusion medium was on average 584 ± 3 mmHg and 7.48 ± 0.01 (n = 137), respectively. The temperature of the incoming perfusate was ~35°C, which yields a calf muscle temperature of ~32°C (22). A period of 15-min equilibration perfusion was in all instances carried out with the hexose-free medium recirculating at a flow of 20 (2-leg perfusions) or 15 ml/min (1-leg perfusions). The initial 25 ml of perfusate passing through the preparation were discarded. At the time of glucose transport measurement the perfusate was exchanged for a new hexose-containing perfusate including specific tracers that reached the arterial catheter at the desired time point. The perfusion was then continued without recirculation. Perfusion pressure in one-leg perfusions (15 ml/min) was 52 ± 2 (n = 4) and 57 ± 3 mmHg (n = 24) in the rested nonstimulated and insulin-stimulated state, respectively. During one-leg perfusions with a perfusate flow of 20 ml/min, the perfusion pressure was 69 ± 4 (n = 12), 76 ± 4 (n = 85), and 72 ± 4 mmHg (n = 12) during insulin stimulation, contractions, and combined stimulation, respectively.

Muscle contractions. In the evaluation of contraction-induced glucose transport, one-leg perfusions were used. After the equilibration perfusion period, electrical stimulation (described below) was initiated at the same time as flow was increased to 20 ml/min. Dependent on the particular experiment, tracer-containing perfusate reached the hindlimb at the onset of contractions or 5, 10, 15, 20, or 30 min later while the hindlimb was still electrically stimulated. Electrical stimulation was performed as described (22). In short, the calf muscles were made to contract isometrically by electrical stimulation of the sciatic nerve with supramaximal trains of 200 ms delivered at a frequency of one train every second. The impulse duration and frequency within the train were 0.1 ms and 100 Hz, respectively. This pattern of stimulation has previously been demonstrated to elicit maximal glucose transport rate (14). The stimulation period was varied from 5 to 35 min.

Insulin stimulation. In the evaluation of insulin-induced glucose transport, two-leg perfusions were used. A maximally effective concentration of insulin (20 mU/ml) was present throughout the whole experiment. Tracer exposure was always preceded by 15 min of preperfusion. After the desired time of tracer exposure had elapsed, the common iliac artery and vein of one hindlimb were tied off and biopsies from the ipsilateral calf muscles were taken as described below. Perfusate flow was then decreased from the 20 to 14.5 ± 0.2 (n = 24) ml/min, resulting in the same arterial pressure as during two-leg perfusions. At the end of the experiment, the perfusion was stopped, and biopsies of the calf muscles were taken from the second leg.

Combined stimulation by insulin and contractions. To evaluate the additive nature of maximal effective insulin and electrical stimulation, experiments as described above were carried out during electrical stimulation with and without a maximal concentration of insulin (20 mU/ml) added to the perfusate. Because the flow rate might affect glucose transport rate, rats perfused with the same flow variation profile as during electrical stimulation were used for the control experiment with maximal insulin stimulation alone.

Perfusate sampling. Oxygen pressure, carbon dioxide pressure, and pH of the perfusate were measured within 5 min after sampling by use of an acid-base analyzer (ABL 30 acid-base analyzer, Radiometer). In addition, a sample of the incoming perfusate needed for calculation of glucose transport was taken just before muscle biopsy sampling and stored at -20°C until analyzed.

Biopsy procedure. Muscle biopsies were taken from three different parts of the calf muscles: the most superficial part of gastrocnemius medialis (consisting mainly of fast-twitch glycolytic fibers), the deep proximal and medial portion of gastrocnemius (consisting mainly of fast-twitch oxidative fibers), and the soleus (consisting mainly of slow-twitch oxidative fibers) (1). The biopsy samples were trimmed of connective tissue, blotted, and freeze clamped with aluminum clamps cooled in liquid nitrogen. The biopsies were stored at - 80°C until analyzed.

Glucose transport. Measurement of muscle glucose transport was performed as described (22). In short, muscle uptake of 3-O-[methyl-14C(U)]-D-glucose or 2-[2,6-3H]deoxy-D-glucose was measured in perchloric acid extracts and corrected for label in the extracellular space as determined by the 3H or 14C counts for mannitol. From the intracellular accumulation of 3-O-[methyl-14C(U)]-D-glucose or 2-[2,6-3H]deoxy-D-glucose, the rate of glucose transport was calculated using a specific activity of hexose determined by the hexose concentration and 3-O-[methyl-14C(U)]-D-glucose or 2-[2,6-3H]deoxy-D-glucose counts in the perfusate. Radioactivity was measured in a liquid scintillation counter (2000 Tri-Carb, Packard Instruments, Downers Grove, IL).

Chemicals. 3-O-[methyl-14C(U)]-D-glucose and D-[1-3H(N)]mannitol were from Du Pont NEN. 2-[2,6-3H]deoxy-D-glucose and D-[1-14C]mannitol were from Amersham International (UK). Human insulin (Actrapid) was from Novo Nordisk (Denmark). All other chemicals were of analytical grade from Sigma Chemical.

Statistics. Data are expressed as means ± SE. Statistical evaluation was done by one-way analysis of variance (ANOVA). When ANOVA revealed significant differences, the groups were identified using a post hoc test corrected for multiple comparisons (Student-Newman-Keuls test). Differences between groups were considered as statistically significant at P < 0.05. Curve fitting to data was performed applying the least-squares method to a linear curve (Y = aX + b), a monoexponential increasing curve [Y = b{1 - exp(-aX)}], or a monoexponential declining curve [Y = exp(-aX) + b] using Jandel Scientific Software (SigmaPlot and SigmaStat).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Despite the fact that mannitol is a hexose highly similar to glucose in molecular weight and composition, it is not specifically transported across the sarcolemma, making it a good choice as extracellular marker in glucose transport studies. The time course for mannitol to reach and equilibrate with the extracellular space in resting perfused rat muscle is depicted in Fig. 1. After 1 min of perfusion, 50-60% of the steady-state space (~0.23 ml/g) is equilibrated by mannitol in all three muscles studied. The mannitol space continued to increase and after 7-11 min reached a plateau that remained largely unchanged for at least 40 min of perfusion.


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Fig. 1.   Mannitol space in resting perfused rat muscles determined at different time points after addition of mannitol to perfusate. triangle , Fast-twitch glycolytic fibers; open circle , fast-twitch oxidative fibers; square , slow-twitch oxidative fibers. Each data point represents mean ± SE of 20 experiments.

The muscle uptake of 8 mM 2-deoxy-D-glucose during maximal insulin stimulation was linear in the range from 1 to at least 40 min in all three muscle fiber types (Fig. 2). Furthermore, maximal insulin-stimulated glucose transport was significantly different (P < 0.05) in the three muscle fiber types, i.e., fast-twitch glycolytic fibers (18 ± 0.6 µmol · g-1 · h-1) < fast-twitch oxidative fibers (32 ± 0.4 µmol · g-1 · h-1) < slow-twitch oxidative fibers (37 ± 0.7 µmol · g-1 · h-1).


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Fig. 2.   Time-dependent 2-deoxy-D-glucose (2-DG) uptake during maximal insulin stimulation (20 mU/ml). Hindlimb was perfused with insulin-containing perfusate for 15 min before tracer exposure. triangle , Fast-twitch glycolytic fibers; open circle , fast-twitch oxidative fibers; square , slow-twitch oxidative fibers. Lines represent best linear fit to data from each muscle group. Correlation coefficient was higher than 0.996 for all 3 curve fits. Each data point represents mean ± SE of 4 experiments.

The other major physiological stimulus increasing muscle glucose transport is muscle contraction. Consequently, we wanted to investigate whether glucose transport measurements during electrical stimulation of the sciatic nerve were valid in the perfused rat hindlimb using 2-deoxy-D-glucose as the glucose analog. In an initial series of perfusions, we studied the time-dependent activation of glucose transport by contractions. These experiments were performed by measurements of muscle 2-deoxy-D-glucose (8 mM) uptake in 5-min intervals after application of electrical stimulation. As shown in Fig. 3, the glucose transport rate was significantly (P < 0.05) lower during the initial 5 min of contractions compared with the subsequent five periods in the two oxidative fibers. In the fast-twitch glycolytic fibers, the initial glucose transport rate only tended (P = 0.09) to be lower than the rates during the subsequent five periods. Because a steady state of 2-deoxy-D-glucose uptake was achieved in all fiber types after 10 min of contractions it was possible to investigate whether 2-deoxy-D-glucose uptake is linear with time during steady-state contractions by exposing the muscle to the tracers after 10 min of contractions. Again, the 2-deoxy-D-glucose uptake was remarkably linear from 1 to at least 15 min of exposure to the tracers in all three muscle fiber types (Fig. 4). Furthermore, the applied electrical stimulation protocol induced significant (P < 0.05) different glucose transport among the three fiber types, i.e., 15 ± 0.3, 43 ± 2, and 31 ± 1 µmol · g-1 · h-1 in fast-twitch glycolytic fibers and in fast- and slow-twitch oxidative fibers, respectively, at steady state.


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Fig. 3.   2-DG uptake measured during last 5 min of electrical stimulation initiated at time 0 and stopped after 5, 10, 15, 20, 25, or 35 min. A: fast-twitch glycolytic fibers; B: fast-twitch oxidative fibers; C: slow-twitch oxidative fibers. Each bar represents mean ± SE of 4 experiments.


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Fig. 4.   Time-dependent 2-DG uptake during electrical stimulation. To secure maximal activation of transport process tracer exposure was preceded by 10 min of precontractions. triangle , Fast-twitch glycolytic fibers; open circle , fast-twitch oxidative fibers; square , slow-twitch oxidative fibers. Lines represent best linear fit to data from each muscle group. Correlation coefficient was higher than 0.993 for all 3 fits. Each data point represents mean ± SE of 4 experiments.

The use of 3-O-methyl-D-glucose may cause underestimation of the glucose transport rate when the transport capacity of the muscle fiber surface membrane is high. To further address this issue, we conducted a series of time course experiments during electrical stimulation similar to the one described previously (Fig. 4), using 3-O-methyl-D-glucose as the glucose analog. As seen from Fig. 5, an underestimation of the glucose transport capacity is evident even when relatively short periods of 3-O-methyl-D-glucose accumulation are used in the measurement of glucose transport rate. This phenomenon was restricted to the two oxidative fiber types (initial transport capacity higher than ~30 µmol · g-1 · h-1), whereas no significant difference compared with 2-deoxy-D-glucose was observed in the glycolytic fibers (initial transport capacity lower than ~15 µmol · g-1 · h-1). The time-dependent glucose transport shown in Fig. 5 is calculated from the average 3-O-methyl-D-glucose uptake in the actual period used for the measurement. Due to the initial higher glucose transport, this calculation procedure overestimates the actual transport rate at a given time point. However, the slope of the 3-O-methyl-D-glucose uptake curve at a certain time point represents the actual 3-O-methyl-D-glucose transport at that particular time point. Thus, by fitting the glucose uptake data for the individual muscle fiber types to a monoexponential curve, we calculated the actual 3-O-methyl-D-glucose transport. These values are also depicted in Fig. 5. Interestingly, the initial glucose transport rate obtained by extrapolation to time 0 using a monoexponential curve fit on these calculated 3-O-methyl-D-glucose transport data is very similar to the values obtained using 2-deoxy-D-glucose (Fig. 5). A similar pattern of time course data as during contractions using 3-O-methyl-D-glucose is observed when the glucose transport capacity is increased by maximal insulin stimulation (data not shown).


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Fig. 5.   Hexose transport measured either as 2-DG (horizontal solid line, data are extracted from Fig. 4) or 3-O-methyl-D-glucose (square ) uptake during electrical stimulation. Stimulation was initiated 10 min before tracer exposure. In addition, actual transport of 3-O-methyl-D-glucose at a certain time point estimated using slope of 3-O-methyl-D-glucose uptake curve at that particular moment, as described in text, is shown (bullet ). A: fast-twitch glycolytic fibers; B: fast-twitch oxidative fibers; C: slow-twitch oxidative fibers. Each data point represents mean ± SE of 4 experiments.

If exposure to 3-O-methyl-D-glucose was infinite, an equilibrium between muscle and perfusate would occur with a maximal accumulation of 3-O-methyl-D-glucose in muscle. From the monoexponential curve fit to the time-dependent 3-O-methyl-D-glucose uptake data, this theoretical maximal 3-O-methyl-D-glucose uptake can be estimated. From the two time course experiments (insulin and electrical stimulation), an apparently similar maximal 3-O-methyl-D-glucose accumulation of 3.4 ± 0.3 µmol/g (n = 6) was calculated for the three muscle fiber types. When this amount of 3-O-methyl-D-glucose is accumulated inside the muscle, the net transport is zero and the 3-O-methyl-D-glucose must occupy a volume of space in which the concentration is 8 mM (as for outside the cells). This volume is ~421 µl/g or only ~75% of the total intracellular water space calculated from the obtained extracellular space of 23% and an estimated total water content of 79% of the muscle wet weight (17).

To demonstrate convincingly that the initial lower 2-deoxy-D-glucose uptake during the initial 5 min of electrical stimulation (Fig. 3) is a phenomenon attributable to the transport and not a glycogenolysis-induced increase in intracellular glucose 6-phosphate causing inhibition of hexokinase, we did a similar experiment as described above (Fig. 3), measuring 3-O-methyl-D-glucose (8 mM) uptake in four 5-min intervals (Fig. 6). Using 5-min periods for 3-O-methyl-D-glucose accumulation will clearly (Fig. 5) underestimate the initial rate of uptake. However, by use of the theoretical maximal 3-O-methyl-D-glucose concentration of 3.4 ± 0.3 µmol/g together with the measured 3-O-methyl-D-glucose uptake in the 5-min accumulation period, the initial transport can be calculated from the monoexponential relationship between time and uptake. These calculated values are also depicted in Fig. 6, together with values obtained using 2-deoxy-D-glucose (extracted from Fig. 3). Comparable, not significantly different initial uptake rates are then obtained using the two analogs in all four 5-min periods.


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Fig. 6.   Time-dependent 3-O-methyl-D-glucose transport measured during last 5 min of electrical stimulation initiated at time 0 and stopped after 5, 10, 15, or 20 min (3-MG). For comparison 2-DG transport during a similar experiment is also shown (extracted from Fig. 3). Furthermore, initial 3-O-methyl-D-glucose transport calculated from obtained value after 5 min of accumulation and an estimated maximal 3-O-methyl-D-glucose concentration of 3.4 µmol/g using a monoexponential time relationship is also shown (3-MG-cal). A: fast-twitch glycolytic fibers; B: fast-twitch oxidative fibers; C: slow-twitch oxidative fibers. Each bar represents mean ± SE of 4 experiments.

The effect of maximal insulin, electrical stimulation, or maximal insulin and electrical stimulation combined on muscle 2-deoxy-D-glucose uptake rate (8 mM) are shown in Fig. 7 together with the basal unstimulated uptake rate. The different interventions were applied during highly similar perfusion protocols including the same flow profile. As expected, the fast- and slow-twitch oxidative fibers had two- to threefold higher responses during all interventions compared with the fast-twitch glycolytic fibers. By combining the two stimuli, we obtained significantly (P < 0.05) higher 2-deoxy-D-glucose uptake compared with insulin or contractions alone in fast- and slow-twitch oxidative fibers but not in fast-twitch glycolytic fibers.


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Fig. 7.   Rate of 8 mM 2-DG uptake in unstimulated state (basal) and during either maximal insulin (Ins), maximal electrical (Cont), or combined electrical and maximal insulin stimulation (Ins + Cont) using identical perfusion protocols in fast-twitch glycolytic fibers (A), fast-twitch oxidative fibers (B), and slow-twitch oxidative fibers (C). Tracer exposure time in all 3 interventions was 10 min and was performed 10 min after initiation of electrical stimulation and/or 25 min after initiation of insulin stimulation. In resting condition uptake of 2-DG was measured during last 30 min of perfusions lasting for 45 min. Each bar represents mean ± SE of 8 experiments. * Significant difference (P < 0.05) from both maximal insulin and electrical stimulation.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Although full equilibration of the mannitol space may be reached a few minutes earlier in in vitro incubated muscles, the time course for the observed equilibration process of mannitol is similar in perfused versus incubated muscle (6). As discussed by Hansen et al. (5), the initial accumulation of molecules in the extracellular space is not uniform in incubated muscle; i.e., an inward gradient exists for a significant period, creating erroneous glucose transport estimates during short time measurements. Probably concluding from this fact together with the slightly slower extracellular equilibration process in perfused muscles, Hansen et al. (4) claimed, without further investigation, that perfused muscles were not suitable for glucose transport measurements. However, as is evident from Figs. 2 and 5, the 2-deoxy-D-glucose time-dependent uptake in the three different muscle fibers studied is highly linear in the range from 1 to at least 40 min during maximal insulin stimulation and from 1 to at least 15 min during contractions. This clearly demonstrates that, despite the non-fully equilibrated extracellular space at early time points (Fig. 1), glucose transport measurements in perfused muscles are valid even when very short exposure times are used. This must imply that only part of the extracellular space is "drained" by the glucose transporters and that this space is equilibrated by mannitol and the D-glucose analog within 1 min. Furthermore, the finding that the maximal intracellular distribution space for 3-O-methyl-D-glucose only constitutes ~75% of the total intracellular water space indicates that several compartments, presumably belonging to different organelles, are not accessible by 3-O-methyl-D-glucose nor probably by 2-deoxy-D-glucose. The very similar initial glucose transport values (Fig. 6) obtained during contraction using either 8 mM 3-O-methyl-D-glucose or 2-deoxy-D-glucose clearly demonstrate that the contraction-induced increase in intracellular glucose 6-phosphate does not cause a shift in the rate-limiting step from transport to the hexokinase-mediated phosphorylation. These data therefore also suggest that contraction-induced activation of the glucose transport system via recruitment of transporters to the plasma membrane is a time-dependent process that seems to be fully completed by the end of the 5th min of stimulation. As indicated by the relatively lower initial level of glucose uptake compared with the maximal level in the slow-twitch oxidative fibers, it seems that with the present stimulation pattern a slower recruitment process is operating in these fibers compared with the other two fiber types.

Several different research groups have used 3-O-methyl-D-glucose in muscle glucose transport measurements using either perfused or in vitro incubated muscles (for review, see Ref. 2). The exposure times varied from 1 to 10 min during conditions in which muscle glucose transport was expected to be maximal. In muscle having a high sarcolemmal glucose transport capacity, e.g., stimulated fast- and slow-twitch oxidative fibers, these glucose transport estimations may therefore have been underestimated, with high transport values being affected relatively more than lower values unless very short exposure times had been used (<= 1 min). Therefore, any observed differences between low and high transport rates will tend to have been underestimated.

In an attempt to avoid disturbances in the glucose transport measurements caused by either the muscle activity per see or changes in flow during contractions, several research groups using either incubated or perfused muscles have chosen to measure muscle glucose transport after contractions (7, 14, 21). However, evidently the relatively fast reversal of contraction-induced glucose transport (half time ~7, 15, and >30 min in slow- and fast-twitch oxidative and fast-twitch glycolytic fibers, respectively; Ref. 15) may cause a significant underestimation of the glucose transport if the measurements are not performed immediately after contractions with a very short tracer exposure time. The present data show that this problem can be fully overcome by performing the measurements during contractions.

The additive nature of maximal effective insulin and electrical stimulation was investigated with identical perfusion protocols to obtain a similar delivery of hexose and insulin during all conditions. In agreement with findings from others (10, 14, 15), a significant additive effect is found in the two oxidative muscle fiber types only (Fig. 7). The lack of additivity in the fast-twitch glycolytic fibers has been reported previously (14, 15) but is in contrast to studies of in vitro incubated epitrochlearis muscles (3, 13). Except for the different model and muscle used, the explanation for this discrepancy is not clear. It could be argued that the incomplete additivity observed could display the limitation of the hexokinase-mediated phosphorylation process rather than the sarcolemmal glucose transport capacity. However, very similar results, i.e., comparing the data relatively, are found when similar experiments are performed with only 1 mM 2-deoxy-D-glucose (data not shown). With 1 mM deoxy-D-glucose, the glucose transport never exceeds the value of 15 µmol · g-1 · h-1 during the three interventions applied in any of the fiber types. At this rate of glucose uptake, the phosphorylation process is not a limiting factor for the glucose transport measurement, as clearly shown by the linear 2-deoxy-D-glucose uptake in muscles estimated to have glucose transport rates up to a least ~43 µmol · g-1 · h-1 (Figs. 2 and 4).

It could be argued that the glucose delivery to the muscle rather than the transport process was a rate-limiting factor for the muscle glucose uptake as indicated by some studies (8, 19). However, because the cell-free perfusate used in the present study has a low oxygen-carrying capacity, the perfusate was infused at relatively high flow rates to secure adequate oxygen delivery. By doing so, a high hexose delivery to the muscles was also achieved. In fact, the rate of 2-deoxy-D-glucose uptake during maximal insulin stimulation was not affected in any of the three muscles investigated (Figs. 2 and 7) by changing the rate of infusion (15 or 20 ml/min per hindlimb). This observation clearly indicates that hexose delivery is not a limiting factor for the glucose transport measurements performed in the present study.

In the present study, we used an erythrocyte-free perfusate that has previously been used by other groups including ourselves at resting conditions (11, 12, 16, 20, 22) as well as during muscle contractions (20, 22). If an appropriately high flow rate is applied in combination with a slight decrease in perfusate temperature to 35°C, the viability of the cell-free hindquarter perfusion seems to be satisfactory even during contractions (2, 22). Furthermore, force development during electrical stimulation is comparable in perfusions with cell-free and erythrocyte-containing perfusate (22).

In summary, the present study clearly demonstrates the suitability of the perfused rat hindlimb model for studying muscle glucose transport. Furthermore, our data illustrate the underestimation of the initial glucose transport rate that may occur when using accumulation of 3-O-methyl-D-glucose in muscle fibers having high glucose transport capacities. Thus 2-deoxy-D-glucose uptake in perfused rat muscles is highly applicable in measurements of the sarcolemmal glucose transport in resting as well as insulin- and contraction-stimulated perfused rat muscles.

    ACKNOWLEDGEMENTS

The authors are grateful to Betina Bolmgren for superior technical assistance.

    FOOTNOTES

This study was supported by the Danish National Research Foundation Grant 504-14.

Address for reprint requests: J. F. P. Wojtaszewski, Copenhagen Muscle Research Centre, August Krogh Institute, 13 Universitetsparken, DK-2100 Copenhagen, Denmark.

Received 2 June 1997; accepted in final form 16 September 1997.

    REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References

1.   Ariano, M. A., R. B. Armstrong, and V. R. Edgerton. Hindlimb muscle fiber populations of five mammals. J. Histochem. Cytochem. 21: 51-55, 1973[Medline].

2.   Bonen, A., M. G. Clark, and E. J. Henriksen. Experimental approaches in muscle metabolism: hindlimb perfusion and isolated muscle incubations. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E1-E16, 1994[Abstract/Free Full Text].

3.   Constable, S., R. Favier, G. D. Cartee, D. Joung, and J. Holloszy. Muscle glucose transport: interactions of in vitro contractions, insulin and exercise. J. Appl. Physiol. 64: 2329-2332, 1988[Abstract/Free Full Text].

4.   Hansen, P., E. Gulve, J. P. Gao, J. Schluter, M. Mueckler, and J. Holloszy. Kinetics of 2-deoxyglucose transport in skeletal muscle: effects of insulin and contractions. Am. J. Physiol. 268 (Cell Physiol. 37): C30-C35, 1995[Abstract/Free Full Text].

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