SPECIAL COMMUNICATION
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
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ABSTRACT |
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
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INTRODUCTION |
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.
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METHODS |
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).
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RESULTS |
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. , Fast-twitch
glycolytic fibers; , fast-twitch oxidative fibers; , slow-twitch
oxidative fibers. Each data point represents mean ± SE of 20 experiments.
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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. , Fast-twitch glycolytic fibers; , fast-twitch
oxidative fibers; , 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.
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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. , Fast-twitch glycolytic fibers; ,
fast-twitch oxidative fibers; , 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.
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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
( ) 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
( ). A: fast-twitch glycolytic
fibers; B: fast-twitch oxidative
fibers; C: slow-twitch oxidative
fibers. Each data point represents mean ± SE of 4 experiments.
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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.
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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.
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 |
DISCUSSION |
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.
 |
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