1 Department of Anatomy, The effects of epinephrine on glucose metabolism
during contractile activity and insulin stimulation were investigated
in fast-twitch (epitrochlearis) and slow-twitch (soleus) muscles from
Wistar rats. All muscles were mounted on contraction apparatuses, and
some muscles were stimulated electrically for 30 min in vitro. Glucose
uptake and glucose phosphorylation were measured with 2-[1,2-3H(N)]deoxy-D-glucose
and glucose transport with
3-O-[methyl-3H]methyl-D-glucose.
D-[1-14C]mannitol
was used to correct for extracellular space. In epitrochlearis, both
contraction and insulin increased glucose transport by threefold, and
combined they showed an additive effect. Epinephrine
(10
glucose 6-phosphate; glucose phosphorylation; glycogen; insulin
INSULIN AND CONTRACTILE ACTIVITY are the two most
important stimuli of glucose uptake in skeletal muscle. Glucose uptake
is, however, regulated in a complex interplay between several
counteracting stimuli. Epinephrine is believed to be the most important
inhibitor. During insulin stimulation, it is well documented that
epinephrine decreases glucose clearance in vivo (32) and, in agreement
with this, epinephrine inhibits insulin-stimulated glucose uptake in skeletal muscles (5, 12, 14).
Although exercise increases the blood concentration of epinephrine
(18), the effect of epinephrine on glucose uptake during contractile
activity has received much less attention than the effect of
epinephrine on insulin-stimulated glucose uptake. The effects of
epinephrine on glucose metabolism during contraction are also unclear.
Glucose utilization requires transport of glucose across the cell
membrane and phosphorylation to glucose 6-phosphate. In most
conditions, glucose transport is believed to be the rate-limiting step
for glucose utilization (30), but the rate-limiting step may switch to
phosphorylation under conditions of high insulin concentration (8, 19).
During epinephrine stimulation, glucose phosphorylation may also become
the rate-limiting step for glucose utilization. Epinephrine stimulates
glycogenolysis and accumulation of glucose 6-phosphate in skeletal
muscles (5, 16, 29), and glucose 6-phosphate is a strong inhibitor of
hexokinase activity (10, 22). Several studies have shown that
epinephrine decreases glucose uptake indirectly by inhibiting
phosphorylation (3, 5). Whether epinephrine reduces transport of
glucose across the membrane is more uncertain (3, 20, 34).
The effect of epinephrine on insulin-stimulated glucose uptake can,
however, not be extrapolated to contraction-stimulated glucose uptake
as several differences exist in the two situations. First, the internal
milieu is quite different in insulin-stimulated resting skeletal muscle
and in contracting muscles; the concentration of several metabolites,
e.g., glucose 6-phosphate, is elevated during contraction. In addition,
insulin and contraction stimulate glucose transport by different
signaling pathways (23). It is therefore possible that epinephrine may
have different effects on glucose uptake during insulin stimulation vs.
contraction.
In this study, we have investigated basic mechanisms for
epinephrine-induced inhibition of glucose metabolism in contracting skeletal muscle. As epinephrine may reduce insulin-stimulated glucose
uptake to a larger extent in oxidative muscles than in glycolytic
muscles (8, 12), the study was performed with a fast-twitch glycolytic
muscle (epitrochlearis) and a slow-twitch oxidative muscle (soleus). We
report here that epinephrine reduces glucose phosphorylation much less
during contractile activity than during insulin stimulation despite
similar glucose 6-phosphate concentrations. Furthermore, epinephrine
did not influence glucose transport across the cell membrane during
contraction or insulin stimulation. Our data therefore suggest that
epinephrine reduces glucose uptake indirectly via glucose
6-phosphate-induced inhibition of glucose phosphorylation in
insulin-stimulated as well as in contracting muscles. Glucose
6-phosphate seems, however, to be a much less powerful inhibitor of
glucose phosphorylation during contractile activity than during insulin
stimulation.
Animals. Male Wistar rats were
obtained from Møllegård Breeding Center (Skrensved,
Denmark). The rats were housed for at least 1 wk before the experiment
in our laboratory animal facilities at a room temperature of 21°C
and subjected to a 12:12-h light-dark cycle (light from 7:00 AM to 7:00
PM). The rats had free access to rat chow (Rat and Mouse Standard Diet;
B & K Universal, Grimston, UK) and water. The experiments were
performed during the light cycle (between 10:00 AM and 2:00 PM), and
the weights of the rats on the day of the experiment were 120-150
g. The experiments and procedures were approved by the local
responsible veterinarian and registered by the Experimental Animal
Board under the Ministry of Agriculture of Norway. The experiments were
therefore conducted in conformity with the laws and regulations
controlling experiments/procedures of live animals in Norway and the
European Convention for the Protection of Vertebrate Animals Used in
Experimental and Other Scientific Purposes (ETS 123).
Muscle preparation and incubation. The
rats were anesthetized with an intraperitoneal injection of 7.5 mg
pentobarbital sodium (50 mg/ml), and the epitrochlearis and soleus
muscles were dissected out. The epitrochlearis muscles were studied
intact, whereas the soleus muscles were divided in two. The muscles
were suspended on a contraction apparatus between two platinum
electrodes at their resting length. The apparatus was then placed in a
test tube, and a gas containing 95%
O2 and 5%
CO2 was continuously gassed
through the incubation buffer as described by Nesher et al. (24).
The muscles were preincubated for 30 min in 3 ml Krebs-Henseleit buffer
containing 5.5 mM glucose, 2 mM sodium acetate, 5 mM HEPES, and 0.1%
bovine serum albumin (fraction V; Sigma) as described earlier (14). All
incubations were performed at 30°C.
Muscle contraction. After
preincubation, some of the muscles were stimulated to contract
isometrically. The muscles were stimulated with impulse trains 200 ms
long at a frequency of 100 Hz (square wave pulses of 0.2 ms duration
and 10 V amplitude) delivered at a rate of one train per 2 s for 30 min. The pulses were generated by a Pulsar 6-bp stimulator (Frederic
Haer) and amplified by equipment built at The Institute of Basic
Medical Sciences, University of Oslo. This stimulation protocol has
been shown to elicit a maximal effect on contraction-stimulated glucose
uptake in epitrochlearis and ~65% of maximal contraction-stimulated
glucose uptake in soleus (unpublished observation).
Glucose uptake and glucose
phosphorylation. For determination of glucose uptake
and glucose phosphorylation, 0.25 µCi/ml
2-[1,23H(N)]deoxy-D-glucose
(30.6 Ci/mmol; NEN) and 0.1 µCi/ml
D-[1-14C]mannitol
(54.5 mCi/mmol; NEN) were added to the buffer (containing 5.5 mM
glucose). Glucose uptake was defined from the intracellular accumulation of
2-[3H]deoxy-D-glucose
during 30 min of incubation assuming similar uptake kinetics for
glucose and 2-deoxyglucose. The muscles were studied while resting or
under contraction during the measurements of glucose uptake and glucose
phosphorylation. Both resting and contracting muscles were incubated
with or without 10 mU/ml insulin (Monotard, Novo Nordisk, Denmark) and
with or without 10
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
6 M) did not influence
glucose transport across the membrane during contractile activity or
insulin stimulation. In the absence of epinephrine, similar glucose
phosphorylation was obtained during contraction and during insulin
stimulation in epitrochlearis (~12 mmol · kg dry
wt
1 · 30 min
1). In the presence of
epinephrine, 9.5 ± 0.6 mmol · kg dry
wt
1 · 30 min
1 glucose was
phosphorylated during contraction, whereas only 2.0 ± 0.3 mmol · kg dry
wt
1 · 30 min
1 was phosphorylated
during insulin stimulation (P < 0.01), despite a similar glucose 6-phosphate concentration. Comparable
results were obtained in soleus. In conclusion, our data suggest that epinephrine inhibits glucose phosphorylation much less during contraction than during insulin stimulation.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
6 M
epinephrine [(
)-epinephrine; Sigma]. The eight
groups are shown in Fig.
1. In all incubations,
0.1% ascorbic acid was added as an antioxidant. After incubations, the
muscles were removed from the contraction apparatuses, blotted on
filter paper, and frozen in liquid nitrogen. The muscles were
freeze-dried and weighed.
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Fig. 1.
Effects of epinephrine on glucose uptake and glucose phosphorylation
(hatched bars) in epitrochlearis muscles
(A) and soleus muscles
(B). Open bars represent
intracellular free glucose. Muscles were incubated 30 min with tracer
amount of
2-[3H]deoxyglucose,
and glucose uptake and glucose phosphorylation were measured with and
without 10 6 M epinephrine
(Adr) in control (Basal), electrically stimulated (El. stim), insulin
stimulated (Ins), and insulin plus electrically stimulated (El. stim + Ins) muscles. Values are means ± SE;
n = 7-24 muscles. Lowercase
letters refer to differences in glucose uptake, and capital letters
refer to differences in glucose phosphorylation.
a,A Significant differences
between muscles incubated with and without epinephrine.
b,B Significantly higher
than El. stim and Ins.
c,C Significantly lower than
El. stim + Adr and El. stim + Ins + Adr.
d,D Significantly lower than
Ins and El. stim + Ins.
e Significantly higher than
El. stim + Adr and Ins + Adr. dw, Dry wt.
The freeze-dried muscles were homogenized in 1 ml of ice-cold 6% perchloric acid (PCA; 2 × 15 s at maximal speed; Polytron PT 1200), and the homogenate was centrifuged (3,000 g for 20 min at 4°C). For determination of glucose uptake, 200 µl of the supernatant were added to 3 ml of scintillation solution (High-ionic Fluor; Packard), mixed, and counted for radioactivity (TRI-CARB 460C; Packard). For determination of glucose phosphorylation, accumulation of 2-[3H]deoxy-D-glucose 6-phosphate was determined after separation on a Dowex-2 column (Dowex 2x8-400; Sigma) in formate form according to Hammerstedt (9). In brief, 700 µl from the supernatant were neutralized (pH 7.0) with ~0.4 ml of 2 M KOH/0.5 M triethanolamine and centrifuged (3,000 g for 10 min at 4°C). One milliliter of the supernatant was placed on a 1.5-ml Dowex-2 ion exchange column. The columns were washed two times with 6 ml deionized water to elute [14C]mannitol and nonphosphorylated 2-[3H]deoxyglucose. The columns were then washed two times with 6 ml of 1 M formic acid/0.3 M ammonium formate to elute 2-[3H]deoxy-D-glucose 6-phosphate. No radioactivity was present in the second wash with water or formic acid. Recovery of [14C]glucose 6-phosphate (54.1 mCi/mmol; NEN) was tested in all experiments and was always >93%. The data are corrected for the actual percent recovery in each experiment. From each eluate, 1 ml was added to 3 ml of scintillation solution (High-ionic Fluor; Packard), mixed, and counted for radioactivity.
Glucose uptake was determined by a different procedure in the studies
of adrenoceptor subtypes mediating the effect of epinephrine. In brief,
muscles were then dissolved in 600 µl of 1 M KOH for 20 min at
70°C. Of the digest, 400 µl were added to 3 ml scintillation solution (High-ionic Fluor; Packard), mixed, and counted for
radioactivity. There was no significant difference in glucose uptake
measured with the KOH and PCA methods. Muscles were divided in two, and glucose uptake was 9.63 ± 0.85 mmol · kg dry
wt1 · 30 min
1 as analyzed after
dissolving in KOH and 9.51 ± 0.78 mmol · kg dry
wt
1 · 30 min
1 after homogenization
in PCA (n = 12). The
coefficient of variation between the methods was 10.9%.
3-O-methylglucose
transport. The rate of glucose
transport was measured with a tracer amount of
3-O-[methyl-3H]methyl-D-glucose,
a glucose analog that is transported across the cell membrane but not
further metabolized. Glucose transport was defined as the intracellular
accumulation of 3-O-methylglucose during the last 10 min of the 30-min stimulation period. The short period of time for measurement of glucose transport was used to avoid
too high accumulation of
3-O-methylglucose intracellularly (34). The muscles were incubated in Krebs-Henseleit buffer identical to
that described above, and the muscles were stimulated electrically or
with insulin (10 mU/ml) in the absence or presence of
106 M epinephrine. For
measurements of glucose transport, the muscles were transferred to a
buffer similar to that used for the first 20 min with addition of 0.25 µCi/ml
3-O-[methyl-3H]methyl-D-glucose
(75.2 Ci/mmol; NEN) and 0.15 µCi/ml
[14C]mannitol. After
incubation, the muscles were blotted on filter paper, frozen in liquid
nitrogen, freeze-dried, and weighed. The muscles were then dissolved in
600 µl of 1 M KOH for 20 min at 70°C. Of the digest, 400 µl
were added to 3 ml of scintillation solution (High-ionic Fluor;
Packard), mixed, and counted for radioactivity.
Metabolites. The concentrations of glucose, glucose 6-phosphate, ATP, phosphocreatine (PCr), and lactate were measured after extraction in 300 µl of 0.6 M PCA for 30 min (the PCA was kept on ice, and the muscles were pressed with a glass pin after 15 min). The extract was neutralized with 100 µl of 2 M KHCO3, the neutralized extract was centrifuged (2,500 g for 10 min at 4°C), and the supernatant was used for analyses. ATP and PCr were analyzed immediately, and the rest of the supernatant was frozen for later analysis of glucose, glucose 6-phosphate, and lactate. All metabolites were analyzed fluorometrically according to Lowry and Passonneau (21).
Glycogen. Glycogen was measured in the muscle sample that had been extracted in PCA. The muscles and the precipitate were dissolved in 500 µl of 1 M HCl, and the glycogen was hydrolyzed for 2.5 h at 100°C and centrifuged (2,500 g for 10 min at 4°C). Glucose units were measured fluorometrically in the supernatant according to Lowry and Passonneau (21).
Calculation of intracellular concentrations of metabolites. The water content in soleus and epitrochlearis was calculated by weighing muscles directly after they were frozen in liquid nitrogen (wet weight) and then after they had been freeze-dried for 24 h (dry weight; Edwards Freeze Dryer; Modulyo Pirani 501). The water content was calculated as the weight reduction in the muscles. In the epitrochlearis muscles the water content was 79.0 ± 0.2% (n = 42) and in soleus 78.5 ± 0.1% (n = 40). We found a significantly higher water content in electrically stimulated epitrochlearis muscles (79.9 ± 0.3%; n = 14; P < 0.0001) than in rested epitrochlearis muscles (78.5 ± 0.1%, n = 28). In the soleus muscles there were no significant differences between contracted (78.6 ± 0.2%, n = 13) and rested (78.4 ± 0.1%, n = 27) muscles.
The extracellular space was determined as the [14C]mannitol space and was calculated to be 0.236 ± 0.004 ml/g wet wt in both soleus and epitrochlearis muscles. There were no differences in extracellular space between rested and contracted muscles.
The intracellular concentration of glucose was calculated using the
formula Ci = [Cm (Cb × E)]/(Mw
E) as
described by Berger et al. (1), where
Ci is millimole glucose per liter cell water, Cm is millimole
glucose per kilogram wet weight,
Cb is millimole glucose per liter
extracellular fluid (5.5 mmol/l), Mw is water content in the muscles
(l/kg wet wt), and E is extracellular space (l/kg wet wt). The
intracellular concentration of glucose 6-phosphate was calculated using
the same formula anticipating no extracellular glucose 6-phosphate and
therefore that Cb = 0.
Statistics. All data are presented as means ± SE. Multiple comparisons were made using analysis of variance, with significant differences determined by Newman-Keuls post hoc analysis. Differences between epitrochlearis and soleus were tested with Student's t-test. Significance was set at the P < 0.05 level.
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RESULTS |
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Glucose uptake and glucose phosphorylation. Glucose uptake and glucose phosphorylation for epitrochlearis and soleus muscles are shown in Fig. 1, A and B, respectively. In epitrochlearis, insulin as well as contraction increased glucose uptake threefold. Furthermore, contraction and insulin had an additive effect on glucose uptake (Fig. 1A). Epinephrine reduced insulin-stimulated glucose uptake by 46% (Fig. 1A). During contraction, epinephrine reduced glucose uptake when insulin was present, whereas no significant inhibitory effect of epinephrine was observed in the absence of insulin (Fig. 1A).
In the basal condition, 2.6 ± 0.2 mmol · kg dry
wt1 · 30 min
1
(n = 13) glucose was phosphorylated in
epitrochlearis, and glucose phosphorylation increased to ~12
mmol · kg dry
wt
1 · 30 min
1 in contracting as well
as in insulin-stimulated muscles. Contraction and insulin had additive
effects on glucose phosphorylation in epitrochlearis (Fig.
1A). Epinephrine reduced glucose
phosphorylation during insulin stimulation and during contractile
activity whether insulin was present or not
(P < 0.05); the reduction, however, differed significantly. Glucose phosphorylation was similar during insulin stimulation and during electrical stimulation in the absence of
epinephrine. In the presence of epinephrine, only 2.0 ± 0.3 mmol · kg dry
wt
1 · 30 min
1 was phosphorylated
during insulin stimulation, whereas 9.5 ± 0.6 mmol · kg dry
wt
1 · 30 min
1 was phosphorylated
during contraction (P < 0.01; Fig.
1A). Insulin did not increase
glucose phosphorylation in epitrochlearis during contractile activity
when epinephrine was present.
In soleus muscles, basal and insulin-stimulated glucose uptake rates were significantly higher than in epitrochlearis muscles (P < 0.02). The contraction-stimulated glucose uptake (the difference between glucose uptake during contraction and under basal conditions) was, on the other hand, higher in epitrochlearis than in soleus (P < 0.01). Epinephrine reduced glucose uptake during insulin stimulation by 37%, whereas epinephrine only reduced glucose uptake by 15-20% in contracting muscles.
Epinephrine decreased glucose phosphorylation in the soleus in all groups (Fig. 1B). Like in the epitrochlearis, epinephrine reduced glucose phosphorylation much more during insulin stimulation than during contractile activity.
Glucose 6-phosphate and intracellular free glucose. Glucose 6-phosphate is a potent inhibitor of hexokinase activity, and epinephrine increased glucose 6-phosphate concentration in both resting and electrically stimulated muscles (P < 0.02; Table 1). The concentration of glucose 6-phosphate was similar in contracting and insulin-stimulated muscles when epinephrine was present. Glucose phosphorylation, on the other hand, differed significantly.
|
As seen in Fig. 1, the glucose uptake exceeds glucose phosphorylation, the difference representing an accumulation of free glucose. Furthermore, we measured the glucose concentration biochemically and corrected for extracellular glucose. No major accumulation of intracellular free glucose occurred in either resting or contracting epitrochlearis muscles incubated without epinephrine (Table 2). Analysis of variance showed significant effects of epinephrine on the intracellular concentration of glucose. Post hoc analysis showed that epinephrine only elevated the intracellular concentration of glucose significantly in insulin-stimulated muscles where the most pronounced reduction of glucose phosphorylation was observed.
|
In the soleus, epinephrine increased the concentration of glucose
6-phosphate in all groups, except when insulin was present during
contraction. Negative values were obtained for intracellular free
glucose concentration in soleus when glucose was measured biochemically
and corrected for mannitol space (values between 0.3 and
0.7 mM in the different groups). Epinephrine, however, increased
the intracellular concentration of free glucose in all groups
(P < 0.02) with the exception of
contracting muscles when insulin was absent (data not shown).
Epinephrine also had little effect on glucose phosphorylation in
contracting soleus muscles.
Glucose transport. Insulin and contraction increased glucose transport (measured with a tracer amount of 3-O-methylglucose) threefold in epitrochlearis (Fig. 2A), an increase similar to that for glucose uptake (Fig. 1A). Furthermore, contractile activity and insulin had an additive effect on glucose transport in epitrochlearis (Fig. 2A). Epinephrine did not influence glucose transport during insulin stimulation or during contractile activity either in the presence or absence of insulin. In the soleus, epinephrine had no influence on glucose transport in any of the groups studied (Fig. 2B).
|
ATP and PCr. Concentrations of ATP and PCr were similar under basal conditions and in insulin-stimulated epitrochlearis muscles, whereas contractile activity decreased ATP and PCr by ~30 and 55%, respectively. Epinephrine had no effect on the concentration of ATP and PCr (Table 3). In the soleus, the decrease in ATP and PCr during contractile activity was similar irrespective of whether epinephrine was present (Table 4).
|
|
Glycogen and lactate. Contractile activity decreased the glycogen concentration in both epitrochlearis (Fig. 3A) and soleus (Fig. 3B). Furthermore, contraction and epinephrine had an additive effect on glycogenolysis in epitrochlearis in the absence of insulin (P < 0.01; Fig. 3A). In the soleus, however, a possible additive effect of contraction and epinephrine on glycogenolysis was not significant (P = 0.07; Fig 3B).
|
Lactate concentration increased during contractile activity in both epitrochlearis and soleus (Table 5). Although epinephrine increased glycogenolysis, similar lactate concentrations were observed in contracting muscles in the presence and absence of epinephrine.
|
- and
-Blockers. The inhibitory
effect of epinephrine on glucose uptake was blocked by timolol (a
-antagonist) in insulin-stimulated (Fig.
4A) and
contracting muscles (Fig. 4B).
Prazosin (an
-antagonist), on the other hand, had no effect on
epinephrine-induced inhibition of glucose uptake either in contracting
or in insulin-stimulated muscles (Fig. 4).
|
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DISCUSSION |
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In this study, we found that epinephrine reduced insulin-stimulated glucose phosphorylation, whereas insulin-stimulated glucose transport across the cell membrane was not influenced. Glucose transport was not influenced by epinephrine during contractile activity. Interestingly, glucose phosphorylation was much less reduced during contractile activity than during insulin stimulation. A major conclusion in the present study is therefore that glucose 6-phosphate is a much less powerful inhibitor of glucose phosphorylation during contractile activity than during insulin stimulation.
Only a few studies have investigated the effect of epinephrine on glucose uptake in skeletal muscle during contraction. Using the perfused rat hindlimb, Richter et al. (31) have reported that epinephrine causes a small increase in glucose uptake during contraction, whereas Chiasson et al. (4) did not find any significant effect of epinephrine on glucose uptake. In contrast, Jansson et al. (13) found that epinephrine reduced glucose uptake in exercising human skeletal muscles.
Epinephrine is also a regulator of blood flow, and Rattigan et al. (27)
have recently shown that norepinephrine reduces glucose uptake via an
-adrenergic regulation of blood flow and a
-adrenergic effect on
the muscle fibers. It is therefore difficult to compare the data from
studies with intact circulation directly with in vitro studies. The
data in the present in vitro study indicate, however, that epinephrine
may cause a small decline in glucose uptake in skeletal muscle
independent of circulation, which is in agreement with Nesher et al.
(24).
Epinephrine reduced insulin-stimulated glucose phosphorylation by 83% in epitrochlearis muscle, which is in agreement with other studies (5, 12, 14). Epinephrine was in fact such an effective inhibitor of insulin-stimulated glucose phosphorylation that glucose phosphorylation was reduced to a level similar to that under basal conditions. In the present study, epinephrine increased the concentration of glucose 6-phosphate to ~1.3 mM when insulin was present, a concentration of glucose 6-phosphate that is sufficient to inhibit hexokinase activity nearly completely in vitro (10, 22). The reduction in glucose phosphorylation during insulin stimulation can therefore be explained by glucose 6-phosphate-induced inhibition of hexokinase activity.
During contraction, epinephrine caused a similar increase in the
concentration of glucose 6-phosphate, and a similar inhibition of
hexokinase should have been expected (22). Surprisingly, a high rate of
glucose phosphorylation still occurred in spite of epinephrine being
present. Glucose phosphorylation was similar in epitrochlearis during
insulin stimulation and contractile activity when epinephrine was
absent. When epinephrine was present during contraction, glucose
phosphorylation was 9.5 mmol · kg dry
wt1 · 30 min
1, whereas only 2.0 mmol · kg dry
wt
1 · 30 min
1 glucose units were
phosphorylated in the presence of epinephrine during insulin
stimulation. This is a dramatic difference and indicates that glucose
6-phosphate is not a potent inhibitor of glucose phosphorylation during
contraction.
The mechanism for the persisting high rate of glucose phosphorylation during contractile activity in the presence of epinephrine remains to be established. In the present study, the concentration of PCr and ATP decreased during contraction, which probably caused an accumulation of Pi (28). The brain isoform of hexokinase (HK I) is Pi regulated, and Pi has been reported to reduce the sensitivity of HK I for glucose 6-phosphate inhibition (26). Regulation of mammalian muscle hexokinase by Pi has not been documented. Karpatkin (17) reported that Pi relieves the inhibition of hexokinase by glucose 6-phosphate in frog skeletal muscle, but Lueck and Fromm (22) did not find any support for that in rat skeletal muscles. It is, however, possible that other metabolic changes that also occur during contraction may reduce the ability of glucose 6-phosphate to inhibit hexokinase activity.
Another possible mechanism for regulation of hexokinase activity is binding of hexokinase to mitochondria (2), a binding that seems to decrease its sensitivity for glucose 6-phosphate-induced inhibition (17). It has been reported that contractile activity increases the binding of hexokinase to mitochondria in skeletal muscle (33), but whether increased binding of hexokinase to mitochondria is the mechanism for the high glucose phosphorylation that occurred during contraction with epinephrine remains to be proven.
To further elucidate the effects of epinephrine on glucose metabolism during contraction, the effect of epinephrine on glucose transport across the membrane was studied with the nonmetabolized glucose analog 3-O-methylglucose. In the present study, epinephrine did not influence glucose transport in either epitrochlearis or in soleus during contraction (Fig. 2). To the best of our knowledge, this is the first time that the effect of epinephrine on glucose transport has been studied during contractile activity, and our data show that epinephrine does not influence glucose transport under those conditions.
Epinephrine was without influence on insulin-stimulated glucose transport in the present study. The effect of epinephrine on glucose transport during insulin stimulation and under basal conditions is controversial. Decreased, unchanged, and increased glucose transport have been reported (3, 5, 34). A recent comprehensive study by Lee and co-workers (20), however, found no effect of epinephrine on insulin-stimulated glucose transport. Epinephrine, therefore, seems to influence neither contraction-stimulated nor insulin-stimulated glucose transport in skeletal muscles.
The hypothesis that epinephrine reduces glucose uptake indirectly by inhibition of glucose phosphorylation is also supported by the increase in concentration of intracellular free glucose during epinephrine stimulation. In agreement with Chiasson et al. (5), we found that epinephrine caused a large accumulation of free glucose during insulin stimulation, both measured biochemically (Table 2) and judged from accumulation of nonphosphorylated 2-deoxyglucose (Fig. 1). The intracellular concentration of free glucose increases when epinephrine reduces glucose phosphorylation without reducing glucose transport. The increase in intracellular free glucose concentration in insulin-stimulated muscles when epinephrine was present therefore supports our measurements of glucose phosphorylation and glucose transport. During contractile activity, on the other hand, epinephrine caused a less pronounced increase in intracellular free glucose, and the increase did not reach statistical significance in epitrochlearis. This is in accordance with the lower reduction in glucose phosphorylation observed in the same groups of muscles. All data in the present study, therefore, suggest that epinephrine inhibits glucose uptake indirectly by inhibiting glucose phosphorylation via increased glucose 6-phosphate concentration. Glucose 6-phosphate is, however, a much less powerful inhibitor of glucose phosphorylation during contractile activity than during insulin stimulation, and the glucose uptake is therefore reduced much less in contracting muscles.
Insulin and contraction had an additive effect on glucose transport in epitrochlearis muscles (Fig. 2A). Several studies have previously reported an additive effect of contractile activity and insulin on glucose transport (7, 11, 25), and it has been suggested that there are two distinct pools of GLUT-4 (6, 30). The present study shows that epinephrine does not influence the additive effect of contractile activity and insulin on glucose transport (Fig. 2A). On the other hand, epinephrine completely abolished the additive effect of insulin on glucose phosphorylation during contraction in epitrochlearis. As the data in the present study suggest that glucose phosphorylation is the rate-limiting step during epinephrine stimulation, it is not surprising that similar amounts of glucose were phosphorylated with and without insulin in contracting muscles when epinephrine was present. The concentrations of glucose 6-phosphate, ATP, and PCr were similar in contracting muscles with and without insulin, and, although insulin increased glucose transport during contraction, glucose phosphorylation was still the rate-limiting step.
In soleus, we found that basal and insulin-stimulated glucose uptake
was higher than in epitrochlearis. This is in agreement with other
studies (7, 11) and is in accordance with the higher GLUT-4 content in
soleus (11). Although the density of -adrenergic receptors is two
times as high in soleus (15), the concentration of glucose 6-phosphate
was lower in soleus than in epitrochlearis during epinephrine
stimulation. The concentration of glucose 6-phosphate was, however,
still sufficient to inhibit glucose phosphorylation substantially. The
relative reduction was also rather similar in soleus and
epitrochlearis, and the higher rate of glucose phosphorylation in
soleus during epinephrine stimulation may probably reflect a higher
hexokinase activity rather than differences in regulation.
Adrenergic stimulation increases the metabolic rate and oxygen consumption in resting skeletal muscle (27). Although epinephrine reduced glucose phosphorylation in resting muscle, it also stimulated glycogenolysis, and the total utilization of carbohydrate was increased in agreement with other studies (24, 29). The increased metabolic rate during epinephrine stimulation makes glycogen the preferred carbohydrate and even decreases the utilization of exogenous glucose in resting muscles. During contractile activity, on the other hand, a large amount of glucose was phosphorylated even though epinephrine was present. Our data indicate that contractile activity in some way overrides the glucose 6-phosphate-induced inhibition of hexokinase activity and allows significant glucose phosphorylation. This may be an important physiological mechanism directing glucose to the active muscles during stressful situations.
In conclusion, glucose transport was not influenced by epinephrine
either during contraction or during insulin stimulation. Interestingly,
epinephrine reduces glucose phosphorylation much less during
contraction than during insulin stimulation. Our data suggest that
epinephrine, via -adrenergic stimulation, reduces glucose uptake
indirectly by glucose 6-phosphate-induced inhibition of glucose
phosphorylation. Glucose 6-phosphate is, however, a much less potent
inhibitor of glucose phosphorylation during contractile activity than
during insulin stimulation.
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ACKNOWLEDGEMENTS |
---|
We thank Idar Bergli and Jesper Franch for excellent technical assistance.
![]() |
FOOTNOTES |
---|
During this study, J. Jensen was a postdoctoral candidate supported by The Research Council of Norway. The study was also supported by The Jahre Foundation and by Nordic Academy for Advanced Study.
Present address of J. Jensen: Department of Physiology, National Institute of Occupational Health, PO Box 8149 Dep., N-0033 Oslo, Norway.
Address for reprint requests: J. Jensen, Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, PO Box 1105 Blindern, N-0317 Oslo, Norway.
Received 25 November 1997; accepted in final form 13 May 1998.
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REFERENCES |
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