Vol.
187,
September
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
16, 1992
COMMUNICATIONS Pages
EPINEPHRINE
ADMINISTRATION BUT REDUCES
STIMULATES
GLUCOSE
685-691
GLUT4 TRANSLOCATION
TRANSPORT
IN MUSCLE
A.Bonen*T L.A. Megeney*, SC. McCarthy?, J.C. McDermottt,
and M.H. Tan?
*Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl TDepartments of Medicine and Biochemistry, Dalhousie University, Halifax, Canada B3H 3J5 Received
July
20,
1992
Summary: Epinephrine opposes glucose transport in muscle. Therefore, we investigated the
effects of epinephrine administration (25pg/lOOg body weight) on glucose transport and glucose transporters in rat muscle. Ninety minutes after epinephrine injection 3-O-methyl glucose transport was reduced (-47%) in perfused muscles of the rat hindlimb. Translocation of the insulinregulatable glucose transporter (GLUT4) in the epinephrine-injected animals was confirmed by the marked increments in the GLUT-4 in the plasma membranes and their concomitant reduction in the intracellular membranes. We speculate a) that it is epinephrine which translocated GLUT4 via a CAMP-linked pathway, and b) that the intrinsic activity reductions are caused either by the glycation of the transporter by the persistent hyperglycemia and/or by epinephrine via the 0 1992Academic Press, Inc. phosphorylation of the GLUT4 transporter protein in muscle.
Skeletal muscle is quantitatively the most important disposal site for glucose in viva, and entry of glucose into this tissue occurs via facilitated transport involving a carrier protein. A major mechanism by which insulin stimulates glucose transport into skeletal muscle involves the translocation of the insulin-regulatable glucose transport protein (GLUT-4) from an intracellular pool to the cell surface (13,17, 22). Increments in the intrinsic activity of the glucose transporter can also stimulate glucose transport into skeletal muscle, particularly after exercise (15,21). It has been proposed that the hormonal/metabolic milieu around a muscle may also alter glucose transport, glucose transporter number and glucose transporter intrinsic activity (8). For example, fasting (8) and streptozotocin-induced diabetes (23) provoke marked changes in a number of different substrates and glucoregulatory hormones, and along with these multiple hormonal/metabolic changes marked alterations in glucose transport, glucose transporter numbers and their intrinsic activities are observed (8,23). Epinephrine administration also provokes marked changes in the hormonal/metabolic milieu (25), and epinephrine is an important counter-regulatory hormone to glucose transport in muscle (9,32). Whether this impediment to glucose transport is due to a reduced number of surface transport proteins or due to a reduction in their intrinsic activities is not known. Therefore, we investigated the effects of epinephrine administration in viva on glucose transport and on glucose transporters in rat skeletal muscle.
685
All
Copyright 8 1992 rights of reproduction
0006-29 I X/92 $4.00 by Academic. Press. Inc. in anp form reserved.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH
COMMUNICATIONS
Materials: All chemicals were obtained from Sigma Chemicals (StLouis), except for [3H]-3-0methylglucose, [3H]-cytochalasin B and [3H]-sorbitol which were purchased from Du Pont-New England Nuclear (Montreal). Porcine monocomponent insulin was a gift from Eli Lilly (Indianapolis). The polyclonal antibody to GLUT-4 was a kind gift from Dr. A. Klip, The Hospital for Sick Children, Toronto, Ontario, Canada. Experimental animals: Male Sprague-Dawley rats (250-300 g body weight) were housed on a 12 hour light/dark cycle with unlimited access to food and water. Animals were randomly assigned to control or epinephrine-injected groups (25ug/lOOg body weight) groups. Both groups were injected intraperitoneally and left for 90min. At that point we determined a) 3-O-methyl glucose transport in the perfused hindlimb muscles or b) glucose transporters in sarcolemmal membranes and intracellular membranes, in two separate groups of animals. Glucose, insulin, free-fatty acids (FFA), epinephrine and nor-epinephrine (5, 25) concentrations were also determined in plasma samples obtained 90 min after epinephrine administration, from animals in b). Skeletal muscle glycogen concentrations (4) were also determined 90 min after epinephrine administration in a separate group of animals. To ensure that all groups were studied at 90 minutes animals were anaesthetized (Somnotol65mg/lOOg weight) at t=70 min and surgicaly prepared for the hindlimb perfusion studies in a) or for the muscle removal for studies in b) Glucose Transport Studies in Perfused Muscles: Ninety minutes after the injections with epinephrine or control injections, perfusion of the hindlimb skeletal muscles were performed as described elsewhere in detail (26, 27).Rates of glucose transport were determined period with [3H]-3-O-methylglucose (3-O-MG) (lOpCi, 5 mM) in the soleus, plantaris, red and white gastrocnemius muscles.The extracellular space were determined as reported elsewhere (27, 16). Membrane preparation and glucose transporter determinations: The procedures described in detail by Klip et al (22) were used to isolate the skeletal muscle plasma and intracellular membranes. Membrane purification, based on 5’ nucleotidase activities, were similar to that reported in the original method (22). Surface glucose transporters in plasma membranes were determined using D-glucose protectable binding of cytochalasin B as has been described elsewhere (22)Westem blotting was performed as described by Douen et al (12). Briefly, intracellular membranes and plasma membranes were subjected to sodium dodecyl sulfatepolyacrylamide electrophoresis on 12% polyacrylamide gels and transferred to nitrocellulose filter membrane for 45 min. Incubations of nitrocellulose filters were performed using anti-GLUT-4 R 820, a polyclonal antibody. Thereafter, the membranes were washed, incubated with r*sI-labelled protein A, dried, and exposed to XAR-5 film. Autoradiographs were quantitated with appropriate software (Scan Analysis, Biosoft, Cambridge, UK) on a Macintosh LC computer. RESULTS Administration of epinephrine (i.p.; 25ug/lOO g body weight) to fed rats provoked changes in circulating substrates and hormones (Table 1). Ninety minutes after epinephrine injection glucose and FFA concentrations were 208% and 180% of the control concentrations, respectively, and epinephrine and nor-epinephrine levels were 2313% and 337% of the control concentrations, repectively. However, the insulin level at t=90 min was reduced to 58% of the control concentration. In addition, epinephrine administration also reduced muscle glycogen depots to 53% (soleus), 45% (plantaris), 46% (red gastrocnemius), and 36.7% (white gastrocnemius) of the control concentrations. Glucose transport rates are known to differ among perfused rat hindlimb muscles (soleus, plantaris and red and white gastrocnemius) (27,30) that are comprised of widely differing fiber types (5). This was also observed in this study (Fig l), although as in previous studies such differences are less pronounced in the absence of insulin (27,30). After 90 min of being exposed to a metabolic
Vol.
187,
TABLE
No.
BIOCHEMICAL
2, 1992
1. Effects of epinepbrine
(25@100
glvcagealymol/e
BIOPHYSICAL
RESEARCH
(E) and norepinephrine
(NE)
wet weieht) red
COMMUNICATIONS
g body weight, i.p.) on muscle glycogen, and on plasma glucose, FFA, insulin,
epinephrine Muwle
AND
Circulatine
white
glucose
FFA
Tr
concentrations insulin
m
control
epinephrine injected
21.5
24.1
24.4
25.6
184.6
k1.7
k2.2
k2.8
k3.1
f5.2
14.6a
10.ga
11.3a
9.4a
384.6a
0.453
k9.4
k19.5
kO.07
f1.5
“epinephrine
group significantly
k2.3
k2.0
E
ml
0.25
4.3
kO.03
k.6
ml
NE ne/ml
0.48
0.32
k.19
k.07
2.5"
Il.12
l.OSa
+.7
T2.3
k.29
different from control group, PcO.05
milieu with high catecholamine, high glucose and high FFA concentrations, and reduced insulin concentrations (Table 1), the rate of 3-O-methyl glucose (3-0-MG) transport was markedly reduced (PcO.05). These reductions were as follows: soleus (-52%), red gastrocnemius (-41.3%), plantaris (-41.3%) and white gastrocnemius (-32.2%) (Fig 1). Scanning densitometry of the western blots showed that the GLUT-4 transporter isoform was increased in the plasma membranes of the epinephrine-treated animals (Fig 2A; P
187,
September
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
16, 1992
COMMUNICATIONS Pages
EPINEPHRINE
ADMINISTRATION BUT REDUCES
STIMULATES
GLUCOSE
685-691
GLUT4 TRANSLOCATION
TRANSPORT
IN MUSCLE
A.Bonen*T L.A. Megeney*, SC. McCarthy?, J.C. McDermottt,
and M.H. Tan?
*Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canada N2L 3Gl TDepartments of Medicine and Biochemistry, Dalhousie University, Halifax, Canada B3H 3J5 Received
July
20,
1992
Summary: Epinephrine opposes glucose transport in muscle. Therefore, we investigated the
effects of epinephrine administration (25pg/lOOg body weight) on glucose transport and glucose transporters in rat muscle. Ninety minutes after epinephrine injection 3-O-methyl glucose transport was reduced (-47%) in perfused muscles of the rat hindlimb. Translocation of the insulinregulatable glucose transporter (GLUT4) in the epinephrine-injected animals was confirmed by the marked increments in the GLUT-4 in the plasma membranes and their concomitant reduction in the intracellular membranes. We speculate a) that it is epinephrine which translocated GLUT4 via a CAMP-linked pathway, and b) that the intrinsic activity reductions are caused either by the glycation of the transporter by the persistent hyperglycemia and/or by epinephrine via the 0 1992Academic Press, Inc. phosphorylation of the GLUT4 transporter protein in muscle.
Skeletal muscle is quantitatively the most important disposal site for glucose in viva, and entry of glucose into this tissue occurs via facilitated transport involving a carrier protein. A major mechanism by which insulin stimulates glucose transport into skeletal muscle involves the translocation of the insulin-regulatable glucose transport protein (GLUT-4) from an intracellular pool to the cell surface (13,17, 22). Increments in the intrinsic activity of the glucose transporter can also stimulate glucose transport into skeletal muscle, particularly after exercise (15,21). It has been proposed that the hormonal/metabolic milieu around a muscle may also alter glucose transport, glucose transporter number and glucose transporter intrinsic activity (8). For example, fasting (8) and streptozotocin-induced diabetes (23) provoke marked changes in a number of different substrates and glucoregulatory hormones, and along with these multiple hormonal/metabolic changes marked alterations in glucose transport, glucose transporter numbers and their intrinsic activities are observed (8,23). Epinephrine administration also provokes marked changes in the hormonal/metabolic milieu (25), and epinephrine is an important counter-regulatory hormone to glucose transport in muscle (9,32). Whether this impediment to glucose transport is due to a reduced number of surface transport proteins or due to a reduction in their intrinsic activities is not known. Therefore, we investigated the effects of epinephrine administration in viva on glucose transport and on glucose transporters in rat skeletal muscle.
685
All
Copyright 8 1992 rights of reproduction
0006-29 I X/92 $4.00 by Academic. Press. Inc. in anp form reserved.
Vol.
187,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
MATERIALS
AND METHODS
RESEARCH
COMMUNICATIONS
Materials: All chemicals were obtained from Sigma Chemicals (StLouis), except for [3H]-3-0methylglucose, [3H]-cytochalasin B and [3H]-sorbitol which were purchased from Du Pont-New England Nuclear (Montreal). Porcine monocomponent insulin was a gift from Eli Lilly (Indianapolis). The polyclonal antibody to GLUT-4 was a kind gift from Dr. A. Klip, The Hospital for Sick Children, Toronto, Ontario, Canada. Experimental animals: Male Sprague-Dawley rats (250-300 g body weight) were housed on a 12 hour light/dark cycle with unlimited access to food and water. Animals were randomly assigned to control or epinephrine-injected groups (25ug/lOOg body weight) groups. Both groups were injected intraperitoneally and left for 90min. At that point we determined a) 3-O-methyl glucose transport in the perfused hindlimb muscles or b) glucose transporters in sarcolemmal membranes and intracellular membranes, in two separate groups of animals. Glucose, insulin, free-fatty acids (FFA), epinephrine and nor-epinephrine (5, 25) concentrations were also determined in plasma samples obtained 90 min after epinephrine administration, from animals in b). Skeletal muscle glycogen concentrations (4) were also determined 90 min after epinephrine administration in a separate group of animals. To ensure that all groups were studied at 90 minutes animals were anaesthetized (Somnotol65mg/lOOg weight) at t=70 min and surgicaly prepared for the hindlimb perfusion studies in a) or for the muscle removal for studies in b) Glucose Transport Studies in Perfused Muscles: Ninety minutes after the injections with epinephrine or control injections, perfusion of the hindlimb skeletal muscles were performed as described elsewhere in detail (26, 27).Rates of glucose transport were determined period with [3H]-3-O-methylglucose (3-O-MG) (lOpCi, 5 mM) in the soleus, plantaris, red and white gastrocnemius muscles.The extracellular space were determined as reported elsewhere (27, 16). Membrane preparation and glucose transporter determinations: The procedures described in detail by Klip et al (22) were used to isolate the skeletal muscle plasma and intracellular membranes. Membrane purification, based on 5’ nucleotidase activities, were similar to that reported in the original method (22). Surface glucose transporters in plasma membranes were determined using D-glucose protectable binding of cytochalasin B as has been described elsewhere (22)Westem blotting was performed as described by Douen et al (12). Briefly, intracellular membranes and plasma membranes were subjected to sodium dodecyl sulfatepolyacrylamide electrophoresis on 12% polyacrylamide gels and transferred to nitrocellulose filter membrane for 45 min. Incubations of nitrocellulose filters were performed using anti-GLUT-4 R 820, a polyclonal antibody. Thereafter, the membranes were washed, incubated with r*sI-labelled protein A, dried, and exposed to XAR-5 film. Autoradiographs were quantitated with appropriate software (Scan Analysis, Biosoft, Cambridge, UK) on a Macintosh LC computer. RESULTS Administration of epinephrine (i.p.; 25ug/lOO g body weight) to fed rats provoked changes in circulating substrates and hormones (Table 1). Ninety minutes after epinephrine injection glucose and FFA concentrations were 208% and 180% of the control concentrations, respectively, and epinephrine and nor-epinephrine levels were 2313% and 337% of the control concentrations, repectively. However, the insulin level at t=90 min was reduced to 58% of the control concentration. In addition, epinephrine administration also reduced muscle glycogen depots to 53% (soleus), 45% (plantaris), 46% (red gastrocnemius), and 36.7% (white gastrocnemius) of the control concentrations. Glucose transport rates are known to differ among perfused rat hindlimb muscles (soleus, plantaris and red and white gastrocnemius) (27,30) that are comprised of widely differing fiber types (5). This was also observed in this study (Fig l), although as in previous studies such differences are less pronounced in the absence of insulin (27,30). After 90 min of being exposed to a metabolic
Vol.
187,
TABLE
No.
BIOCHEMICAL
2, 1992
1. Effects of epinepbrine
(25@100
glvcagealymol/e
BIOPHYSICAL
RESEARCH
(E) and norepinephrine
(NE)
wet weieht) red
COMMUNICATIONS
g body weight, i.p.) on muscle glycogen, and on plasma glucose, FFA, insulin,
epinephrine Muwle
AND
Circulatine
white
glucose
FFA
Tr
concentrations insulin
m
control
epinephrine injected
21.5
24.1
24.4
25.6
184.6
k1.7
k2.2
k2.8
k3.1
f5.2
14.6a
10.ga
11.3a
9.4a
384.6a
0.453
k9.4
k19.5
kO.07
f1.5
“epinephrine
group significantly
k2.3
k2.0
E
ml
0.25
4.3
kO.03
k.6
ml
NE ne/ml
0.48
0.32
k.19
k.07
2.5"
Il.12
l.OSa
+.7
T2.3
k.29
different from control group, PcO.05
milieu with high catecholamine, high glucose and high FFA concentrations, and reduced insulin concentrations (Table 1), the rate of 3-O-methyl glucose (3-0-MG) transport was markedly reduced (PcO.05). These reductions were as follows: soleus (-52%), red gastrocnemius (-41.3%), plantaris (-41.3%) and white gastrocnemius (-32.2%) (Fig 1). Scanning densitometry of the western blots showed that the GLUT-4 transporter isoform was increased in the plasma membranes of the epinephrine-treated animals (Fig 2A; P
