Biochimica et Biophysica Acta, 1094(1991) 217-223
2.I,7
© 1991ElsevierScience PublishersB.V. All rights reserved0167-4889/91/$03.511 ADONIS 0167488991002343
Insulin-like, action o f catecholamines and Ca 2÷ to stimulate
glucose transport and GLUT4 translocation in perfused rat heart S t e p h e n R a t t i g a n , G e o f f r e y J. A p p l e b y a n d Michael G. Clark Department of Biochemistry. Unit'ersity of Tasmania. Hobart (Australia)
(Received 30 January 19911 (Revisedmanuscriptreceived4 June lOOt) Key words: Catecholamine;Calciumion; Glucosetransport:Insulin;GLUT4;(Rat heart) The uptake of 2-deoxyglucose by per'fused rat hearts was compared to the distribution of the insulin-regulatable glucose transporter (GLUT4) in membrane preparations from the same hearts. The hearts were treated with the a-adrenergic combination of epinephrine + propranolol, the/].adrenergic agonist isoproterenol, high (8 raM) Ca z" concentrations, insulin and the alpha adrenergic combination or insulin alone. Epinephrine (1 pM) + propranolol (10/zM), isoproterenol (10 /~M), high Ca z+, insulin (1 /zM) + epinephrine (! IzM) + propranolol (10 /zM) and insulin (1 /zM) each led to an increase in 2-deoxyglucose uptake and a shift in the recovery of the GLUT4 from a high-speed pellet membrane fraction (putatively intracellular) to a low-speed pellet membrane fr~ction (putatively sarcolemmal). There were significant correlations (r = -0.673, P < 0.001) between the stimulation of 2.deoxyglucose uptake and the loss of GLUT4 from the intraceiiuiar membr,me fraction, or tile inc-.~ase in the sarcolemmal fraction. The data provide evidence that the GLUT4 is translocated by agents that stimulate glucose transport in heart, and therefore this mechanism is not restricted tc ~ ~ulin.
Introduction Glucose transport by perfused rat heart is regulated by insulin and catecholamines [1-6]. The catecholamine regulation is mediated by a Ca2+-dependent process for both or- and ~-adrenergic mechanisms [46]. The mechanism of increase in glucose transport appears to be similar to other insulin-sensitive tissues such as adipocytes [7] and skeletal muscle [8,9] and involves the translocation of glucose transporter protein from an intracellular pool to the sarcolemma. Evidence for this has come from studies by Watanabe et al. [10] who observed that glucose transport activiW reconstituted into vesicles was increased in sarcolemreal vesicles after insulin treatment of rat heart. Also increased glucose transporters in sarcolemmal preparations from insulin-treated hearts have been observed by ,.--'ytochalasin B binding [6,11,12]. Furthermore, cateehoiamine or high Ca 2+ perfusion of rat heart also leads to increased amounts of glucose transporter protein present in sarcolemmal preparations [6].
Recently it has been reported that in insulin-sensitive tissues a unique form of glucose transporter protein (GLUT4) exists and it is this protein that is translocated in response to insalin stimulation [13]. Molecular cloning of GLUT4 and analysis of mRNA from a number of tissues has shown that GLUT4 appears to be predominantly expressed in skeletal muscle, white and brown adipose tissue and heart [14]. The aim of the present study was to investigate whether the insnlin-regulatable glucose transporter (GLUT4) was also translocated by agents, other than insulin, that increase glucose transport in the perfused rat heart. Experimental procedures Animal~
Male rats (230-270 g body weight) of the HoodedWistar strain, maintained ad libitum on a standard laboratory chow diet [15] were used for these experiments. Materials
Correspondence: M,G. Clark, Departmentof Biochemistry,Universityof Tasmania, GPO Box 2.52C,Hobart, 7001, Australia.
DL-Propranolol hydrochloride, L-epinephrine bitartrate and L-isoproterenoI-HCI were obtained from
218 Sigma Chemical Co (St. Louis, MO). 2-Deoxy-D-[13H]glucose and [U-14C]sucrose were obtained from Amersham Australia Pty Ltd. Porcine insulin (Actrapid MC, I00 units/ml) was obtained from Novo Industri A/S.
Heart perfusions Three hearts per membrane preparation were perfused in the Langendorff manner using a system based on that of Williamson [16]. The perfused buffer was Krebs-Henseleit bicarbonate buffer containing 0.05 mM EDTA and 5 mM glucose (unless indicated otherwise) and the perfusion temperature was 37 °C. The hearts were preperfused at a constant pressure of 60 mmHg for 10 min in a non-recirculating manner prior to commencing the experiment. To determine 2-deoxyglucose uptake the three hearts were then perfused with 75 ml of buffer containing 0.5 mM 2-deoxy-o-[1all]glucose (0.5 p.Ci), 0.5 mM [U-J4C]sucrose (0.25 /.LCi), no glucose and the agonist. The concentration of CaCI 2 was usually 1.27 raM, but where indicated was 8 raM. A sample of the perfusate was taken at the end of the perfusion (15 rain) and the aH/m4C ratio was determined. The hearts were removed from the apparatus, blotted dry, trimmed free of vessels and fat, weighed and then used for membrane preparation.
Heart membrane preparation Membrane preparations from the hearts were prepared essentially as described by Watanabe et al. [10]. The hearts were minced and homogenized in 45 ml of ice-cold buffer A (0.25 M sucrose, 10 mM Tris-HCl, pH 7.5) for 30 s with a Silverson homogenizer. The homogenate was filtered through two layers of gauze and further homogenized in a Dounce tissue grinder (8 strokes). A 0.5 ml aliquot of this homogenate was taken for 2-deoxy-D-[1-SH]glucose uptake determination, while the rest was centrifuged in a Sorval SS-34 rotor (4500 × g for 10 min at 2°C). The supernatant (S1) was stored on ice for further processing (see below). The pellet was suspended in 30 ml of buffer A, homogenized with the Silverson and Dounce homogenizers as before, and centrifuged again for 10 min at 4500 × g. The pellet was discarded, while the supernatant ($3) and the previously saved supernatant (S1) were separately centrifuged for 30 min at 23 500 X g. The supernatant ($3) that was obtained from fraction 82 was discarded, while the supernatant ($4) from fraction ~,~ was stored on ice for further processing (see below). The two pellets obtained from the above centrifugation were combined and suspended in 2 ml of ice-cold buffer A. The suspension was placed on top of a linear sucrose density gradient that was prepared by the method of Luthe [17] in a centrifuge tube for a Beckman SW 25.1 rotor. The sucrose concentration in the gradient was 20.0% (w/w) at the top and 40%
(w/w) at the bottom;the sucrose was buffered with 10 mM Tris-HCl (pH 7.5). The density gradient centrifugation was carried out for 180 min at 65000×g, average. After the centrifugation, the sucrose solution was collected from the top and divided into eight fractions (4.0 ml each). The fractions were diluted with 14 ml of 10 mM Tris-HCl (pH 7.5) and centrifuged in a Beckman 70 Ti rotor (150000 × g for 60 min at 2°C). The supernatant was discarded while the pellet (Watanabe et al. [10]: plasma membrane-rich fraction) was retained. Fraction $4 left in ice at an earlier step was also centrifuged for 60 rain at 150000×g. The resultant supernatant was discarded while the pellet (Watanabe et al. [10]: 1)6 fraction, high-speed pellet fraction) was retained. The high-speed pellet fraction and the sucrose density fractions (5 to 8) were suspended in 1 ml of 10 mM Tris-HCi (pH 7.5) and the sucrose density fractions 1-4 were suspended in 0.5 ml of 10 mM Tris-HC! (pH 7.5). The fractions were stored frozen at - 2 0 ° C until used for determination of marker enzymes or glucose transporter.
2-Deoxyglucose uptake determination The (0.5 ml) aliquot of homogenate was centrifuged for 15 min at 8000×g at 2°C. The pellet was discarded and the supernatant was counted for radioactivity. The 2-deoxyglucose uptake was calculated from the following equation 2-deoxyglueose uptake (nmol/min per g) = (3Hdpra in hearts - (14Cdpm in hearts x3Hdp,~/14Cdp m ratio in peffm~te)) / ( w e t wt. (g) of hearts × spec. radioact, of 2-deoxy-o-[ 1- all]glucose ( d p m / n m o l ) )
(1)
Glucose transporter quantitation Membrane fractions (150/~1) were solubilized in an equal volume of 60 mM Tris-HCi (pH 6.8), 2% SDS, 10% glycerol, 0.02% bromophenol blue, 2.5% mercaptoethanol. The fractions (sucrose density fractions 3-8 and high-speed pellet, 10 #1) were then subjected to SDS-PAGE on a 10% gel using the system of Laemmli [18]. The proteins were electrophoretically transferred to Millipore lmmobilon PVDF membrane. The membrane was then blocked with milk protein, and incubated with rabbit polyclonai serum to the C-terminus of the insulin regulatable glucose iransporter at a dilution of 1 : 500. The membrane was then incubated with 1:100 dilution of I~I-labelled goat anti-rabbit IgG antisera (Dupont, New England Nu-
219 TABLE 1 2-Oeoxy.o-[I-JHJglucose uptake by per]used rat heart Three hearts per experiment were perfused as described in the experimental section. The values shown are means+S.E, for the number of experimentsindicatedin brackets. Statisticaldifferences were determinedby an unpairedStudent's t-test. Treatment None I /zM epb~ephrine + l0 p,I~ propranolol I0/zM isoproterenol 8mMCa z+ I p,M insulin+ I p,M epinephrine + 10/zM prooranolol 1 p,M insulin
2-Deoxy-D-[I-3H]glucoseuptake (nmol/min per g wet wt3 26.3±6.3 (5) 98.9+ 12.2 * * (4) 56.1± 6.9 * (4) 84.4+8.6 ** (4) 127.5+16.1** (4) 121.8± 19.8 ** (5)
* P < 0.02 sigm(icantlydifferent from no treatment. ** P < 0.001 significantlydifferent from no treatment. clear). Radiolabeiled bands were visualized by autoradiography using Hyperfilm-MP and the 43 kDa band was cut from the membrane and counted for radioactivity. The labelling of the GLUT4 band was found to be proportional over a protein range of 0-100 ~ g (data not shown). However, labelling varied with each blot and thus the labelling of each fraction per mg of protein was expressed as a percentage of the total labelling on each membrane.
Marker enzyme assays Total proteins were measured after TCA precipitation by the method of Lowry c t a l . [19] and expressed as nag of protein per fraction. 5'-Nucleotidase was the marker used for plasma membranes and was determined by the method of Avruch and Wallach [20] and expressed as nmol AMP hydrolysed per min per fraction. UDP-Gal: N-acetylglucosamine galactosyltransferase was assayed by the method of Fleischer and Fleischer [21] as described by Kono et al. [22] and expressed as nmol UDP-Gal hydrolysed per rain per fraction. Results
The data on 2-deoxy-D-[1-3H]glucose uptake and membrane distribution of GLUT4 was obtained from each group of three perfused hearts and, therefore, allowed an assessment of the correlation between glucose transport and translocation of GLLIT4 within the same preparation.
2-Deoxyglucose uptake Hearts were perfused with six treatments, which represented situations of differing glucose transport rate. Hearts were perfused with no additions at normal
(1.25 mM) Ca 2. concentrations to represen~ basal glucose transport rates. For stimulated glucose transport states, hearts were perfused wit~t 1 ~ M epinephrine + 10 /.LM propranolol (ot-agonist combination), 10 /.LM isoproterenol (/3-agonist), 8 mM Ca2+,l/xM insulin + 1 # M epinephrine + 10 p,M propranolol or 1 # M insulin. The results of the 2-deoxy-D-[l-3H]glucose uptake for these perfusions are shown in Table 1. All treatments significantly increased the uptake of 2-deoxyglucose into the hearts. The greatest increase occurred with the combination of a-agnnist and insulin and represented a 4.8-fold increase over basal values. Although the alpha combination of epinephrine plus propranohi increased the uptake 3.8-fold and insulin 4.6-told, there was no additive effect when insulin, epinephrine and propranolol was used (4.8-fold).
Marker enzymes The protein content, 5'-nucleotidase and galactosyltransferase activity for the membrane preparations from the basal perfused hearts are shown in Table II. There were no significant differences in the recovery of protein, 5'-nucleotidase and galaetyosyl transferase activity for the membrane preparations from epinephrine + propranoloL isoproterenoi, high Ca 2+, epinephrine + propranolol + insulin or insulin perfused hearts compared to the basal hearts (data not shown). The enrichment of 5'-nucleotidase and galactosyl transferase showed a similar pattern for the two enzymes with the major enrichment occurring in the first three sucrose density gradient fractions and the P6 fraction. Watanabe et al. [10] using a similar membrane preparation also observed that the enrichment patterns of 5'-nucleot.;dase and galactosyitransferase in the sucrose density gradient were superimposed. However, their gradient was fractionated from the bottom and they observed the greater enrichment in the second and third fraction which would correspond to the sixth and seventh fraction in this study. The differences observed in the sucrose density gradient fractions between the two studies are probably related to the different rotors and centrifugntion conditions used. Glucose transporter The distribution of the insulin-regulatable glucose transporter within the sucrose gradient and P6 fraction was assessed by SDS-PAGE and Western blotting and detection using a rabbit pobjelonal serum to the Co terminus and tZSl-labeiled goat anti-rabbit IgG. Only sucrose gradient fractions SG3 to SG8 were used as the protein content of fractions SGI and SG2 was too low to obtain reliable values. The results for the b a ~ l and stimulated hearts are shown in Table lit. Significant differences between basal and all stimulated hemts were only found for the P6 fraction and the total of the sucrose density gradient fractions.
220 TABLE II
Protein, 5'.naeleotidase and galactosyhransferase activity of homogenate arid membrane fractions from basal perfnsed hearts (no addition, n = 5) Membranes were prepared from three perfuscd hearts per experiment as de~ribcd in the experimental section. The protein, 5'-nucleotidase activity and galactosyl transferase activities were determined in the whole homogenate of the hearts (HEM), the sucrose density gradient fractions ( S G I - 8 ) and the high-speed pellet fraction (P6). The values shown are means 4- S.E. for five experiments. Also shown is the distribution and enrichment compared to the whole homogenate.
HeM Protein (rag/fraction) S.E. Distribution (%) 5'-Nucleotidase (nmol/min per fraction) S.E. Distribution (%) Enrichment Galactosylt rans ferase (omol/min per fraction) S.E. Distribution (%) Enrichment
SGI
SG2
SG3
SG4
SG5
SG6
SG7
SG8
P6
323 17 100
0.07 0.02 0.02
0.16 0.05 0.05
0.34 0.08 0.l l
0.52 0.09 0.16
1.02 0.08 0.32
2.54 0.42 0.79
4.12 0.57 1.28
1.38 0.47 0.43
4.99 0.42 1.54
4215 224 100 1.0
II 3 0.26 12.0
49 tO 1.16 23.5
67 l0 1.59 15.1
68 lI 1.61 10.0
119 15 2.82 8.9
139 14 3.30 4.2
126 18 2.99 23
47 15 1.12 2.6
295 43 7.00 4,5
21.6 1.7 100 1.0
0.16 0.07 0.74 34.2
0.50 0.14 2.31 46.7
0.63 0.11 2.92 27.7
0.76 0.12 3.52 21.9
1.41 0.22 6.53 20.7
1.39 0.27 6.44 8.2
1.26 0.28 5.83 4.6
0.78 0.32 3.61 8.5
1.65 0.21 7.64 4.9
'Significantly less ( P < 0 . 0 5 ) GLUT4 was found in the SG3 fraction of basal hearts compared to the insulinstimulated hearts. There were no significant differences observed between the other sucrose density frac-
tions. However, a trend suggesting translocation of GLUT4 to the SG3 fraction was evident. These changes in the GLUT4 labelling of the membrane fraction appeared to be related to the stimula-
TABLE III
GLUT4 content of heart membrane fractions Membranes were prepared from control, epinephrine+propranolol, isoproterenol, high Ca 2+, epinephrine+propanolol+insulin, and insulin perfused hearts as described in the experimental section. G L U T 4 content was determined by Western blotting tzSI-labelled second antibody as described in the experimental section. The values shown are means+ S.E. for the number of perfusions as indicated. Statistical differences were determined by an unpaired Student's t-test. Treatment
Sucrose density gradients fraction 3
4
5
6
7
8
Total 3-8
P6 fraction
None (n = 5)
34.9 :t: 6.0
22.2 :l: 3.3
7.2 1.1
7.0 2.7
5.9 1.6
6.6 2.8
83.7 + 0.9
16.3 :[:2.1
Epinephrine + propranolol (n = 4)
50.4 ± 7.4
25.6 6.1
6.8 1.2
4.0 1.0
2.8 0.2
0.3 0.3
89.8 * 4-1.8
I0.1 * 4-1.8
Isoproterenul (n = 4)
47.4 ± 7. 8
24.7 4.0
4.7 0.8
4.7 1.3
5.2 1.1
2.5 1.7
89.3 * 4-1.8
10.7 * 4-1.8
High Ca z+ (n = 4)
41.5 :t=8.8
23.7 5.1
4.6 0.2
7.2 4.2
9.1 5.8
4.5 2.3
90.6 ** 4-1.9
9.4 ** 4-1.9
Insulin + epinenhrine + prol3;anolol (n = 4)
47.9 + 11.2
24.3 5.3
4.7 1.0
5.6 2.2
4.7 2.4
5.8 2.7
93.0 * ** 4-1.5
7.0 *** 4-1.5
Insulin (n=5)
54.5 * :1:2.0
21.4 4.0
6.0 0.5
3.9 l.l
3.2 3.0 .. !:0.. .... 1.5
92.1 *** 4-1.2
7.9 *** 4-1.2
(,
< 0.05; ** P < 0.01;
** P
< 0.001 significantlydifferentfrom no treatment.
221 r : *0.673 P < 0.001
and was the same as we ha~e previously observed [5]. The concentration of stimulatory agents was sufficient to produce maximal responses for each agent [5]. The effects of maximal doses of alpha adrenergic agents and insulin are additive when glucose uptake and lactate output arc measured in the perfused heart [5]. However, the !ack of additivity on 2-deoxvglucose uptake suggests that glucose transport is fully stimulated by insulin and thus transport is not limiting glucose metabolism in this situation. This is further supported by the findings with the distribution of the GLUT4 in the membrane fractions (discussed below).
• e
E t-
""
10C
50 o
®
~o
Membrane preparation
g
~o
~ P = - 0.673 P < 0.001
o -o
15c
oJ
5(:
• •
o°
~
o
,~
o, is
•i,
1=5I c o u n t s
o
2'o
in m e m b r a n e f r a c t i o n
Fig. I. Correlation between G L U T 4 in the sucrose density gradient fractions (top panel) and the P6 fraction V (bottom panel) and
2-deoxyglucnseuptake. The percentagedistributionof GLUT4 on Western blots of membrane preparationsfrom perfused hearts and the 2-deo~glucoseuptakefrom the same hearts were determinedas described in experimentalprocedures.Heartswere perfusedwith no additions(o). I /~M epinephrine+ l0 ~M propranolol(e), l0 ~M isopmterenol (B), 8 mM Ca2+ (A), I ,ttM iosulil.~-I ttM epinephrine+10 ;tM pmpranolol(v) or l ~M insulin(O). Each valuerepresentsa singleperfusion. tion of 2-deoxyglucose uptake as -- significant (P < 0 , ~ 1 ) negative correlatio.-, was observed between GLUT4 labelling in P6 fraction and 2-deoxyglucose uptake and ~ s i t i v e correlation with the total of the sucrose density gradient fractions and 2-deoxyglucose uptake (Fig. 1). Discussion
2-Deoxyglucose uptake The stimulation of 2-deoxyglucose uptake (Table 1) by epinephrine + propranolol, isoprotcrenol, high Ca 2+, insulin + epinephrine + propranolol and insulin
Obtaining purified membrane preparations from rat heart is a very difficult process and a number of different techniques have been attempted [10,11,2225]. In this study the method described by Watanabe et al. [10] was used as they had demonstrated the translocation of glucose transport (by recnnstitution methods) in response to insulin and Wheeler [24] had used the method to show the ~.ranslocation of glucose transporters (by cytochalasin B binding) in response to anoxia in the heart. This method consists of differential centrifugation and sucrose density centrifugation. The characterization of the membrane fractions obtained have been ext~:nsively discussed by Watanabe et al. [10]. The sucrose density fractions appear to be enriched with T-tubules and sarcolemma, while the high-speed pellet fraction (P6) is enriched with low-density membranes and golgi apparatus [10}. However, it is not possible to quantitate the cross-contamination as there is no suitable enzyme marker for the recruitable membrane pool of glucose transporters [I0,28], and it is now apparent that many marker enzymes are also translocated [29,30].
Glucose transporter Stimulation of 2-deoxyglucose uptake in the rat heart was associated with alterations in the distribution of the GLUT4 in the ntembrane fractions. There was a signific~.ni decrease in the recovery from the high-speed pellet (P6) and increases in the sucrose density gradient fractions (Table liD. This parallels the changes observed by Watanabe et al. [10] for reconstituted glucose transport activity and is similar to changes in cytochalasin B binding in response to insulin [6,1i,12] and anoxia [24]. The decrease of GLUT4 from the P6 fraction and increase in the composite of the sucrose density gradient fractions was significantlycorrelated to the change: in the 2-deoxyglucose uptake (Fig. 1). This suggests that the increase in glucose transport is a result of the translocation of the GLUT4. However, because of the large cross-contamination of the membrane preparations it is not possible to quantitate the changes. Thus,
222 it is unknown whether the translocation can fully account for the changes in 2-deoxyglucose uptake or an intrinsic change in the activity of the transporter has also occurred. It is apparent from these results that stimulation with a-adrenergic or /3-adrenergic agents, or alterations in Ca 2+ concentration in the perfusate, lead to ~imilar changes in GLUT4 recovery in membrane preparations as perfusion with insulin. Similar findings have been observed for the effect of insulin and contraction (exercise) in skeletal muscle [31]. Contraction of skeletal musc!e causes similar changes as a-adrenergic agents or the perfusion of high Ca z+ in the heart. They result in increased glucose uptake [4,9,32] and increased cytoplasmic Ca 2+. In contrast the effect of fl-adrenergic agents in the heart are different from those that occur in the other tissues that contain the GLUT4. In adipocytes, /3-adrenergic agents have no effect on the transloeation of the G L U T 4 [33]. Also fl-adrenergic agents inhibit the insulin effects on glucose transport without affecting the insulin-induced translocation [34]. Recently it has been reported that /3-adrenergic agents cause the phosphorylation of the GLUT4 and this may account for the inhibition of glucose transport caused by these agents in adipose and skeletal muscle tissue [33]. The process in the heart is clearly different as /3-adrenergic stimulation leads to both a increase in glucose transport (Table I) and the translocation of the GLUT4 (Fig. 1 and Table liD. The reason for this is not dear, but may relate to the fact that /3-adrenergic stimulation in the heart mediates its effect on glucose metabolism by increasing intracellular Ca z+ [4-6] or because of the increased metabolic demand by the increased contractility. It is unknown whether/3-adrenergic stimulation in the heart leads to phosphorylation of GLUT4 in the heart. It is interesting to note that the combination of insulin and a-adrenergic stimulation, which are additive on glucose uptake and lactate output in the heart [5], did not result in further transloeation of GLUT4 than that observed for insulin. This could be interpreted as indicating a modification of the intrinsic activity of the transporter as has been suggested in skeletal muscle [28,29,35,36]. However, in the heart there was also no additivity of insulin and a-adrenergic stimulation on 2-deoxyglucose uptake. Thus the results suggest that glucose transport is fully stimulated by insulin and becomes no longer rate-limiting for other agents that further stimulate glucose metabolism in the heart [5]. In conclusion, these results demonstrate that agents that increase glucose transport in the rat heart, such as a-adrenergic agonists, /3-adrenergie agonists ¢,r high Ca 2+ peffusions have a similar mechanism of action as insulin. They all cause a redistribution of the insulinregulatable glucose transporter from a high-speed pel-
let membrane fraction to a low-speed pellet membrane fractiou. Acknowledgements We are grateful to Dr. David James, Washington University Medical School, St. Louis, for providing us with the antibody to the insulin-regulatable glucose transporter and for helpful suggestions. W e are also grateful to Mr. M. Glancy for his technical assistance. This work was supported by grants from the National Health and Medical Research Council.
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223 28 Hirshman, M.F., Goodyear, L.J., Wardzala, L.J., Horton, E.D. and Horton, E.S. (1990) J Biol. Chem. 265, 987-991. 29 Omatsu-Kanbe, M. and Kitasato, H. (1990) Biochem. J. 272, 727-733. 30 Klip, A., Ramlal, T., Douen, A.G., Burdctt, E., Young, D., Cartee, G.D. and Hollosz~, J.O. (1988) FEBS Lett. 238, 419-423. 31 Douen A.G., Ramlal, T., Cartee, G.D. and Klip, A. (1990) FEBS Left. 261, 256-260. 32 Richter, E ~. (1984) Acta Physiol. Scand. Suppl. 528, 1-42.
33 James, D.E., Hiken, J. and Lawrence, J.C. ~,1989) Proc. Natl. Acad. Sci. USA 86, 8368-8372. 34 Cushman, S.W. and Wardzala, L.J. (1980) J. Biol. Chem. 255, 4758-4762. 35 Sternlicht, E., Barnard, RJ. and Grimditch, G.K. (1988) Am. J. Physiol. 254. E633-E638. 36 Stemlicht, E., Bamard, R.J. and Grimditch, G.IL (1989) Am. J. Physiol. 256, E227--E230.
2.I,7
© 1991ElsevierScience PublishersB.V. All rights reserved0167-4889/91/$03.511 ADONIS 0167488991002343
Insulin-like, action o f catecholamines and Ca 2÷ to stimulate
glucose transport and GLUT4 translocation in perfused rat heart S t e p h e n R a t t i g a n , G e o f f r e y J. A p p l e b y a n d Michael G. Clark Department of Biochemistry. Unit'ersity of Tasmania. Hobart (Australia)
(Received 30 January 19911 (Revisedmanuscriptreceived4 June lOOt) Key words: Catecholamine;Calciumion; Glucosetransport:Insulin;GLUT4;(Rat heart) The uptake of 2-deoxyglucose by per'fused rat hearts was compared to the distribution of the insulin-regulatable glucose transporter (GLUT4) in membrane preparations from the same hearts. The hearts were treated with the a-adrenergic combination of epinephrine + propranolol, the/].adrenergic agonist isoproterenol, high (8 raM) Ca z" concentrations, insulin and the alpha adrenergic combination or insulin alone. Epinephrine (1 pM) + propranolol (10/zM), isoproterenol (10 /~M), high Ca z+, insulin (1 /zM) + epinephrine (! IzM) + propranolol (10 /zM) and insulin (1 /zM) each led to an increase in 2-deoxyglucose uptake and a shift in the recovery of the GLUT4 from a high-speed pellet membrane fraction (putatively intracellular) to a low-speed pellet membrane fr~ction (putatively sarcolemmal). There were significant correlations (r = -0.673, P < 0.001) between the stimulation of 2.deoxyglucose uptake and the loss of GLUT4 from the intraceiiuiar membr,me fraction, or tile inc-.~ase in the sarcolemmal fraction. The data provide evidence that the GLUT4 is translocated by agents that stimulate glucose transport in heart, and therefore this mechanism is not restricted tc ~ ~ulin.
Introduction Glucose transport by perfused rat heart is regulated by insulin and catecholamines [1-6]. The catecholamine regulation is mediated by a Ca2+-dependent process for both or- and ~-adrenergic mechanisms [46]. The mechanism of increase in glucose transport appears to be similar to other insulin-sensitive tissues such as adipocytes [7] and skeletal muscle [8,9] and involves the translocation of glucose transporter protein from an intracellular pool to the sarcolemma. Evidence for this has come from studies by Watanabe et al. [10] who observed that glucose transport activiW reconstituted into vesicles was increased in sarcolemreal vesicles after insulin treatment of rat heart. Also increased glucose transporters in sarcolemmal preparations from insulin-treated hearts have been observed by ,.--'ytochalasin B binding [6,11,12]. Furthermore, cateehoiamine or high Ca 2+ perfusion of rat heart also leads to increased amounts of glucose transporter protein present in sarcolemmal preparations [6].
Recently it has been reported that in insulin-sensitive tissues a unique form of glucose transporter protein (GLUT4) exists and it is this protein that is translocated in response to insalin stimulation [13]. Molecular cloning of GLUT4 and analysis of mRNA from a number of tissues has shown that GLUT4 appears to be predominantly expressed in skeletal muscle, white and brown adipose tissue and heart [14]. The aim of the present study was to investigate whether the insnlin-regulatable glucose transporter (GLUT4) was also translocated by agents, other than insulin, that increase glucose transport in the perfused rat heart. Experimental procedures Animal~
Male rats (230-270 g body weight) of the HoodedWistar strain, maintained ad libitum on a standard laboratory chow diet [15] were used for these experiments. Materials
Correspondence: M,G. Clark, Departmentof Biochemistry,Universityof Tasmania, GPO Box 2.52C,Hobart, 7001, Australia.
DL-Propranolol hydrochloride, L-epinephrine bitartrate and L-isoproterenoI-HCI were obtained from
218 Sigma Chemical Co (St. Louis, MO). 2-Deoxy-D-[13H]glucose and [U-14C]sucrose were obtained from Amersham Australia Pty Ltd. Porcine insulin (Actrapid MC, I00 units/ml) was obtained from Novo Industri A/S.
Heart perfusions Three hearts per membrane preparation were perfused in the Langendorff manner using a system based on that of Williamson [16]. The perfused buffer was Krebs-Henseleit bicarbonate buffer containing 0.05 mM EDTA and 5 mM glucose (unless indicated otherwise) and the perfusion temperature was 37 °C. The hearts were preperfused at a constant pressure of 60 mmHg for 10 min in a non-recirculating manner prior to commencing the experiment. To determine 2-deoxyglucose uptake the three hearts were then perfused with 75 ml of buffer containing 0.5 mM 2-deoxy-o-[1all]glucose (0.5 p.Ci), 0.5 mM [U-J4C]sucrose (0.25 /.LCi), no glucose and the agonist. The concentration of CaCI 2 was usually 1.27 raM, but where indicated was 8 raM. A sample of the perfusate was taken at the end of the perfusion (15 rain) and the aH/m4C ratio was determined. The hearts were removed from the apparatus, blotted dry, trimmed free of vessels and fat, weighed and then used for membrane preparation.
Heart membrane preparation Membrane preparations from the hearts were prepared essentially as described by Watanabe et al. [10]. The hearts were minced and homogenized in 45 ml of ice-cold buffer A (0.25 M sucrose, 10 mM Tris-HCl, pH 7.5) for 30 s with a Silverson homogenizer. The homogenate was filtered through two layers of gauze and further homogenized in a Dounce tissue grinder (8 strokes). A 0.5 ml aliquot of this homogenate was taken for 2-deoxy-D-[1-SH]glucose uptake determination, while the rest was centrifuged in a Sorval SS-34 rotor (4500 × g for 10 min at 2°C). The supernatant (S1) was stored on ice for further processing (see below). The pellet was suspended in 30 ml of buffer A, homogenized with the Silverson and Dounce homogenizers as before, and centrifuged again for 10 min at 4500 × g. The pellet was discarded, while the supernatant ($3) and the previously saved supernatant (S1) were separately centrifuged for 30 min at 23 500 X g. The supernatant ($3) that was obtained from fraction 82 was discarded, while the supernatant ($4) from fraction ~,~ was stored on ice for further processing (see below). The two pellets obtained from the above centrifugation were combined and suspended in 2 ml of ice-cold buffer A. The suspension was placed on top of a linear sucrose density gradient that was prepared by the method of Luthe [17] in a centrifuge tube for a Beckman SW 25.1 rotor. The sucrose concentration in the gradient was 20.0% (w/w) at the top and 40%
(w/w) at the bottom;the sucrose was buffered with 10 mM Tris-HCl (pH 7.5). The density gradient centrifugation was carried out for 180 min at 65000×g, average. After the centrifugation, the sucrose solution was collected from the top and divided into eight fractions (4.0 ml each). The fractions were diluted with 14 ml of 10 mM Tris-HCl (pH 7.5) and centrifuged in a Beckman 70 Ti rotor (150000 × g for 60 min at 2°C). The supernatant was discarded while the pellet (Watanabe et al. [10]: plasma membrane-rich fraction) was retained. Fraction $4 left in ice at an earlier step was also centrifuged for 60 rain at 150000×g. The resultant supernatant was discarded while the pellet (Watanabe et al. [10]: 1)6 fraction, high-speed pellet fraction) was retained. The high-speed pellet fraction and the sucrose density fractions (5 to 8) were suspended in 1 ml of 10 mM Tris-HCi (pH 7.5) and the sucrose density fractions 1-4 were suspended in 0.5 ml of 10 mM Tris-HC! (pH 7.5). The fractions were stored frozen at - 2 0 ° C until used for determination of marker enzymes or glucose transporter.
2-Deoxyglucose uptake determination The (0.5 ml) aliquot of homogenate was centrifuged for 15 min at 8000×g at 2°C. The pellet was discarded and the supernatant was counted for radioactivity. The 2-deoxyglucose uptake was calculated from the following equation 2-deoxyglueose uptake (nmol/min per g) = (3Hdpra in hearts - (14Cdpm in hearts x3Hdp,~/14Cdp m ratio in peffm~te)) / ( w e t wt. (g) of hearts × spec. radioact, of 2-deoxy-o-[ 1- all]glucose ( d p m / n m o l ) )
(1)
Glucose transporter quantitation Membrane fractions (150/~1) were solubilized in an equal volume of 60 mM Tris-HCi (pH 6.8), 2% SDS, 10% glycerol, 0.02% bromophenol blue, 2.5% mercaptoethanol. The fractions (sucrose density fractions 3-8 and high-speed pellet, 10 #1) were then subjected to SDS-PAGE on a 10% gel using the system of Laemmli [18]. The proteins were electrophoretically transferred to Millipore lmmobilon PVDF membrane. The membrane was then blocked with milk protein, and incubated with rabbit polyclonai serum to the C-terminus of the insulin regulatable glucose iransporter at a dilution of 1 : 500. The membrane was then incubated with 1:100 dilution of I~I-labelled goat anti-rabbit IgG antisera (Dupont, New England Nu-
219 TABLE 1 2-Oeoxy.o-[I-JHJglucose uptake by per]used rat heart Three hearts per experiment were perfused as described in the experimental section. The values shown are means+S.E, for the number of experimentsindicatedin brackets. Statisticaldifferences were determinedby an unpairedStudent's t-test. Treatment None I /zM epb~ephrine + l0 p,I~ propranolol I0/zM isoproterenol 8mMCa z+ I p,M insulin+ I p,M epinephrine + 10/zM prooranolol 1 p,M insulin
2-Deoxy-D-[I-3H]glucoseuptake (nmol/min per g wet wt3 26.3±6.3 (5) 98.9+ 12.2 * * (4) 56.1± 6.9 * (4) 84.4+8.6 ** (4) 127.5+16.1** (4) 121.8± 19.8 ** (5)
* P < 0.02 sigm(icantlydifferent from no treatment. ** P < 0.001 significantlydifferent from no treatment. clear). Radiolabeiled bands were visualized by autoradiography using Hyperfilm-MP and the 43 kDa band was cut from the membrane and counted for radioactivity. The labelling of the GLUT4 band was found to be proportional over a protein range of 0-100 ~ g (data not shown). However, labelling varied with each blot and thus the labelling of each fraction per mg of protein was expressed as a percentage of the total labelling on each membrane.
Marker enzyme assays Total proteins were measured after TCA precipitation by the method of Lowry c t a l . [19] and expressed as nag of protein per fraction. 5'-Nucleotidase was the marker used for plasma membranes and was determined by the method of Avruch and Wallach [20] and expressed as nmol AMP hydrolysed per min per fraction. UDP-Gal: N-acetylglucosamine galactosyltransferase was assayed by the method of Fleischer and Fleischer [21] as described by Kono et al. [22] and expressed as nmol UDP-Gal hydrolysed per rain per fraction. Results
The data on 2-deoxy-D-[1-3H]glucose uptake and membrane distribution of GLUT4 was obtained from each group of three perfused hearts and, therefore, allowed an assessment of the correlation between glucose transport and translocation of GLLIT4 within the same preparation.
2-Deoxyglucose uptake Hearts were perfused with six treatments, which represented situations of differing glucose transport rate. Hearts were perfused with no additions at normal
(1.25 mM) Ca 2. concentrations to represen~ basal glucose transport rates. For stimulated glucose transport states, hearts were perfused wit~t 1 ~ M epinephrine + 10 /.LM propranolol (ot-agonist combination), 10 /.LM isoproterenol (/3-agonist), 8 mM Ca2+,l/xM insulin + 1 # M epinephrine + 10 p,M propranolol or 1 # M insulin. The results of the 2-deoxy-D-[l-3H]glucose uptake for these perfusions are shown in Table 1. All treatments significantly increased the uptake of 2-deoxyglucose into the hearts. The greatest increase occurred with the combination of a-agnnist and insulin and represented a 4.8-fold increase over basal values. Although the alpha combination of epinephrine plus propranohi increased the uptake 3.8-fold and insulin 4.6-told, there was no additive effect when insulin, epinephrine and propranolol was used (4.8-fold).
Marker enzymes The protein content, 5'-nucleotidase and galactosyltransferase activity for the membrane preparations from the basal perfused hearts are shown in Table II. There were no significant differences in the recovery of protein, 5'-nucleotidase and galaetyosyl transferase activity for the membrane preparations from epinephrine + propranoloL isoproterenoi, high Ca 2+, epinephrine + propranolol + insulin or insulin perfused hearts compared to the basal hearts (data not shown). The enrichment of 5'-nucleotidase and galactosyl transferase showed a similar pattern for the two enzymes with the major enrichment occurring in the first three sucrose density gradient fractions and the P6 fraction. Watanabe et al. [10] using a similar membrane preparation also observed that the enrichment patterns of 5'-nucleot.;dase and galactosyitransferase in the sucrose density gradient were superimposed. However, their gradient was fractionated from the bottom and they observed the greater enrichment in the second and third fraction which would correspond to the sixth and seventh fraction in this study. The differences observed in the sucrose density gradient fractions between the two studies are probably related to the different rotors and centrifugntion conditions used. Glucose transporter The distribution of the insulin-regulatable glucose transporter within the sucrose gradient and P6 fraction was assessed by SDS-PAGE and Western blotting and detection using a rabbit pobjelonal serum to the Co terminus and tZSl-labeiled goat anti-rabbit IgG. Only sucrose gradient fractions SG3 to SG8 were used as the protein content of fractions SGI and SG2 was too low to obtain reliable values. The results for the b a ~ l and stimulated hearts are shown in Table lit. Significant differences between basal and all stimulated hemts were only found for the P6 fraction and the total of the sucrose density gradient fractions.
220 TABLE II
Protein, 5'.naeleotidase and galactosyhransferase activity of homogenate arid membrane fractions from basal perfnsed hearts (no addition, n = 5) Membranes were prepared from three perfuscd hearts per experiment as de~ribcd in the experimental section. The protein, 5'-nucleotidase activity and galactosyl transferase activities were determined in the whole homogenate of the hearts (HEM), the sucrose density gradient fractions ( S G I - 8 ) and the high-speed pellet fraction (P6). The values shown are means 4- S.E. for five experiments. Also shown is the distribution and enrichment compared to the whole homogenate.
HeM Protein (rag/fraction) S.E. Distribution (%) 5'-Nucleotidase (nmol/min per fraction) S.E. Distribution (%) Enrichment Galactosylt rans ferase (omol/min per fraction) S.E. Distribution (%) Enrichment
SGI
SG2
SG3
SG4
SG5
SG6
SG7
SG8
P6
323 17 100
0.07 0.02 0.02
0.16 0.05 0.05
0.34 0.08 0.l l
0.52 0.09 0.16
1.02 0.08 0.32
2.54 0.42 0.79
4.12 0.57 1.28
1.38 0.47 0.43
4.99 0.42 1.54
4215 224 100 1.0
II 3 0.26 12.0
49 tO 1.16 23.5
67 l0 1.59 15.1
68 lI 1.61 10.0
119 15 2.82 8.9
139 14 3.30 4.2
126 18 2.99 23
47 15 1.12 2.6
295 43 7.00 4,5
21.6 1.7 100 1.0
0.16 0.07 0.74 34.2
0.50 0.14 2.31 46.7
0.63 0.11 2.92 27.7
0.76 0.12 3.52 21.9
1.41 0.22 6.53 20.7
1.39 0.27 6.44 8.2
1.26 0.28 5.83 4.6
0.78 0.32 3.61 8.5
1.65 0.21 7.64 4.9
'Significantly less ( P < 0 . 0 5 ) GLUT4 was found in the SG3 fraction of basal hearts compared to the insulinstimulated hearts. There were no significant differences observed between the other sucrose density frac-
tions. However, a trend suggesting translocation of GLUT4 to the SG3 fraction was evident. These changes in the GLUT4 labelling of the membrane fraction appeared to be related to the stimula-
TABLE III
GLUT4 content of heart membrane fractions Membranes were prepared from control, epinephrine+propranolol, isoproterenol, high Ca 2+, epinephrine+propanolol+insulin, and insulin perfused hearts as described in the experimental section. G L U T 4 content was determined by Western blotting tzSI-labelled second antibody as described in the experimental section. The values shown are means+ S.E. for the number of perfusions as indicated. Statistical differences were determined by an unpaired Student's t-test. Treatment
Sucrose density gradients fraction 3
4
5
6
7
8
Total 3-8
P6 fraction
None (n = 5)
34.9 :t: 6.0
22.2 :l: 3.3
7.2 1.1
7.0 2.7
5.9 1.6
6.6 2.8
83.7 + 0.9
16.3 :[:2.1
Epinephrine + propranolol (n = 4)
50.4 ± 7.4
25.6 6.1
6.8 1.2
4.0 1.0
2.8 0.2
0.3 0.3
89.8 * 4-1.8
I0.1 * 4-1.8
Isoproterenul (n = 4)
47.4 ± 7. 8
24.7 4.0
4.7 0.8
4.7 1.3
5.2 1.1
2.5 1.7
89.3 * 4-1.8
10.7 * 4-1.8
High Ca z+ (n = 4)
41.5 :t=8.8
23.7 5.1
4.6 0.2
7.2 4.2
9.1 5.8
4.5 2.3
90.6 ** 4-1.9
9.4 ** 4-1.9
Insulin + epinenhrine + prol3;anolol (n = 4)
47.9 + 11.2
24.3 5.3
4.7 1.0
5.6 2.2
4.7 2.4
5.8 2.7
93.0 * ** 4-1.5
7.0 *** 4-1.5
Insulin (n=5)
54.5 * :1:2.0
21.4 4.0
6.0 0.5
3.9 l.l
3.2 3.0 .. !:0.. .... 1.5
92.1 *** 4-1.2
7.9 *** 4-1.2
(,
< 0.05; ** P < 0.01;
** P
< 0.001 significantlydifferentfrom no treatment.
221 r : *0.673 P < 0.001
and was the same as we ha~e previously observed [5]. The concentration of stimulatory agents was sufficient to produce maximal responses for each agent [5]. The effects of maximal doses of alpha adrenergic agents and insulin are additive when glucose uptake and lactate output arc measured in the perfused heart [5]. However, the !ack of additivity on 2-deoxvglucose uptake suggests that glucose transport is fully stimulated by insulin and thus transport is not limiting glucose metabolism in this situation. This is further supported by the findings with the distribution of the GLUT4 in the membrane fractions (discussed below).
• e
E t-
""
10C
50 o
®
~o
Membrane preparation
g
~o
~ P = - 0.673 P < 0.001
o -o
15c
oJ
5(:
• •
o°
~
o
,~
o, is
•i,
1=5I c o u n t s
o
2'o
in m e m b r a n e f r a c t i o n
Fig. I. Correlation between G L U T 4 in the sucrose density gradient fractions (top panel) and the P6 fraction V (bottom panel) and
2-deoxyglucnseuptake. The percentagedistributionof GLUT4 on Western blots of membrane preparationsfrom perfused hearts and the 2-deo~glucoseuptakefrom the same hearts were determinedas described in experimentalprocedures.Heartswere perfusedwith no additions(o). I /~M epinephrine+ l0 ~M propranolol(e), l0 ~M isopmterenol (B), 8 mM Ca2+ (A), I ,ttM iosulil.~-I ttM epinephrine+10 ;tM pmpranolol(v) or l ~M insulin(O). Each valuerepresentsa singleperfusion. tion of 2-deoxyglucose uptake as -- significant (P < 0 , ~ 1 ) negative correlatio.-, was observed between GLUT4 labelling in P6 fraction and 2-deoxyglucose uptake and ~ s i t i v e correlation with the total of the sucrose density gradient fractions and 2-deoxyglucose uptake (Fig. 1). Discussion
2-Deoxyglucose uptake The stimulation of 2-deoxyglucose uptake (Table 1) by epinephrine + propranolol, isoprotcrenol, high Ca 2+, insulin + epinephrine + propranolol and insulin
Obtaining purified membrane preparations from rat heart is a very difficult process and a number of different techniques have been attempted [10,11,2225]. In this study the method described by Watanabe et al. [10] was used as they had demonstrated the translocation of glucose transport (by recnnstitution methods) in response to insulin and Wheeler [24] had used the method to show the ~.ranslocation of glucose transporters (by cytochalasin B binding) in response to anoxia in the heart. This method consists of differential centrifugation and sucrose density centrifugation. The characterization of the membrane fractions obtained have been ext~:nsively discussed by Watanabe et al. [10]. The sucrose density fractions appear to be enriched with T-tubules and sarcolemma, while the high-speed pellet fraction (P6) is enriched with low-density membranes and golgi apparatus [10}. However, it is not possible to quantitate the cross-contamination as there is no suitable enzyme marker for the recruitable membrane pool of glucose transporters [I0,28], and it is now apparent that many marker enzymes are also translocated [29,30].
Glucose transporter Stimulation of 2-deoxyglucose uptake in the rat heart was associated with alterations in the distribution of the GLUT4 in the ntembrane fractions. There was a signific~.ni decrease in the recovery from the high-speed pellet (P6) and increases in the sucrose density gradient fractions (Table liD. This parallels the changes observed by Watanabe et al. [10] for reconstituted glucose transport activity and is similar to changes in cytochalasin B binding in response to insulin [6,1i,12] and anoxia [24]. The decrease of GLUT4 from the P6 fraction and increase in the composite of the sucrose density gradient fractions was significantlycorrelated to the change: in the 2-deoxyglucose uptake (Fig. 1). This suggests that the increase in glucose transport is a result of the translocation of the GLUT4. However, because of the large cross-contamination of the membrane preparations it is not possible to quantitate the changes. Thus,
222 it is unknown whether the translocation can fully account for the changes in 2-deoxyglucose uptake or an intrinsic change in the activity of the transporter has also occurred. It is apparent from these results that stimulation with a-adrenergic or /3-adrenergic agents, or alterations in Ca 2+ concentration in the perfusate, lead to ~imilar changes in GLUT4 recovery in membrane preparations as perfusion with insulin. Similar findings have been observed for the effect of insulin and contraction (exercise) in skeletal muscle [31]. Contraction of skeletal musc!e causes similar changes as a-adrenergic agents or the perfusion of high Ca z+ in the heart. They result in increased glucose uptake [4,9,32] and increased cytoplasmic Ca 2+. In contrast the effect of fl-adrenergic agents in the heart are different from those that occur in the other tissues that contain the GLUT4. In adipocytes, /3-adrenergic agents have no effect on the transloeation of the G L U T 4 [33]. Also fl-adrenergic agents inhibit the insulin effects on glucose transport without affecting the insulin-induced translocation [34]. Recently it has been reported that /3-adrenergic agents cause the phosphorylation of the GLUT4 and this may account for the inhibition of glucose transport caused by these agents in adipose and skeletal muscle tissue [33]. The process in the heart is clearly different as /3-adrenergic stimulation leads to both a increase in glucose transport (Table I) and the translocation of the GLUT4 (Fig. 1 and Table liD. The reason for this is not dear, but may relate to the fact that /3-adrenergic stimulation in the heart mediates its effect on glucose metabolism by increasing intracellular Ca z+ [4-6] or because of the increased metabolic demand by the increased contractility. It is unknown whether/3-adrenergic stimulation in the heart leads to phosphorylation of GLUT4 in the heart. It is interesting to note that the combination of insulin and a-adrenergic stimulation, which are additive on glucose uptake and lactate output in the heart [5], did not result in further transloeation of GLUT4 than that observed for insulin. This could be interpreted as indicating a modification of the intrinsic activity of the transporter as has been suggested in skeletal muscle [28,29,35,36]. However, in the heart there was also no additivity of insulin and a-adrenergic stimulation on 2-deoxyglucose uptake. Thus the results suggest that glucose transport is fully stimulated by insulin and becomes no longer rate-limiting for other agents that further stimulate glucose metabolism in the heart [5]. In conclusion, these results demonstrate that agents that increase glucose transport in the rat heart, such as a-adrenergic agonists, /3-adrenergie agonists ¢,r high Ca 2+ peffusions have a similar mechanism of action as insulin. They all cause a redistribution of the insulinregulatable glucose transporter from a high-speed pel-
let membrane fraction to a low-speed pellet membrane fractiou. Acknowledgements We are grateful to Dr. David James, Washington University Medical School, St. Louis, for providing us with the antibody to the insulin-regulatable glucose transporter and for helpful suggestions. W e are also grateful to Mr. M. Glancy for his technical assistance. This work was supported by grants from the National Health and Medical Research Council.
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