From the $University of Melbourne, Department of Medicine, Royal Melbourne Hospital and the Wommonwealth Scientific and. Industrial Research Organization ...
Vol. 267, No. 10, Ieaue of April 5, pp. 7021-7025,1992 Printed in U.S. A .
THEJOURNALOF BIOLOGICAL CHEMISTRY
0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.
Cyclic AMP Acutely Stimulates Translocation of the Major Insulinregulatable Glucose Transporter GLUT4* (Received for publication, November 4,1991)
Ashraf S. M. KeladaSO, S. Lance Macaulayn, and Joseph ProiettoSII From the $Universityof Melbourne, Department of Medicine, Royal Melbourne Hospital and the WommonwealthScientific and Industrial Research Organization, Divisionof Bwmolecular Engineering, Parkville, Victoria3050, Australia
Facilitatedglucosetransportacrossplasma mem- although at a much reduced concentration compared to branes is mediated by a family of transporters GLUT4 (2). (GLUT1-GLUTS) that have different tissue distribuIsoproterenol, a ,&adrenergic agonist, has been shown to tions and K,,, values for transport. It has been shown modulate glucose transport in rat adipocytes (3-7). It stimuthat insulin stimulates glucose transport in fat and lates glucose transport at low concentrations (10 nM), but muscle tissues by causing the redistribution of one of inhibits transportat higher concentrations (1000nM) (3). The these proteins(GLUT4)frominsidethe cell tothe mechanism for the stimulation of transport is not known. plasma membrane. Previous studies have shown that However, incubation of adipocytes with high concentrations agents that change CAMPlevels are able to modulate of isoproterenol causes phosphorylation of a serine residue on glucose transport in fat cells. The aim of this study was the C-terminal end of GLUT4 and inhibitsglucose transport to investigate the mechanisms responsible for modu- without decreasing the transporternumber (8).This suggests lation of glucose transport by CAMP.2-Deoxyglucose that phosphorylation decreases the intrinsic activity of the transport and insulin-regulatable glucose transporter glucose transporter molecule. Since isoproterenol is a &ago(GLUT4) immunoreactivity in plasma and low density nist, this modulation of glucose transport is probably CAMPmicrosomal membranes were measured in adipocytes mediated. Indeed, it has been shown in rat adipocytes that incubated for 30 min with insulin or dibutyryl-CAMP a high concentration (5 mM)of dibutyryl-cyclicAMP (Bt2cAMP).Low concentrations of Bt2cAMP (10 PM) (Bt2cAMP) inhibits both basal and maximally insulin-stimincreased2-deoxyglucoseuptakebytranslocating ulated glucose transport rates (3). GLUT4 from low density microsomal membranes to The aim of this study was to investigate the mechanism of the plasma membranes. BtzcAMP at 1000 PM inhibited glucose transporter modulation by CAMP. glucose transport below basal but further increased translocation of GLUT4. The effect ofBtzcAMPon EXPERIMENTALPROCEDURES translocation was additive to thatof 7 nM insulin. We Materials concludethatinratadipocytes, Bt2cAMP acutely translocates GLUT4 but inhibits its activity to transBradford protein assay reagent, acrylamide and Dowex 2-X8,200port glucose. 400 mesh (Cl- form) were purchased from Bio-Rad. Adenosine, aden-
The oxidation of glucose is a major source of energy for mammalian cells. Transport of glucose into thecell is accomplished by membrane-associated carrier proteins that bind and transfer it across the plasma membrane lipid bilayer. Glucose carriers or transporters are expressed in most if not all mammalian cells. Five functional glucose transporter isoforms have been identified which have distinct tissue distributions and biochemical properties (1). Adipocytes predominantly contain the GLUT4 isoform which is responsible for most, if not all, of the insulin-stimulated uptakeof glucose in these tissues (2). This action of insulin is accomplished via a redistribution of GLUT4 transporters from low density microsomes to theplasma membrane, although the exact mechanism which regulates thisredistribution is still unclear. Adipocytes are also known to express the GLUT1 isoform * This project was supported by a program grant from the National Health andMedical Research Council of Australia and from a grantin-aid from the Diabetes Australia Research Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. 5 This work fulfills portions of the Ph.D. thesis requirements. (1 Wellcome Australia Senior Research Fellow.
osine monophosphate (AMP), Bt*cAMP, 8-bromo-CAMP,bovine insulin, bovine serum albumin (radioimmunoassay grade), pepstatin A, phenylmethylsulfonyl fluoride, N-acetylglucosamine, and UDP-galactose were purchased from Sigma. Dinonyl phthalate was purchased from BDH Chemicals (Kilsyth, Victoria, Australia). Nitrocellulose was purchased from Schleicher and Schuell (Dassel, Federal Republic of Germany). Leupeptin was purchased from Boehringer Mannheim (GmbH, F.R.G.). Beckman Ready-SOLV EP scintillant was purchased from Beckman Instruments Inc. (Galway, Ireland). [2-3H] Adenosine monophosphate, uridine diphospho-D-[U-“Clgalactose, and 2-deoxy-~-[U-’~C]glucose were purchased from Amersham International (Buckinghamshire, England). Collagenase (Type 5) was purchased from Worthington Biochemical. lZ5I-Protein A was a generous gift from the Walter and Eliza Hall Institute, Parkville, Australia.
Animals Male Sprague-Dawley rats (150-200 g) were purchased from Monash University Animal Facility (Clayton, Australia) and werefed standard laboratory chow (Barastoc Products, Pakenham,Australia).
Methods Rat Epididymal Adipocytes-Adipocytes were isolated by the collagenase digestion procedure of Rodbell (9) in modified Krebs-Ringer bicarbonate/Hepes’ buffer, pH 7.4 (containing 1.15 mM Ca2+, 3% bovine serum albumin, and 5 mM glucose). The number of cells in each preparation was determined using a hemocytometer. Following digestion, cells were distributed into 20-ml polypropylene vials, 6 ml/
’The abbreviations used are: Hepes, 4-(2-hydroxyethyl)-l-piperazineethansulfonic acid; LDM, low density microsomal membrane.
7021
7022
Acute BtzcAMP-stirnuluted GLUT4 Translocation
vial, approximately 1-2 X lo6 cells/ml (in Krebs-Ringer bicarbonate/ Hepes buffer containing 3% bovine serum albumin, 0.1 mM glucose, and 200 nM adenosine) in an atmosphere of carbogen (5% COz and 95% 02)with shaking (100 rpm), a t 37 "C. Incubations were initiated by the addition of agonists to vials incubated in triplicate for 30 min unless indicated otherwise. After this time, 400-p1 aliquots were removed from each vial for determination of glucose transport rate. The remainder of the cells from each triplicate incubation were pooled, washed, and fractionated to yield plasma and microsomal membrane fractions as described below. Glucose Transport-2-Deoxyglucose uptake was used as the index of glucose transport and was measured as in Ref. 10. Following the 30-min exposure to agonists, the assay was initiated in the 400-pl at 0.25 aliquots by the addition of 0.1 mM 2-deoxy-~-[U-'~C]glucose pCi/tube andincubated for a further 5min. The assay was terminated by spinning aportion of the cells through dinonyl phthalate. Antibody Preparation-A peptide comprising the last 12 amino acids of the C-terminal region of GLUT4 from the sequence published by James etal. (11)was synthesized and conjugated to keyhole limpet hemocyanin by Dr. B. Kemp, Institute of Medical Research, St. Vincent's Hospital, Melbourne. The conjugated peptide was used to immunize a rabbit once each month. Serum was tested for specific reactivity to membranes containing GLUT4 (fat cells) by immunoblotting 10 days after each immunization (12, 13) using lZ5I-Protein A detection as described below. After the eighth injection, a specific antiserum (R1159)was obtained that recognized GLUT4. The GLUT4 antibody detected a 46-kDa molecular mass protein in membranes from adipocytes and muscle tissues but not liver, erythrocytes, or brain. The level of this 46-kDa protein increased in the plasma membrane fraction in parallel with a decrease in its level in the low density microsomal fraction (consistent with translocation of the protein) following treatment of fat cells with insulin (Fig. 3). Fractionation of Membrane-Plasma and microsomal membranes were prepared from the incubated cells by differential ultracentrifugation as described by Simpson et al. (14)and Smith et al. (15), and modified as follows. Incubated cells were washed twice in buffer containing the appropriate corresponding agonists and homogenized (14 strokes at setting 7, equivalent to 1400 rpm), on a Janke and Kunkel homogenizer (Staufen, F.R.G.), in a buffer containing 20mM Tris, 1mM EDTA, and 0.25 M sucrose, pH 7.4. All steps were carried out at 4 "C using this buffer. The homogenate was centrifuged in a The infranatant (fatBeckman JA-20 rotor for 2 min at 6000 X gmaX. free cell homogenate) was withdrawn from under the fat layer and centrifuged for 15 min at 31,000 X g,,,. The supernatantwas decanted and saved for preparation of the microsomal membrane fractions. The pellet was resuspended and centrifuged at 2400 rpm (in anLKB, Bromma, 2161 Midispin R) for 10 min, and the supernatantobtained was centrifuged at 31,000 X g,,, for 20 min. The pellet was resuspended, and the plasma membrane fraction was obtained by centriffor 60 min on a 32% sucrose cushion. The ugation at 95,000 X gmaX plasma membrane fraction was washed once by resuspension in buffer for 20 min. The plasma membrane and centrifugation at 33,000 X gmaX pellet was resuspended in buffer containinga protease inhibitor mixture (400 p~ phenylmethylsulfonyl fluoride, 4 pM bacitracin, 10 pg/ml pepstatin A, and 10 pg/ml leupeptin). The low density microsomal membrane fraction was obtained from the initial supernatant first by centrifugation at 65,000 X g, for 15 min, yielding a pellet of high density microsomal membranes, and thenby recentrifugation of the 65,000 X gmaX supernatant at210,000 X g,,, for 75 min. The latter pellet, the low density microsomal membrane fraction, was resuspended in buffer containing the protease inhibitor mixture. All samples were stored at -70 "C. Protein Determination-Protein was determined by the Coomassie Brilliant Blue method described by Bradford (16) using crystalline bovine serum albumin as a standard anddye reagent supplied by BioRad. 5'-Nucleotidase Assay--5'-Nucleotidase was used as the plasma membrane marker and was assayed by the method described by Avruch and Wallach (17). UDP-Galactose:N-AcetylglucosamineGalactosyltransferase-UDPga1actose:N-acetylglucosaminegalactosyltransferase was used as the low density microsomal membrane marker and was assayed by modification of the method described by Fleischer (18). Twenty pl of membrane suspension (20-100 pg of protein) was incubated with 20 pl of 0.2 M sodium cacodylate, pH 6.5, 3 pl of 1 M MnC12,3 p1 of 1 M mercaptoethanol, 5 pl of 10% (v/v) Triton X-100, 3 p1 of 1 M Nacetylglucosamine, and 15 p1 of 10 mM UDP-galactose containing UDP-["C]galactose (1pCi/gmol). The incubation was carried out for
60 min at 37 "C and terminated by adding 17 p1 of 0.3 M EDTA, pH 7.4. The reaction mixture was passed through a column of Dowex 2X8, 200-400 mesh in the C1- form and washed with 2 ml of water. All of the eluate was collected in a scintillation vial and mixed with 5 ml of scintillant. For each sample, a control tube was run, in which all ingredients were present except N-acetylglucosamine. This value represented galactose released. The difference between the tubes in which acceptor was present or absent represented the calculated transferase activity. Three experiments were carried out tocharacterize the membranes and validate the fractionation procedure. The specific association of 5'-1iucleotidase enzyme activity with the adipocyte plasma membrane has been established by Stanley and Luzio (19). According to Suzuki and Ron0 (20), UDP-ga1actose:N-acetylglucosaminegalactosyltransferase, a marker enzyme characteristic of membranes of the Golgi apparatus, ismost enriched in the low density microsomal membrane fraction.TableIdemonstrates that there was aconsistent 20% contamination of 5'-nucleotidase in the LDM membrane fraction and an approximate 20% contamination of galactosyltransferase activity in the plasma membrane fraction. This extent of contamination is within acceptable limits for this procedure (14, 15, 21, 22). Table I1 shows that therecovery of 5'-nucleotidase in the plasma membrane fraction was not altered by treatment with either insulin or Bt2cAMP. Electrophoretic and Zmmunoblotting Analysis-Proteins in the plasma and low density microsomal membrane fractions were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 10% gels using the system of Laemmli (23). The proteins were electrpphoretically transferred to nitrocellulose sheets overnight at 0.15 A and immunoblotted using a modification of the methods described by Towbin et al. (12) and Burnette (13). The nitrocellulose sheets were blocked with 5% skim milk and incubated for 1 h with the polyclonal antibody to GLUT4 described above (R1159). The sheets were then incubated with '"I-Protein A for 1 h. Autoradiography was used to detect immunolabeled bands which were quantitated afterslicing by counting in ay counter. Statistical Analysis Statistical significance was determined using Student's t test for paired data. RESULTS
Effects of BtzcAMP on 2-Deoxyglucose Transport-As expected, insulinstimulated2-deoxyglucosetransport above basalin a concentration-dependentmannerwith a 100% increase at 0.07 nM ( p < 0.001) and a 230% increase at 7 nM ( p < 0.001) (Fig. 1).The relatively modest fold stimulation with insulin is partly explained by the addition of 200 nM TABLEI 5'-Nucleotidase and UDP-galactose:N-acetylglucosamine galactosyltransferase activities expressed as a percentage of their respective highest specific activity observed within each preparation Each value is the average - f S.E. of three separate membrane preparations. PM, plasma membranes. Fraction
5"Nucleotidase Galactosyltransferase (mean f S.E.) (mean f S.E.) %
Homogenate PM LDM
20.2 & 3.8 20.4 f 5.6 100
21.5 f 2.1 100 19.8 +- 2.3
TABLE I1 5'-Nucleotidase activities expressed as a percentage of their respective highest specific activity observed within each preparation Each value is the average f S.E. of two separate membrane preparations. PM, plasma membranes. I
Fraction
Basal (mean k S.E.)
7.0 nM Insulin (mean f S.E.)
Homogenate PM LDM
16.9 f 8.3 100 30.4 f 1.7
10.4 f 1.4 100 25.7 f 0.8
1000 p~ Bt,cAMP (mean k S.E.)
%
16.5 f 5.4 100 30.2 f 2.1
Bt2cAMP-stimulated Translocation Acute GLUT4
7023 TABLE111 2-Deoxyglucose transport in isolated rat adipocytes and fold stimulation of GLUT4 immunoreactivity membranes of in plasma fat cells incubated with insulin or a combinationof insulin and IO p M RtncAMP Adipocytes were isolated and incubated with either no additions (basal) or the agonists indicated for 30 min in the presence of 200 nM adenosine. An aliquot was taken to measure 2-deoxyglucose uptake as described under “Experimental Procedures,” and the remainder of the cells were fractionated to yield membranes. plasma The plasma membranes were subjected to SDS-PAGE followed by protein transfer to nitrocellulose and immunoblot analysis using antibody R1159 as described under “Experimental Procedures.” There was no statistically significantdifference between 2-deoxyglucosetransport or fold stimulation of transporter number between insulin alone and insulin plus 10 p M Bt2cAMP.
1200
1000 800 600 400
200
Lla3aI
INS
INS
0.07
7.0 nM
nM
B12cAMP B12cAMP 10 CM
loo0 pM
GLUT4
FIG.1. 2-Deoxyglucose uptake in response to insulin and BtZcAMP. Adipocytes were isolated and incubated with either no additions (basal) or the agonists indicated, for 30 min in the presence of 200 nM adenosine. An aliquot was taken tomeasure 2-deoxyglucose transport as described under “Experimental Procedures.” Data are mean +- S.E. of n = 11-15 experiments (*, p < 0.05; **, p < 0.001 versus basal).
1200
2-Deoxyglucose transport ( n = 5)
(-fold stimulation) ( n = 4)
pmol/1O6 cells/
min
Basal 0.07 nM insulin 0.07 nM insulin + 10 p M BtzcAMP 7.0 nM insulin 7.0 nM insulin 10 p M BtZcAMP
+
T
immunoreactivity
257 f 40 472 f 78 587 f 136
2.40 f 0.59 3.44 & 1.27
939 f 101 826 f 103
4.46 f 1.45 5.78 ? 1.87
1
PM 46-
LDM
”
B a s aIlN S
INS
INS
INS
0.07
0.07
7.0
7.0
nM
nM
nM
nM
+
+
1000
BIZCAMP 1000
PM
PM
BIZCAMP
4t
Basal
INS cAMP INS 7.0 nM nM 0.07
10
pM
cAMP 1000 pM
FIG.3. Autoradiograph of plasma and low density micro-
the combination of 0.07 or 7 nM insulin and 1000 p~ BtZcAMP. Adipocytes were isolated and incubated with either no additions (basal) or the agonists indicated, for 30 min in thepresence of 200 nM adenosine. An aliquot was taken tomeasure 2-deoxyglucose transport as described under “Experimental Procedures.” Data are mean f S.E. of n = 11-13 experiments (*, p < 0.05 versus 0.07 nM insulin ( I N S ) ;* *, p < 0.001 versus 7 nM insulin).
somal membranes showing translocation of GLUT4 in response to insulin and Bt2cAMP. Adipocytes were treated as described in Fig. l, an aliquot was taken to measure 2-deoxyglucose transport, and the remainder of the cells were fractionated to yield plasma membranes and low density microsomal membranes. These were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by protein transfer to nitrocellulose and immunoblot analysis using antibodyR1159 as described under “Experimental Procedures.”
adenosine to the incubation medium. This nucleotide lowers endogenous cAMP levels and has been shown to increase glucose transport (24), thus reducing the fold stimulation above basal. A t a concentrationof 10 p ~ BtzcAMP , increased 8-deoxyglucose transport, but the effect was modest (39% above basal, p < 0.05, Fig. 1).In contrast, 1000 p~ BtzcAMP inhibited 2-deoxyglucosetransport below basal ( p < 0.05, Fig. 1).1000 p~ BtzcAMP significantly inhibited insulin-stimulated 8-deoxyglucose uptake at both 0.07 ( p < 0.05) and 7 nM ( p < 0.001) insulin (Fig. 2). The stimulatory effect of 10 p~ Bt2cAMP on 2-deoxyglucose transport was not additive to that of either concentration of insulin (Table 111). Effects of Bt2cAMP on GLUT4 Distribution-The major finding of the present study is that BtzcAMP, like insulin, causes the acute translocationof GLUT4 from the low density
microsomal membranes to the plasma membrane. Fig. 3 shows an autoradiograph from one experiment demonstrating the Bt2cAMP and insulin-mediated increase in transporter protein in theplasma membranes and a corresponding decrease in the LDM membrane fraction suggesting translocation of GLUT4 in response to acute exposure to each of these compounds. Fig. 4 shows the pooled data from 9-10 experiments. Bt2cAMPcaused a dose-dependent increase in GLUT4 on the plasma membrane. At 10 p~ Bt2cAMP, the fold stimulation was modest (1.5-fold; p = 0.06 versus basal), but correlated with the modest but significant increase of 2-deoxyglucose transport at this BtzcAMP concentration (Fig. 1).BtcAMP a t 1000 p~ and 0.07 nM insulin induced a similar degree of translocation. However, 2-deoxyglucose transport in cells incubated with 1000 p~ Bt2cAMP was markedly reduced com-
FIG.2. 2-Deoxyglucose uptake in response to insulin and
Acute Bt2cAMP-stimulatedGLUT4 Translocation
7024
0.07
7.0
10
nM
nM
VM
1000 pM
FIG. 4. Mean f S.E. fold stimulation of GLUT4 in plasma membranes in response to insulin and Bt2cAMP. The experiment was conducted as described in the legend to Fig. 3 and under “Experimental Procedures.” Data represent means & S.E. of n = 910 experiments (* *, p < 0.01 versus basal).
”
Basal
INS 0.07
nM
INS 0.07 nM nM
7.0
nM
+
Bt2cAMP
Bt2cAMP 1000 PM
1000
FIG. 6. Mean f S.E. fold stimulation of GLUT4 in plasma membranes in response to insulin and the combination of insulin and Bt2cAMP. Data representmeans & S.E. of n = 7 experiments (#, p < 0.05 vers’sus 0.07 nM insulin ( I N S ) ;+, p < 0.05 versws 7 nM insulin).
PM
LDM
Ih’S
7.0
+
PM
46-
INS
.
was inhibited by 8-bromo-cAMP, but thisoccurred at a higher concentration (10 mM).
46-
DISCUSSION Basal
INS
INS
0.07
0.077.0 nM
nM
PM
+
cAMP loo0 PM
INS 7.0 nM
INS nM
+
cAMP loo0
FIG. 5. Autoradiograph of plasma and low density microsomal membranes showing the additive effect of insulin (INS) and BtzcAMPon translocation of GLUT4 from the low density microsomal membrane to the plasma membrane. The experiment was performed as described in the legend to Fig. 2 and under “Experimental Procedures.”
pared to 0.07 nM insulin ( p < 0.001, Fig. l ) , suggesting that Bt2cAMP causes a decrease inthe intrinsicactivity of GLUT4. The effects of 1000 PM Bt2cAMP on GLUT4 translocation were additive to that of insulin, both in the appearance of GLUT4 in the plasma membranes and the disappearance in the low density microsomal membrane fraction (Fig. 5). Fig. 6 shows the average fold stimulation from seven experiments. 1000 PM BtzcAMP was additive to both 0.07 nM ( p < 0.05) and 7.0 nM ( p < 0.05) insulin. Although the trendwas similar, translocation in response to 10PM Bt2cAMPwas not additive to that induced by insulin at either concentration (Table111). Stimulation of 2-deoxyglucose transport and GLUT4 translocation was maximal with 7.0 nM insulin (2-deoxyglucose transport: 1038 & 45 uersus 1096 +. 20 pmol/106 cells/min and GLUT4 translocation: 2.9 f 0.6 uersus 2.7 f 0.5-fold for 7.0 and 70.0 nM insulin, respectively, n = 3). To exclude the possibility that theeffects of Bt,cAMP were unique to this compound, three experiments were performed with 8-bromo-CAMP.1000 PM 8-bromo-CAMPcaused a 2.6 & 0.6-fold stimulation of GLUT4 transport above basal ( n= 3) which is similar to the fold stimulation induced by 1000 PM Bt2cAMP (Fig. 4). Like Bt2cAMP, 2-deoxyglucose transport
The principal finding of this study is that Bt2cAMPinduces the acute translocation of GLUT4 transporters from the low density microsomal fraction to the plasma membrane in rat adipocytes. The effect of 1000 p~ Bt,cAMP is of a similar magnitude to that of 0.07 nM insulin, but considerably less thanthe maximal insulin-induced translocation. Furthermore, BtzcAMP-inducedtranslocation is additive to that caused by maximal insulin-stimulatedtranslocation. The mechanism by which Bt,cAMP causes translocation of GLUT4 is unclear. It is likely that theprocess of translocation involves the phosphorylation of proteins relatedto movement of intracellular organelles rather thanphosphorylation of the glucose transporter protein itself. We have shown here that Bt2cAMP and maximal insulin-induced translocation are additive, suggesting that insulin and cAMP act via different mechanisms. Recently, Slot et al. (25) using immunolocalization techniques showed that in rat brown adipose tissues 99% of GLUT4 is found intracellularly in the basal state and that insulin caused the redistribution of GLUT4 to thecell surface, so that during insulin stimulation, 40% of GLUT4 is found on the plasma membrane. They also reported that in the presence of insulin, there was an increase of GLUT4 labeling in the coated pits, coated vesicles, and small vacuoles suggesting that, like cell surface receptors, GLUT4 is actively recycled through early endosomes in these cells. These authors proposed that in the basal state, GLUT4 could be continuously excluded from the plasma membrane by endocytosis. This raises the possibility that insulin could increase the number of glucose transporters on the plasma membrane by either inhibiting endocytosis or stimulating exocytosis. On the basis that insulin increased GLUT4in the endocytic pathway, they concluded that insulin acts mainly by stimulating exocytosis (25). Because BtzcAMP and insulin-stimulated translocation may have different mechanisms, it is possible that Bt2cAMP could cause the redistribution of GLUT4
Acute Bt2cAMP-stimulated GLUT4 Translocation by inhibiting endocytosis rather than stimulating exocytosis. Alternatively, maximal insulin stimulation may not saturate the capacity for exocytosis of GLUT4 or theremay be different pools of GLUT4 within the cell that respond to different agonists. Unlike insulin, the stimulatory “insulin-like” action of 1000 PM Bt2cAMP on GLUT4 translocation is accompanied by a simultaneous inhibition of glucose transport, suggesting that Bt2cAMP decreases the intrinsic activity of GLUT4. This latter effect is thought to be due to theability of BhcAMP to phosphorylate GLUT4 by activation of CAMP-dependentprotein kinase. Lawrence et al. (8) have shown that addition of Bt2cAMP leads to phosphorylation of a serine residue in the C-terminal region of the protein (serine 488) and have postulated that this phosphorylation might result in decreased intrinsic activity of the transporter. In thepresent study, low concentrations (10 PM) of Bt2cAMP stimulated glucose transport. This increase in glucose transport was associated with a modest increase in GLUT4 protein in the plasma membrane fraction. Thus, at low concentrations, the effect of Bt2cAMP on stimulation of GLUT4 translocation is not accompanied by inhibition of intrinsic activity which apparently requires higher levels of the nucleotide. The effects of Bt2cAMP on stimulation of GLUT4 translocation and inhibition of glucose transport at a high concentration may explain the contradictory literature onthe effects of the &agonist isoproterenol on glucose transport in rat adipocytes. Isoproterenol has been reported to both stimulate and inhibit glucose transport in rat adipocytes (3, 26, 27). Furthermore, it has been shown that catecholamines inhibit glucose transport in the presence of adenosine deaminase, but not in the presence of adenosine (5). Adenosine, which is released into the medium by fat cells, activates the Gi regulatory protein of adenylate cyclase resulting in a decrease in cAMP levels. In thepresence of adenosine deaminase, which metabolizes adenosine, thereare higher concentrations of cAMP both basally and following stimulation with P-agonists (7). Therefore, it is possible that, in the presence of adenosine, low concentrations of isoproterenol stimulate glucose transport by causing a modest increase in cAMP levels which we show in this study stimulate translocation of GLUT4. At higher isoproterenol concentrationsor in the presence of adenosine deaminase, the cAMP levels achieved are higher, resulting in adecrease in intrinsic activity of the transporters, thus inhibiting transport. The experiments described in this paper were all performed in thepresence of excess adenosine (200 nM) in order to lower endogenous cAMP levels. It has been shown that both GLUTl and GLUT4 transportersarepresent in adipocytes with thelatter isoform constituting approximately 95% of the total transporterpopulation (2,28). Following insulin stimulation, both transporters are translocated, but GLUT4 is the major transporter species responsible for glucose transport (2). In the present study, GLUTl immunoreactivity was not measured. In chronic experiments in 3T3-Ll adipocytes, Clancy and Czech (29) have shown no increase of GLUTl inplasma membranes after a 4-h incubation with 1000 FM Bt2cAMP. After an 18-h incubation, there was an increase in GLUTl in plasma membranes without a concomitant decrease in the LDM fraction suggesting the stimulation of new transporter synthesis. In
7025
contrast, translocation of GLUT4 was found at both time points. The acute effects of Bt2cAMP on GLUTl distribution remain to be investigated. In conclusion, we have shown that, in rat adipocytes, BtzcAMP causes a dose-dependent acute translocation of GLUT4 and confirm that at high concentration this effect is modulated by an inhibition of the intrinsic activity of the transporter to transportglucose. Acknowledgments-We are grateful to Dr. Bruce Kemp of the Institute of Medical Research, St. Vincent’s Hospital, Melbourne, for synthesizing and conjugating the 12-amino acid peptide from the Cterminal end of the GLUT4 protein. We wish to thank Wanvick Atkinson for his excellent technical assistance. REFERENCES 1. Bell, G. I., Kayano, T., Buse, J. B., Burant, C. F., Takeda, J., Lin, D., Fukumoto, H., and Seino, S. (1990) Diabetes Cure 13,198208 2. Zorzano, A., Wilkinson, W., Kotliar, N., Thoidis, G., Wadzinkski, B. E., Ruoho, A. E., and Pilch, P. F. (1989) J. Biol. Chem. 264, 12358-12363 3. Kashiwagi, A., Huecksteadt, T. P., and Foley, J. E. (1983) J. Biol. Chem. 258,13685-13692 4. Kirsch, D. M., Baumgarten, M., Deufel, T., Rinninger, F., Kemmler, W., and Haring, H. U. (1983) Biochern. J. 216,737745 5. Green, A. (1983) FEBS Lett. 1 5 2 , 261-264 6. Joost. H. G., and Goke. R. (1984) FEBS Lett. 167.5-9 7. Joost; H. G:, Goke, R.’, and Ste’infelder, H. J. (1985) Biochem. Pharmacol. 34,649-653 8. Lawrence, J. C., Hiken, J. F., and James, D. E. (1990) J . Biol. Chem. 265,2324-2332 9. Rodbell, M. (1964) J. Biol. Chern. 239, 375-380 10. Olefsky, J. M. (1978) Biochem. J. 172, 137-145 11. James, D. E., Strube, M., and Mueckler, M. (1989) Nature 3 3 8 , 83-87 12. Towbin, H., Staehelin, T., and Gordon, J . (1979) Proc. Natl. Acud. Sci. U. S. A. 76,4350-4354 13. Burnette, W. N. (1981) Anal. Biochem. 1 1 2 , 195-203 14. Simpson, I. A., Yver, D. R., Hissin, P. J., Wardzala, L. J., Karnieli, E., Salans,L. B., and Cushman, S. W. (1983) Biochirn. Biophys. Acta 763,393-407 15. Smith, M.M., Robinson, F.W., Watanabe, T., and Kono, T. (1984) Biochirn. Biophys. Acta 7 7 5 , 121-128 16. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 17. Avruch, J., and Wallach, D. F. H.(1971) Biochim. Biophys. Acta 233,344-347 18. Fleischer, B. (1974) Methods Enzyrnol. 2 9 , 180-191 19. Stanley, K. K., and Luzio, J. P. (1979) Biochem. SOC.Trans, 7 , 361-362 20. Suzuki, K., and Kono, T. (1980) Proc. Nutl. Acud. Sci. U. S. A. 77,2542-2545 21. Guerre-Millo, M., Lavau, M., Horne, J . S., and Wardzala, L. J . (1985) J. Biol. Chem. 2 6 0 , 2197-2201 22. Kono, T., Suzuki, K., Dansey, L. E., Robinson, F. W.,and Blevins, T. L. (1981) J. Biol. Chern. 2 5 6 , 6400-6407 23. Laemmli, U.K. (1970) Nature 227,680-685 24. Taylor, W.M., and Halperin, M.L. (1979) Biochern. J. 178, 381-389 25. Slot, J. W., Geuze, H. J., Gigengack, S., Lienhard, G. E., and James, D. E. (1991) J . Cell Biol. 1 1 3 . 123-135 26. Smith, U., Kuroda, M., and Simpson, I:A. (1984) J. Biol. Chern. 259,8758-8763 27. Ludvigsen, C., Jarett, L., and McDonald, J. M. (1980) Endocrinology 106, 786-790 28. Oka, Y., Asano, T., Shibasaki, Y., Kasuga, M., Kanazawa, Y., and Takaku, F. (1988) J. Biol. Chem. 263,13432-13439 29. Clancy, B. M., and Czech, M. P. (1990) J. Biol. Chern. 265, 12434-12443
