Isoproterenol stimulates phosphorylation of the insulin-regulatable ...

Proc. Nati. Acad. Sci. USA Vol. 86, pp. 8368-8372, November 1989 Biochemistry

Isoproterenol stimulates phosphorylation of the insulin-regulatable glucose transporter in rat adipocytes (insulin receptor/insulin action/catecholamines/cAMP-dependent protein kinase)

DAVID E. JAMES*, JEFFREY HIKENt,

AND

JOHN C. LAWRENCE, JR.t

Departments of *Cell Biology and Physiology and tPharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Saint Louis, MO 63110

Communicated by Stuart Kornfeld, August 10, 1989

Molecular cloning has led recently to the identification of a number of glucose transporter species among various tissues (15-18). The human erythrocyte glucose transporter was first cloned from Hep G2 cells (17). This transporter is expressed in many tissues including muscle, fat, and central nervous tissue (16, 19). However, the Hep G2-type glucose transporter is not the major insulin-sensitive glucose transporter in muscle and adipose tissue (20). These cells express a unique glucose transporter [insulin-regulatable glucose transporter (IRGT)] that undergoes insulin-dependent translocation to the cell surface (15, 20). The amino acid sequences of the IRGT and Hep G2-type glucose transporter are 65% identical (17). The most striking differences between the two transporters occur within presumed intracellular domains. In the case of the IRGT sequence, these regions contain several potential phosphorylation sites that are not present in the Hep G2-type glucose transporter (17). In the present study, we have investigated the effects of insulin and isoproterenol on the phosphorylation and subcellular distribution of IRGT.

We have examined the acute effects of insulin ABSTRACT and isoproterenol on the phosphorylation state of the insulinregulatable glucose transporter (IRGT) in rat adipocytes. The IRGT was immunoprecipitated from either detergentsolubilized whole-cell homogenates or subcellular fractions of 32P-labeled fat cells and subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The 32P-labeled IRGT was detected by autoradiography as a species ofapparent Mr 46,000. Insulin stimulated translocation of the IRGT from low-density microsomes to the plasma membrane but did not affect phosphorylation of the transporter in either fraction. Isoproterenol inhibited insulin-stimulated glucose transport by 40% but was without effect on the subcellular distribution of the transporter in either the presence or absence of insulin. Isoproterenol stimulated phosphorylation of the IRGT 2-fold. Incubating cells with dibutyryl-cAMP and 8-bromo-cAMP also stimulated phosphorylation 2-fold, and the transporter was phosphorylated in vitro when IRGT-enriched vesicles were incubated with cAMP-dependent protein kinase and [Y32P]ATP. These results suggest that isoproterenol stimulates phosphorylation of the IRGT via a cAMP-dependent pathway and that phosphorylation of the transporter may modulate its ability to transport glucose.

MATERIALS AND METHODS Isolation and Incubation of Adipocytes. Adipocytes were prepared by incubating epididymal fat pads of 190 to 210-g Sprague-Dawley rats with crude collagenase (type I, lot 46J057, Cooper Biomedical) by the method of Rodbell (21). Cells were incubated essentially as described (22). The incubation medium contained 135 mM NaCl, 5.4 mM KCl, 1.4 mM MgSO4, 1.4 mM CaC12, 0.18 mM sodium phosphate, 3% (wt/vol) bovine serum albumin (fraction V, lot 58F0581, Sigma), and 10 mM Hepes (pH 7.4). Cells were suspended (5 ml of medium per g of original adipose tissue) in medium and incubated at 37°C for 2 hr with 32p; (0.1 mCi/mi; 1 Ci = 37 GBq). This time is sufficient to achieve steady-state labeling of [y-32P]ATP (22). Cells were then incubated with insulin and other agents for 1-30 min before homogenization. Under these conditions, neither insulin nor isoproterenol affects the specific activity of [y-32P]ATP (22).

Glucose transport is the first step in the complex pathway of intracellular glucose utilization, and it is rate limiting for glucose uptake in muscle and fat (reviewed in refs. 1 and 2). Furthermore, it is a focal point for acute regulation of glucose metabolism by hormones such as insulin and epinephrine and other stimuli. Insulin has been shown to stimulate the movement of glucose transporters from an intracellular domain to the cell surface, and this appears to be a major mechanism for activation of glucose transport (3-8). However, this may not be the only mechanism for acutely regulating glucose transport. /3-Adrenergic agonists, such as epinephrine or isoproterenol, inhibit insulin-stimulated glucose transport in both muscle and adipose tissue (reviewed in refs. 9-12). That such inhibition was not accompanied by a corresponding change in the subcellular distribution of the glucose transporter, as measured by cytochalasin B binding (4), led to the conclusion that isoproterenol modifies the intrinsic ability of the carrier protein to transport glucose (10). In previous experiments in which an anti-human erythrocyte glucose transporter antibody was used to perform immunoprecipitations from 32plabeled rat (13) and 3T3-L1 (14) adipocytes, phosphorylation of the transporter was not detected either in control cells (13, 14) or in cells incubated with insulin (13, 14) or isoproterenol (13). Consequently, it has been concluded that the inhibition of transport in response to isoproterenol does not involve phosphorylation of the transporter (13).

Preparation of Cells for Immunoprecipitation. When assessing the effects of agents on total transporter phosphorylation, cells were homogenized in buffer A, which contained 100 mM NaF, 2 mM sodium pyrophosphate, 10 mM EDTA, 2 mM EGTA, and 50 mM Tris HCl (pH 7.8 at 23°C). Total membranes were pelleted by centrifugation at 150,000 x g for 90 min at 4°C. Pellets were solubilized in 100 ,ul of buffer A containing 1% sodium dodecyl sulfate (SDS) and frozen at -70°C before immunoprecipitation. Buffer A contains inhibitors of both phosphatases and kinases. To be certain that the inhibitors blocked posthomogenization changes in the phosphorylation state of the IRGT, some experiments were performed in which cells were homoge-

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Abbreviations: IRGT, insulin-regulatable glucose transporter; LDM, low-density microsomes; HDM, high-density microsomes; PM, plasma membrane; mAb, monoclonal antibody.

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Biochemistry: James et al. nized in buffer A containing 1% SDS. Inclusion of SDS ensured rapid denaturation of proteins and complete extraction of the glucose transporter. The same amounts of 32Plabeled transporter were recovered by using both methods. When subcellular fractionation was performed, adipocytes were rinsed once at 370C in incubation medium prepared without CaC12, MgSO4, or albumin (hormones and other constituents were present where appropriate). The cells were then homogenized in buffer B, which contained 25 mM NaF, 1 mM sodium pyrophosphate, 1 mM EDTA, 0.5 mM EGTA, 250 mM sucrose, and 1 mM ammonium molybdate (pH 7.4). Sodium vanadate (1 mM) was included in some experiments. Plasma membranes (PM), high-density microsomes (HDM), low-density microsomes (LDM), and mitochondria/nuclei were prepared essentially as described (23). Membrane fractions were suspended in buffer B and stored overnight at -700C. Immunoprecipitation. The protein contents of membrane samples were measured by using bicinchoninic acid (24) and adjusted to the same value. SDS-solubilized membranes (100 sul; 100-200 Ag of protein) were added to 900 Al of buffer B containing 1% Triton X-100 and 100 mM NaCl. Samples were incubated for 30 min at 22°C and then centrifuged for 5 min at 13,000 x g. Supernatants were retained for immunoprecipitation with a monoclonal antibody (mAb 1F8) previously shown to be specific for the IRGT (15). The mAb was first immobilized by incubation at 220C with goat anti-mouse IgG coupled to Sepharose beads (Cappel Laboratories; 1 ,ug of secondary antibody per 1 ,ug of mAb 1F8) suspended in buffer B containing 2% bovine serum albumin. After 60 min the beads were pelleted by centrifugation and washed three times with buffer B containing 1% Triton X-100. Beads were then added to solubilized membrane samples (typically 125 ,1 of beads per mg of membrane protein). After incubation for 60 min at 22°C, the samples were centrifuged at 13,000 x g for 10 s to pellet the beads containing the immune complexes. No detectable IRGT (assayed by immunoblotting; see below) remained in the supernatant. The samples were washed four times with buffer B containing 1% Triton X-100 (1 ml per wash) before SDS sample buffer (50 ,l) was added to elute IRGT (25). Electrophoretic Analysis. Samples were subjected to SDS/ PAGE (7.5% polyacrylamide resolving gel) by the method of Laemmli (25). In some experiments, 32P-labeled samples were transferred to nitrocellulose sheets (Schleicher & Schuell). Autoradiograms were prepared by exposing nitrocellulose sheets or dried gels to preflashed X-Omat AR film (Kodak) and Cronex Lightning Plus enhancing screens at -700C. Relative amounts of 32P-labeled IRGT were determined by optical density scanning of the autoradiograms. Direct measurement of the 32p content of immunoprecipitated IRGT was accomplished by scintillation counting of gel slices containing the 32P-labeled protein. A rabbit polyclonal antibody (R820) specific for a 12-amino acid peptide based on the deduced carboxyl-terminal sequence of the IRGT (15) was used to detect IRGT by immunoblotting. The antiserum (20 gg of IgG per ml) was prepared in phosphate-buffered saline (PBS) containing 1% Triton X-100 and 1% powdered milk (pH 7.4; Carnation) for 1 hr at 220C. The sheets were washed three times (10 min each) with PBS containing 1% Triton X-100 and then were incubated with 1251-labeled protein A (2 ,uCi/ml; Amersham). The sheets were then washed four times (10 min each) with PBS containing Triton X-100 and dried. The amount of 1251 present in the IRGT band under these conditions was large relative to the 32p present. Therefore, 32P did not interfere with quantitation of 1251 by optical density scanning of autoradiograms. This was confirmed by measuring 1251 emissions in slices of nitrocellulose (containing the IRGT) with a y counter. The use of R820 for

Proc. Natl. Acad. Sci. USA 86 (1989)

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immunoblotting instead of mAb 1F8 eliminated a problem in assessing the amount of IRGT. 1251-labeled protein A does not bind to mAb 1F8 under these conditions. Thus, the heavy chain of mAb 1F8, which migrates close to the IRGT in 7.5% acrylamide gels, did not interfere with the detection or quantitation of 1251 associated with IRGT. To determine whether the amount of detected 1251 accurately reflected the amount of IRGT immunoprecipitated, adipocytes were labeled with [35S]methionine (26) in the presence and absence of insulin. PM, LDM, HDM, and mitochondria/nuclei fractions were isolated, and immunoprecipitations were performed with mAb 1F8. Samples of the immunoprecipitated IRGT were then divided equally and subjected to SDS/PAGE. One gel was impregnated with 1 M sodium salicylate to enable detection of the 35S-labeled transporter (27); the other was used for immunoblotting with R820. Based on optical density scanning of autoradiograms, the ratios of 35S/125I remained constant among the subcellular fractions obtained from insulin- and noninsulin-treated cells. These results indicate that immunoblotting provides an accurate measure of the relative amount of IRGT.

RESULTS Phosphorylation of IRGT in Rat Adipocytes. To investigate the phosphorylation of the IRGT, fat cells were incubated in medium supplemented with 32p;. After homogenizing the cells, membrane fractions were prepared by high-speed centrifugation, and immunoprecipitations were performed with mAb 1F8. A phosphorylated protein with an apparent molecular weight of 46,000 was detected by autoradiography after SDS/PAGE (see, for example, Fig. la). The mobility of the phosphorylated species corresponded exactly with that of the IRGT detected by immunoblotting with R820 (Fig. lb). In other experiments (not shown here), we found that this phosphorylated species could be immunoprecipitated with the R820 antisera but not with a nonimmune IgG or an antibody against the Hep G2-type glucose transporter. These results strongly suggest that the Mr 46,000 species is the IRGT and that under basal conditions it is phosphorylated. It has not been possible to directly measure the number of IRGT molecules immunoprecipitated. However, by using measurements of glucose-inhibitable cytochalasin B binding sites as an estimate of transporter number (4, 28) and assuming that the specific activity of 32p in the transporter was equal to that of the intracellular pool of [y-32P]ATP, we have calculated that there is at least 0.2 mol of phosphate per mol of IRGT. It should be emphasized that this is an approximation and could represent an underestimate of the true stoichiometry. Irrespective of this, a significant proportion of the intracellular pool of IRGT appears to be phosphorylated, suggesting that phosphorylation of this protein may be of physiological relevance. Effect of Insulin on Phosphorylation of IRGT. Insulin stimulates the translocation of the IRGT from the LDM fraction of the cell to the PM (15). Therefore, it seemed possible that insulin affected phosphorylation of IRGT within a specific compartment of the cell. To investigate this possibility, membrane fractionation was performed after incubating 32p_ labeled cells in the absence or presence of insulin. The specific activity of transporters in the PM of insulin-treated cells was no different from that in the LDM of nonstimulated cells (Fig. ic). Whereas there was no difference in the specific activity of the IRGT between LDM from nonstimulated cells and PM from insulin-treated cells, the specific activity of the IRGT in the PM from nonstimulated cells was higher. Because of the low amount of transporters in this fraction obtained from nonstimulated cells, we cannot rule out the possibility that phosphorylation of this small subset (i.e., 5-10% of the total; see Fig. lb) of transporters was affected

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Proc. Natl. Acad. Sci. USA 86 (1989)

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FIG. 2. Stimulation of IRGT phosphorylation by f-adrenergic agonists. Rat adipocytes were incubated with 32P; and 2.5 ,Ag of adenosine deaminase per ml for 2 hr. Incubations were continued for 10 min in the absence [control (CON)] or presence of 1 A±M isoproterenol (ISO), 100 /uM methoxamine (MET), or 10 uM epinephrine (EPI). Adipocytes were homogenized in buffer A, and samples were centrifuged at 150,000 x g for 90 min. The pellets, which contained total cellular IRGT, were solubilized with SDS, and IRGT was immunoprecipitated with mAb 1F8. Immunoblotting indicated that under these conditions the same amount of transporter per unit of protein was present in each treatment group. After SDS/PAGE, autoradiograms were prepared by exposing gels to film for 24 hr at -70°C. Autoradiograms were scanned for optical density, and the peak area corresponding to the IRGT band was measured. Radioactivity in each sample was calculated by using peak areas of radioactive standards. The results represent the cpm of [32P]IRGT per g of adipose tissue and are the means SEM of five experiments. *, Not significantly different from the control by Dunnett's t test (29), but P < 0.01 compared with the control as determined by Student's t test; **, P < 0.01 compared with the control as judged by analysis of variance and Dunnett's t test for comparing multiple values to a single control (29). ±

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FIG. 1. Effect of insulin on phosphorylation of the IRGT. Rat adipocytes were incubated with 32p; (0.1 mCi/ml) for 2 hr. Cells were incubated for a further 20 min in the absence (lanes -) or presence (lanes +) of insulin (200 milliunits/liter and then homogenized. Subcellular fractionation was performed to obtain mitochondria/ nuclei (M/N), HDM, LDM, and PM. The 32P-labeled IRGT was immunoprecipitated from each fraction by using mAb 1F8. Samples were split into equal parts, and these were subjected to SDS/PAGE with a 7.5% resolving gel. (a) Autoradiogram of a dried gel. (b) Detection of IRGT (after transfer of proteins to nitrocellulose) by using the anti-peptide antibody R820 and 125I-labeled protein A. (c) Specific activity of IRGT (means SEM) from seven experiments. Immunoprecipitated transporter was subjected to SDS/PAGE and transferred to nitrocellulose. An autoradiogram of the nitrocellulose was prepared to quantitate [32P]IRGT. The nitrocellulose sheets were then incubated with R820 and 1251-labeled protein A to estimate the relative amounts of transporter. Autoradiograms of the 32P- and 1251-labeled species were scanned for optical density, and areas beneath the peaks corresponding to the IRGT were determined. Specific activities in arbitrary units were calculated by dividing the values for 32p peaks by those of 1251 peaks. To compare results from different experiments, the specific activity of the transporter in the LDM of control cells was assigned a value of 1. ±

by insulin. In other experiments we investigated the effects of incubating cells with insulin for different times (1-30 min) and with increasing concentrations (2.5-2500 milliunits/liter) of the hormone. To be sure that phosphorylation/dephosphorylation was inhibited, cells were homogenized in buffer containing SDS before immunoprecipitation. In agreement with the subcellular fractionation studies (Fig. 1c), no effect of insulin on IRGT phosphorylation was detected. Stimulation of IRGT Phosphorylation by Adrenergic Agonists and cAMP Derivatives. In contrast to insulin, /3adrenergic agonists were found to increase phosphorylation of IRGT. In the experiments shown in Fig. 2, cells were incubated for 10 min with different adrenergic agonists. The /3-adrenergic agonist isoproterenol stimulated the phosphorylation of IRGT -'2-fold. Epinephrine, which binds to both

a- and ,B-adrenergic receptors, also stimulated phosphorylation of IRGT, although its effect was slightly less than that of isoproterenol. Methoxamine, an a-adrenergic receptor agonist, was without effect on IRGT phosphorylation (Fig. 2). These data indicate that IRGT phosphorylation may be stimulated via ,3-adrenergic receptor activation. Results from subcellular fractionation experiments indicated that isoproterenol stimulated the phosphorylation of IRGT in both the LDM and PM fractions. In the experiments presented in Fig. 3, cells were first incubated with insulin for 10 min, a time sufficient to stimulate translocation of transporters to the PM. Incubations were then continued for an additional 5 min in the absence or presence of isoproterenol. Isoproterenol stimulated phosphorylation of IRGT 2-fold both in the LDM and in the PM (Fig. 3a). Isoproterenoldependent phosphorylation of IRGT was significantly enhanced by including adenosine deaminase (2.5 ,ug/ml) in the incubation medium. Many of the effects of A3-adrenergic agonists are mediated by increased intracellular cAMP (30, 31). To investigate further the possible role of cAMP on increasing IRGT phosphorylation, cells were incubated with the cAMP derivatives 8-bromo-cAMP and dibutyryl-cAMP. Both cAMP derivatives increased IRGT phosphorylation -2-fold (Fig. 4). In contrast, 8-bromoadenosine monophosphate and butyrate were without effect (Fig. 4). cAMP-dependent protein kinase mediates many of the actions of cAMP in eukaryotic cells (30, 31). Hence, it seemed possible that the IRGT was a substrate for cAMPdependent kinase. To test this hypothesis, IRGT-enriched LDM were incubated with [y-32P]ATP in the absence or presence of the catalytic subunit of cAMP-dependent protein kinase. The catalytic subunit catalyzed the phosphorylation of the IRGT (Fig. 3a). These results suggest that IRGT is a


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FIG. 3. Effect of isoproterenol and adenosine deaminase on IRGT phosphorylation in insulin-treated cells. 32P-labeled cells were incubated with insulin (200 milliunit/liter) for 15 min followed by a 5-min incubation with no other additions (lanes 3 and 6), with isoproterenol (lanes 4 and 7), or isoproterenol and adenosine deaminase (2.5 jLg/ml) (lanes 5 and 8). Cells were then homogenized, and subcellular fractions were prepared. LDM (lanes 3-5) or PM (lanes 6-8) fractions (100 ,ug of protein each) were solubilized, and IRGT was immunoprecipitated with mAb 1F8. Samples were subjected to SDS/PAGE. (a) Autoradiogram of a dried gel showing [32P]IRGT (lanes 3-8). (b) Autoradiogram of the corresponding region of an immunoblot prepared with the anti-peptide antiserum R820 and 1251-labeled protein A. In b, an aliquot of rat adipocyte microsomal membrane was electrophoresed and immunoblotted as a standard (lane 1). In a, samples of IRGT-enriched LDM vesicles (5 ,ug) prepared from unlabeled cells as described (23) were incubated in 10 mM Hepes (final volume, 100 ,l; pH 7.4) containing 1 mM EDTA, 0.1 mM EGTA, 250 mM sucrose, 6 mM MgCl2, and 0.2 mM [y-32P]ATP (3000 cpm/pmol) for 1 hr in the absence or presence of the catalytic subunit (0.4 ,M) of cAMP-dependent protein kinase from beef heart. After 1 hr the reaction was terminated by adding 100 ,ul of 200 mM NaF and 10 mM EDTA. Immunoprecipitation was performed with mAb 1F8, and samples were subject to SDS/PAGE. An autoradiogram (12-hr exposure) of the dried gel was prepared, and the region surrounding the IRGT phosphorylated in the absence (lane 2) or presence (lane 1) of the catalytic subunit is shown.

direct substrate for cAMP-dependent kinase but do not exclude possible indirect actions of the kinase. Effect of Isoproterenol on Subcellular Distribution of IRGT. One mechanism by which isoproterenol-dependent phosphorylation of IRGT might result in inhibition of insulinstimulated cellular glucose transport is by inhibiting translocation of transporters to the cell surface. To address this issue, cells were incubated with hormones before subcellular fractionation. The relative amount of IRGT in each fraction was measured after immunoprecipitation by immunoblotting with R820. In the absence of insulin, the IRGT was located predominantly in the LDM, and this distribution was unaffected when cells were incubated with 1 ,uM isoproterenol for 5 min (Fig. 5). Insulin stimulated the translocation of IRGT to the PM. Isoproterenol did not affect the amount of IRGT detected in the PM after insulin treatment.

DISCUSSION Insulin-dependent glucose transport in muscle and fat is most likely mediated by the IRGT because it appears to be the predominant transporter in these tissues, and it undergoes insulin-dependent translocation to the plasma membrane (15,

FIG. 4. Increased phosphorylation of IRGT by cAMP derivatives. Adipocytes were incubated with 32P for 2 hr followed by a 10-min incubation with no addition (CON), 5 mM 8-bromo-cAMP (8BrcA), 5 mM 8-bromo-AMP (8BrA), 5 mM dibutyryl-cAMP (dbcA), or 5 mM butyrate (but). The IRGT was immunoprecipitated after homogenization and solubilization of the total membrane fractions as described in Fig. 2. Means + SEM for five experiments are shown. **, P < 0.01 compared with control (29).

20). The present results show that the IRGT is phosphorylated in fat cells. By using 32P-labeled epitrochlearis muscle (results not shown), we have found that the IRGT is also phosphorylated in skeletal muscle. Insulin is perhaps the most important regulator of glucose transport (1, 2). It is now evident that a major mechanism by which insulin stimulates glucose transport involves translocation of glucose transporters from inside the cell to the PM (3-7, 20). Protein phosphorylation has been implicated in insulin action (32, 33). Thus, it was of interest to examine whether the regulation of glucose transport by insulin involved phosphorylation of IRGT. The results presented here suggest that the ability of insulin to stimulate translocation of the glucose transporter from the LDM fraction to the PM does not involve phosphorylation of the transporter per se. However, an important role for phosphorylation in the activation of glucose transport by insulin cannot be excluded. In fact, the recent observation that glucose transport is increased by incubating adipocytes with okadaic acid, an *S- A

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FIG. 5. Failure of isoproterenol to affect insulin-dependent translocation of the IRGT. Adipocytes were incubated in low phosphate medium containing 2.5 ,ug of adenosine deaminase per ml for 2 hr. Incubations were continued as follows: 20 min without additions, 20 min with insulin (INS; 200 milliunits/liter), 15 min without additions followed by 5 min with 1 ,uM isoproterenol (ISO), or 15 min with insulin and a further 5 min with insulin plus isoproterenol. LDM and PM were prepared. The IRGT was immunoprecipitated from each fraction by using mAb 1F8, and samples were subjected to SDS/ PAGE. Proteins were transferred to nitrocellulose sheets, which were incubated with the anti-peptide antiserum R820 and 125I-labeled protein A. Autoradiograms were prepared, and the relative amounts of IRGT were determined by comparing peak areas obtained from optical density scanning. Each value was normalized to the amount of IRGT in LDM from control cells, which was assigned a value of 1.0. Means SEM of five experiments are shown. ±

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inhibitor of type I and Ila phosphatases, suggests that protein phosphorylation might be linked to activation of transport (34). This could potentially involve phosphorylation of other proteins in, or associated with, the intracellular vesicles containing the glucose transporter. Isoproterenol has been shown to inhibit glucose transport in both muscle and adipose tissue (9, 10, 12, 13). Our results indicate that these effects might be mediated via phosphorylation of IRGT. The magnitude of the isoproterenolmediated increase in IRGT phosphorylation (2-fold, Figs. 2 and 3) was within the range of its inhibitory effect on cellular glucose transport (data not shown; refs. 9 and 10). Furthermore, adenosine appears to be a major determinant of the magnitude of the effect of isoproterenol on inhibiting glucose transport and stimulating phosphorylation of IRGT (Fig. 3a). This nucleoside accumulates in the incubation medium throughout the course of the incubation and inhibits the effect of isoproterenol on glucose transport. It has been shown (10) that the effect of isoproterenol on inhibiting transport can be restored by adding adenosine deaminase. At the relatively high cell concentrations used in the present studies, the effect of isoproterenol on stimulating IRGT phosphorylation was similarly sensitive to adenosine deaminase (Fig. 3a). In particular, adenosine deaminase appeared to be required to demonstrate effects of isoproterenol on IRGT phosphorylation in the PM. Isoproterenol did not significantly alter the subcellular distribution of the IRGT in either the absence or presence of insulin (Fig. 5). These results agree with previous findings that the concentration of PM glucose transporters, as measured by cytochalasin B binding, was unaffected by isoproterenol (10). Under the conditions of the experiments described in Fig. 5, insulin was found to stimulate 2deoxyglucose uptake an average of 23-fold, whereas in the presence of isoproterenol, the insulin effect was reduced -50%o. Based on these findings we suggest that stimulation of IRGT phosphorylation by isoproterenol may decrease the intrinsic transport rate of the glucose transporter. Phosphorylation of IRGT was also increased by incubating adipocytes with derivatives of cAMP (Fig. 4). Together with the ability of cAMP-dependent kinase to phosphorylate IRGT in vitro (Fig. 3a), these data strongly suggest that the effects of isoproterenol on stimulation of IRGT phosphorylation are mediated by cAMP-dependent protein kinase. The surrounding sequence of amino acids is important in determining whether a particular serine/threonine will be phosphorylated by this kinase. The consensus sequence for cAMP-dependent protein kinase is (Arg or Lys)-Xaa-(Ser or Thr) (31). While future studies will be required to identify sites of phosphorylation in the IRGT, it is interesting that the putative cytoplasmic domains of the IRGT contain several consensus sites for cAMP-dependent protein kinase (for example, Ser-243, Thr-351, and Ser-497). The fact that these sites are not present in the Hep G2-type glucose transporter might explain the inability to detect phosphorylation of the Hep G2-type glucose transporter with isoproterenol (13). We thank Kerri James for artwork. Support for this work was provided by grants from the National Institutes of Health (AR34815) to J.C.L., the Juvenile Diabetes Foundation to D.E.J., and the Washington University Diabetes Research and Training Center.

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