Proc. Natl. Acad. Sci. USA Vol. 88, pp. 11500-11504, December 1991 Biochemistry

Phosphorylation state of the GLUT4 isoform of the glucose transporter in subfractions of the rat adipose cell: Effects of insulin, adenosine, and isoproterenol (lipolytic hormones/antilipolytic hormones/cyclic AMP-dependent protein kinase)

H. NISHIMURA*, J. SALTIS*t, A. D. HABBERFIELD*t, N. B. GARTY§¶, A. S. GREENBERG§, S. W. CUSHMAN*, C. LONDOS§, AND 1. A. SIMPSON*II *Experimental Diabetes, Metabolism and Nutrition Section, Diabetes Branch, and NMembrane Regulation Section, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892

Communicated by Rachmiel Levine, September 24, 1991 (received for review July 8, 1991)

ABSTRACT The acute effects of insulin, adenosine, and isoproterenol on the activity, subcellular distribution, and phosphorylation state of the GLUT4 glucose transporter isoform were investigated in rat adipocytes under conditions carefully controlled to monitor changes in cAMP-dependent protein kinase (A-kinase) activity. In contrast to GLUT1, which has not been shown to be phosphorylated even when cells are exposed to any of the above agents, GLUT4 was partially phosphorylated (0.1-0.2 mol/mol) when the activity of the A-kinase was suppressed, and remained unchanged in response to insulin. Isoproterenol elicited a 64% inhibition of insulinstimulated glucose transport activity in the absence, but not the presence, of adenosine receptor agonists. However, in either the presence or the absence of agonists, A-kinase was activated as assessed by examining the phosphorylation of the major adipocyte A-kinase substrate, perilipin. Similarly, under either condition, phosphorylation of GLUT4 was enhanced 1.4-fold in the intracellular membranes, but no significant change was observed in the plasma membrane. In the absence of adenosine receptor agonists, isoproterenol exerted a small (14%) but significant inhibition of the insulin-induced translocation of GLUT4 but had no effect on the translocation of GLUT1. Thus, changes in the phosphorylation state and/or subcellular distribution of GLUT4 cannot account for the inhibition of insulin-stimulated glucose activity induced by isoproterenol.

Insulin stimulates glucose transport activity in rat adipose cells primarily by inducing the translocation of the transporter isoforms GLUT1 and GLUT4 from an intracellular location to the plasma membrane (1-4). GLUT1 is widely distributed whereas GLUT4, the predominant form in adipose cells (4-6), is found only in tissues where insulin regulates glucose transport activity (i.e., white and brown adipose tissue, heart, and skeletal muscle; for review see refs. 7 and 8). Hereafter, insulin-stimulated glucose transport activity will be abbreviated as transport activity. A second level of transport regulation is exerted by agents that modulate adenylyl cyclase and lipolysis (9-11). Lipolytic agents (isoproterenol, glucagon, and corticotropin) inhibit transport activity, but only in the absence of antilipolytic agents (adenosine, nicotinic acid, and prostaglandin E1). Further, Smith et al. (12) and Kuroda et al. (13) demonstrated that both the transport inhibition by lipolytic agents and the augmentation by antilipolytic agents did not result from changes in transporter location. An unresolved issue is whether the changes in transport activity mediated by these various agents are related to the changes in cAMP. Currently, two distinct mechanisms have

been proposed. Kuroda et al. (13) demonstrated an apparent dissociation between isoproterenol-mediated stimulation of cAMP-dependent protein kinase (A-kinase) and inhibition of transport activity, suggesting that changes in A-kinasemediated phosphorylation are not responsible for transport inhibition. This was supported by Joost et al. (14), who showed that the GLUT1 isoform of the glucose transporter was not phosphorylated in response to insulin, adenosine, or isoproterenol. More recently, however, with the advent of anti-GLUT4 antibodies, James et al. (15) and Lawrence et al. (16) have demonstrated that (i) GLUT4 is phosphorylated "in intact cells," (ii) GLUT4 is a "cell-free" target of A-kinase, and (iii) "cell-free" phosphorylation is augmented by lipolytic agents. Hence, they proposed that transport activity in the adipose cells is regulated by an A-kinase-dependent phosphorylation of GLUT4. We have compared transport activity and the phosphorylation state of GLUT4 under conditions carefully controlled to monitor simultaneous changes in transport activity and A-kinase activity. We confirmed that GLUT4 is phosphorylated and that isoproterenol mediates a small increase in GLUT4 phosphorylation. This enhanced phosphorylation is confined to intracellular membranes and occurs both in the presence and in the absence of adenosine receptor activation, but isoproterenol inhibits transport activity only in the absence of adenosine receptor agonists, suggesting that phosphorylation does not explain the inhibition of transport activity.

METHODS Animals and Cell Preparation. Adipose cells were isolated from 50-56 male rats (170-200 g, CD strain, Charles River Breeding Laboratories) as described (17). The cells (0.8-1.2 x 106 per ml) were preincubated for 90 min at 37°C in the presence or absence of 32p1 [0.1 mCi (3.7 MBq)/ml] in a Krebs-Ringer medium, pH 7.4, containing 2.5 mM glucose, 10 mM sodium bicarbonate, 0.1 mM sodium phosphate, 30 mM Hepes, and 5% (wt/vol) albumin. To suppress A-kinase Abbreviations: A-kinase, cAMP-dependent protein kinase; PIA, (R)-N6-(1-methyl-2-phenylethyl)adenosine; 8DPCPX, 8-cyclopentyl-1,3-dipropylxanthine. tPresent address: Baker Medical Research Institute, P.O. Box 348, Prahran, Victoria 3181, Australia. tPresent address: Amgen Incorporated, Amgen Center, Thousand Oaks, CA 91320. VPresent address: The Weizmann Institute of Science, Rehovot, Israel. I1To whom reprint requests should be addressed at: Experimental Diabetes, Metabolism and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Building 10, Room 5N102, National Institutes of Health, Bethesda, MD 20892.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

11500

-20m'in

Biochemistry: Nishimura

et

al.

Proc. Nati. Acad. Sci. USA 88 (1991)

activity, 200 nM adenosine and 10 nM (R)-N6-(1-methy1-2phenylethyl)adenosine (PTA) were included in the incubation medium. After centrifugation through dinonylphthalate and resuspension in an equal volume of the above medium without 32p,, the cells were subjected to the protocols outlined below.

Cell Incubation and Processing. Both control and radiolabeled cells were incubated in parallel and subjected to the protocols outlined in Fig. 1. Specific methods are described below. Following removal of 200-gl aliquots for transport

activity determinations (18), the remaining cells were fractionated.

Preparation of Subcellular Membrane Fractions. Cells from each incubation were centrifuged through dinonylphthalate (5 ml) and resuspended in 20 ml of homogenization buffer (20 mM Tris/1 mM EDTA/255 mM sucrose, pH 7.4) containing phosphatase inhibitors (0.2 mM vanadate, 10 MM sodium fluoride, and 10 mM sodium pyrophosphate) at 17'C. The cells were homogenized and plasma membranes and intracellular membranes were prepared (17). For the reasons noted by Shibata et al. (19), the intracellular membranes correspond directly to the membranes erroneously named "low-densitymicrosomes" (20). Membrane protein was as-

sayed using bicinchoninic acid by a modification of the method of Smith etal. (21) according to the manufacturer's instructions (Sigma). Cytochalasin B binding was determined in the subcellular fractions from unlabeled cells (17). To confirm reproducible fractionation, membranes were analyzed for GLUTi and GLUT4 by Western blotting. Immunoprecipitation of Glucose Transporters. Immunoprecipitation of GLUT4 from radiolabeled cellswas as described (14), with 1 mg of solubilized membrane protein and either a mouse monoclonal antibody (1F8, a gift from Paul Pilch, Boston University) or a rabbit antibody raised against the 15-amino acid C-terminal sequence of GLUT4 (a gift from Hoffman LaRoche). The bound complex was washed sequentially with SDS- and NaCl-containing buffers (22). The immune complex was eluted with sample buffer and 80%6 of the sample subjected to SDS/PAGE (23). The gels were dried and autoradiographed at -700C for 12 hr. 32P-labeled glucose transporter was quantitated by cutting out the band for Cerenkov counting. To determine the distribution of GLUT4 the remaining 20% was Western blotted using IF8 and Nash

Protocol 1

IJFinemnU

Protocol 2

f Insulin --o-

Protocol 3

Protocol 4

--

a

-.-30mn- -- -

-

-

I

ISO(1gM) ADA(0.5U/mQ)

8DPCPX(lliM)

ISO(1AR 1

Insulin In -

-

II-I-labeled sheep anti-mouse IgG. Transporters were quantitated by y counting of excised bands. Assessment of A-Kinase Activity. A-kinase activity was assessed by viewing the behavior of the major adipocyte A-kinase substrate, perilipin, in SDS/PAGE. This protein is phosphorylated in unstimulated cells (quiescent A-kinase) and migrates in SDS/PAGE as a 62-kDa protein, but it gains an additional 5 mol of phosphate per mol as the ±cAMP A-kinase activity ratio rises to 0.35-0.5, at which point it migrates as a 65/67-kDa doublet (24, 25). Thus, the migration patterns of the various phosphorylation species of perilipin provide a sensitive indicator of A-kinase activity (25). Determination of ATP-Specific Radioactivity. Two hundred microliters of the cell suspension was added to 22P1I of 25% on ice. The perchloric acid, mixedwerevigorously, andforplaced precipitated samples centrifuged 10 min at 10,000 X g at 40C, and 100 Al of supernatant was added to 100ul of solution consisting of 4.2 M KOH, 1 M Hepes (pH 7.6), and water (1:2:7). ATP was determined in 10-/u aliquots of a 1:10 dilution of the neutralized supernatants by luminometry in a Packard Picolite luminometer with a firefly luciferein/ luciferase kit (Analytical Luminescence. Laboratories, San Diego, CA). Aliquots of the neutralized supernatant were assayed also for ATP labeled in they position by quantitatively transferring the terminal phosphate to the A-kinase peptide substrate Kemptide, with an excess of A-kinase [purified from rat adipocytes (26)]. The A-kinase reaction mixture contained 100 Kemptide, 20 mM Mops (pH 7.0), 16 mM magnesium acetate, 4 mM dithiothreitol, 10 cAMP, and usually 20 /4 of the neutralized supernatant. The reaction was initiated with A-kinase and conducted for 1 hr at 30°C to ensure complete transfer. The reactions were was purified

,uM

,uM

terminated and 32P-labeled peptide specific activity of ATP was determined to be. -.500 cpm/ pmol, which is consistent with previous observations (28,29). (27).

Calculations.

Statistical

The

significance was tested with one-

way analysis of variance followed by Duncan's multiple range test and a paired Student's t test, as appropriate, and differences were accepted as significant at the P < 0.05 level.

RESULTS Since assessments of cellular

phosphorylation require

=90

reduced Pi concentration (28), min of incubation with 32P; atofa these we tested the incubation conditions on

consequences glucose transport activity (Table 1). Prolonged incubation enhanced both basal and insulin-stimulated transport activity while blunting the inhibitory response to isoproterenol from 46.0 ± 1.4% to 28.0 + 0.6 (P