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The Effects of Wortmannin on Rat Skeletal Muscle

DISSOCIATION OF SIGNALING PATHWAYS FOR INSULIN- AND CONTRACTION-ACTIVATED HEXOSE TRANSPORT (∗)
  • Jih-I Yeh
    Affiliations
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Eric A. Gulve
    Affiliations
    G. D. Searle & Company, St. Louis, Missouri 63167
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  • Lucia Rameh
    Affiliations
    Division of Signal Transduction, Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02115
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  • Morris J. Birnbaum
    Correspondence
    Howard Hughes Medical Inst., University of Pennsylvania School of Medicine, Clinical Research Bldg., Rm. 322, 415 Curie Blvd., Philadelphia, PA, 19104-6148.
    Affiliations
    Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115
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  • Author Footnotes
    ∗ This work was supported by National Institutes of Health Grant DK39519 (to M. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
      Both the anabolic hormone insulin and contractile activity stimulate the uptake of glucose into mammalian skeletal muscle. In this study, we examined the role of phosphatidylinositol 3-kinase (PI 3-kinase), a putative mediator of insulin actions, in the stimulation of hexose uptake in response to hormone and contraction. Phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate accumulate in skeletal muscle exposed to insulin but not hypoxia, which mimics stimulation of the contractile-dependent pathway of hexose transport activation. The fungal metabolite wortmannin, an inhibitor of PI 3-kinase, completely blocks the appearance of 3′-phospholipids in response to insulin. Moreover, wortmannin entirely prevented the increase in hexose uptake in muscle exposed to insulin but was without effect on muscle stimulated by repetitive contraction or hypoxia. These results support the view that PI 3-kinase is involved in the signaling pathways mediating insulin-responsive glucose transport in skeletal muscle but is not required for stimulation by hypoxia or contraction. Furthermore, these data indicate that there exist at least two signaling pathways leading to activation of glucose transport in skeletal muscle with differential sensitivities to wortmannin.

      INTRODUCTION

      Insulin, which increases glucose uptake into muscle and adipocytes while decreasing the hepatic production of glucose, is the most important hormone involved in the regulation of hexose metabolism in mammals. Even though 90% of hexose disposal after an oral glucose load occurs in skeletal muscle, the mechanisms by which this is regulated remain poorly understood. In addition to insulin, muscle contraction and hypoxia also increase glucose transport(
      • Barnard R.J.
      • Youngren J.F.
      ,
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ). The acceleration of hexose uptake into skeletal muscle upon exposure to insulin, contractile activity, or hypoxia, which mimics that pathway activated by contraction, is accompanied by a redistribution of the “insulin-responsive” facilitated glucose transporter isoform, GLUT4, from an intracellular compartment to the plasma membrane(
      • Barnard R.J.
      • Youngren J.F.
      ,
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ). Presumably, the increase in cell surface transporter in response to any one of these physiological stimuli accounts for much of the augmentation in hexose uptake. Nonetheless, it is unclear if contraction and hypoxia utilize the same signaling pathways as does insulin to activate glucose uptake. Previous studies have implicated some diversity in the means by which glucose uptake is regulated in muscle(
      • Barnard R.J.
      • Youngren J.F.
      ). The effects of maximal stimulation by contraction and hypoxia are non-additive, whereas activation by insulin and hypoxia are fully or partially additive(
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ,
      • Nesher R.
      • Karl I.E.
      • Kipnis D.M.
      ). This has been interpreted as indicative of alternative mechanisms by which hypoxia or contraction on the one hand and insulin on the other stimulate glucose transport but has not led to significant insight as to the level at which the pathways differ(
      • Barnard R.J.
      • Youngren J.F.
      ). Thus far, no defined pharmacological agent has been identified that unambiguously abolishes accelerated transport in response to one stimulus while preserving that in response to the other (
      • Henriksen E.J.
      • Sleeper M.D.
      • Zierath J.R.
      • Holloszy J.O.
      ,
      • Cartee G.D.
      • Briggs-Tung C.
      • Holloszy J.O.
      ).
      Insulin, as well as other growth factors, rapidly stimulates the enzyme PI 3-kinase
      The abbreviations used are: PI 3-kinase
      phosphatidylinositol 3-kinase
      KHB
      Krebs-Henseleit buffer
      PtdIns(3,4)P2
      phosphatidylinositol 3,4-bisphosphate
      PtdIns(3,4,5)P3
      phosphatidylinositol 3,4,5-triphosphate
      2-DG
      2-deoxyglucose
      IRS-1
      insulin receptor substrate 1.
      (
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Ruderman N.B.
      • Kapeller R.
      • White M.F.
      • Cantley L.C.
      ,
      • Kelly K.L.
      • Ruderman N.B.
      • Chen K.S.
      ,
      • Chen K.S.
      • Friel J.C.
      • Ruderman N.B.
      ,
      • Folli F.
      • Saad M.J.A.
      • Backer J.B.
      • Kahn C.R.
      ). The binding of insulin to its receptor leads to tyrosine phosphorylation of the insulin receptor substrate, IRS-1, which in turn binds to and activates PI 3-kinase via the SH2 domain of its regulatory subunit, p85(
      • White M.F.
      • Kahn C.R.
      ). An increase in the activity of the PI 3-kinase catalytic subunit, p110, results in the accumulation of phosphatidylinositol derivatives phosphorylated at the D-3 position(
      • Cantley L.C.
      • Auger K.R.
      • Carpenter C.
      • Duckworth B.
      • Graziani A.
      • Kapeller R.
      • Soltoff S.
      ,
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ). Recently, the demonstration that the fungal metabolite wortmannin inhibits both the activation of PI 3-kinase as well as the insulin-dependent translocation of GLUT4 transfected into Chinese hamster ovary cells and glucose transport in adipocytes has led to the suggestion that the lipid kinase is an obligate intermediate in the insulin-signaling pathway(
      • Kanai F.
      • Ito K.
      • Todaka M.
      • Hayashi H.
      • Kamohara S.
      • Ishii K.
      • Okada T.
      • Hazeki O.
      • Ui M.
      • Ebina Y.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Okada T.
      • Kawano Y.
      • Sabakibara T.
      • Hazeki O.
      • Ui M.
      ). Moreover, the insulin-dependent binding of PI 3-kinase to IRS-1 is decreased in several animal models of insulin resistance(
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ,
      • Saad M.J.A.
      • Folli F.
      • Kahn J.A.
      • Kahn C.R.
      ). However, the studies using antagonists of the PI 3-kinase have not distinguished whether their inhibitory effect was being exerted at the level of the insulin signal transduction pathway or more directly on vesicle-mediated redistribution of hexose carriers. In this study, we explored the role of PI 3-kinase in mediating the stimulation of hexose uptake into skeletal muscle in response to several distinct, physiological stimuli.

      MATERIALS AND METHODS

       Treatment of Animals and Muscle Preparation

      Male Sprague-Dawley rats obtained from Charles River Laboratories were maintained on a diet of rat chow and water. Food was restricted to 4 g/animal after 6 p.m. the night prior to an experiment. Animals weighing 75-100 g were anesthetized with an intraperitoneal injection of sodium pentobarbital (8 mg/100 g of body weight), and the epitrochlearis, a thin muscle that is highly suitable for incubation in vitro, or hemidiaphragm muscle with ribs attached was dissected out intact(
      • Nesher R.
      • Karl I.E.
      • Kaiser K.E.
      • Kipnis D.M.
      ,
      • Goldberg A.L.
      • Martel S.B.
      • Kushmerick M.J.
      ,
      • Kipnis D.M.
      ). The latter muscle was trimmed from the ribs at the end of the incubations.

       Hexose Uptake

      Muscles were incubated in shaking water baths in stoppered 25-ml Pyrex flasks gassed with 5% CO2, 95% O2 unless indicated otherwise. The treatment of muscle for each experimental condition is summarized in Table 1. The incubation media consisted of KHB (116 mM NaCl, 4.6 mM KCl, 1.16 mM KH2PO4, 25.3 mM NaHCO3, 2.5 mM CaCl2•2H2O, 1.16 mM MgSO4•7H2O) and 0.2% bovine serum albumin (RIA grade, ICN Biochemicals) with the following additions: for the preincubation, 32 mM mannitol and 8 mMD-glucose; during the rinse period, 40 mM mannitol; for the measurement of hexose uptake, 39 mM14C-labeled D-mannitol (0.1 μCi/ml, DuPont NEN) and 1 mM3H-labeled 2-deoxy-D-glucose (1.5 μCi/ml, DuPont NEN). In some experiments, hexose transport activity was assayed using 8 mM3H-labeled 3-O-methyl-D-glucose (1.5 μCi/ml, American Radiolabeled Chemicals, Inc.) and 32 mM14C-labeled D-mannitol as described(
      • Young D.A.
      • Uhl J.J.
      • Cartee G.D.
      • Holloszy J.O.
      ). After incubation, each muscle was transferred to a 75 × 15-mm glass tube containing 1 ml of distilled water, boiled for 10 min, and mixed with EcoScint (National Diagnostics, Atlanta, Georgia) for quantitation of associated radioactivity in a Packard liquid scintillation analyzer. The calculation of extracellular space and 2-deoxy-D-glucose uptake rate was done as described(
      • Young D.A.
      • Uhl J.J.
      • Cartee G.D.
      • Holloszy J.O.
      ). The use of 2-deoxy-D-glucose to measure hexose transport activity in isolated skeletal muscle has been discussed(
      • Hansen P.A.
      • Gulve E.A.
      • Holloszy J.O.
      ). Muscles were stimulated to contract tetanically in vitro, in the presence or absence of wortmannin (Sigma) by a protocol identical to that described by Henriksen et al.(
      • Henriksen E.J.
      • Sleeper M.D.
      • Zierath J.R.
      • Holloszy J.O.
      ).

       Analysis of Phospholipids

      Muscles were rinsed and incubated in phosphate-free KHB plus 32 mM mannitol, 8 mM glucose, and 0.2% bovine serum albumin for 15 min, followed by incubation of four epitrochlearis muscles in a 25-ml Erlenmeyer flask containing 3 ml of phosphate-free KHB and 1.5 mCi/ml [P]phosphoric acid (9313 Ci/mmol, ICN) for a total of 4.5 h. Muscles were exposed to drugs or hypoxia in the presence of [P]phosphoric acid according to the protocol described in Table 2. At the end of the incubation, each sample was homogenized, and phospholipids were extracted, deacylated, and analyzed by high pressure liquid chromatography as described(
      • Whitman M.
      • Downes C.P.
      • Keeler M.
      • Keller T.
      • Cantley L.
      ,
      • Vlahos C.J.
      • Matter W.F.
      ). The data are normalized to the total dpm in phosphatidylinositol 4-monophosphate plus phosphatidylinositol 4,5-bisphosphate.

      RESULTS

       Wortmannin Blocks Insulin-stimulated Hexose Uptake in Isolated Rat Skeletal Muscle

      Insulin (300 nM) stimulated the uptake of 2-DG into isolated rat epitrochlearis muscle about 4-fold; preincubation with 1 μM wortmannin completely blocked the increase of 2-hexose uptake by insulin (Fig. 1A). In the series of experiments shown in Fig. 1A, wortmannin reduced the level of 2-DG accumulation to values below that observed in the absence of insulin or inhibitors. However, this was somewhat variable, and in muscles with low basal transport activity, wortmannin reduced uptake no further (See Fig. 3). Since the accumulation of 2-DG depends on phosphorylation as well as transport, an equivalent series of experiments was performed with the non-phosphorylatable sugar, 3-O-methylglucose. The complete inhibition of 3-O-methylglucose uptake into epitrochlearis muscle by wortmannin serves as proof that the drug blocks the insulin-dependent transport of sugar and not a subsequent metabolic step (Fig. 1B). Moreover, the blockade was not limited to the epitrochlearis muscle, as wortmannin prevented the insulin-dependent increase in 3-O-methylglucose transport in rat diaphragm (Fig. 1B). The inhibitory effect of wortmannin on hexose uptake was concentration-dependent, with an IC occurring at about 10-30 nM and complete blockade at 1 μM (Fig. 2).
      Figure thumbnail gr1
      Figure 1:Effect of wortmannin on insulin-stimulated hexose uptake in isolated rat skeletal muscle. A, 2-DG uptake into isolated rat epitrochlearis muscle was measured as described in the text in the absence (basal) or presence of 300 nM insulin for 30 min, with or without pretreatment with 1 μM wortmannin for 30 min. B, 3-O-MG uptake of rat epitrochlearis and hemidiaphragm muscles was measured under the same conditions as in A. The basal uptake into muscles averaged 0.15 μmol/ml/10 min. Data presented are the mean ± S.E. of eight muscles for epitrochlearis and seven muscles for the hemidiaphragm muscles.
      Figure thumbnail gr3
      Figure 3:The effect of wortmannin on contraction-stimulated 2-DG uptake in muscle. Isolated rat epitrochlearis muscles were treated or not with 1 μM wortmannin, followed by 10 tetanic contractions elicited by stimulating at 50 Hz for 10 s at 1 contraction/min for 10 min. Muscles were stimulated to contract in the absence or presence of wortmannin. 2-DG uptake rate was then measured as described under “Materials and Methods.” Data are presented as mean ± S.E. of 5-7 muscles. The difference in the accumulation of hexose with and without wortmannin was not significant (p = 0.65).
      Figure thumbnail gr2
      Figure 2:Dose-response curve of wortmannin inhibition of insulin-stimulated 2-DG uptake in isolated rat epitrochlearis muscle. Isolated muscles were treated with the indicated concentrations of wortmannin for 30 min, followed by incubation in the absence () or presence (•) of 300 nM insulin for 30 min. 2-DG uptake was then measured as described under “Materials and Methods.” Data are the mean ± S.E. of seven muscles except for wortmannin alone, which is the average of two muscles.

       Wortmannin Does Not Block the Increase in Hexose Uptake Stimulated by Muscle Contraction or Hypoxia

      As previously reported(
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ), tetanic contraction stimulated hexose uptake into isolated rat epitrochlearis muscle (Fig. 3). Wortmannin, at the same concentration that completely blocked insulin-stimulated 2-DG uptake, was without effect on hexose accumulation in response to contraction. In the same experiment, the contractile force produced in response to repetitive electrical stimulation was decreased 40-50% by 1 μM wortmannin (data not shown). Because wortmannin did not alter contraction-activated hexose transport activity, its effect on tension generation is not a confounding factor. Nevertheless, to avoid the complications of difference in contractile force, the effect of wortmannin on hexose uptake stimulated by hypoxia was also examined. Previously published work supports the view that hypoxia and contraction activate glucose transport via the same pathways(
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ), but muscles incubated under hypoxia do not generate significant tensions. As shown in Fig. 4, hypoxia increased the accumulation of 2-DG in rat epitrochlearis muscle about 4-fold, and this was unaffected by 1 μM wortmannin. The exposure of hypoxic muscle to a maximally effective concentration of insulin increased 2-DG uptake to a level greater than that achieved by hypoxia alone; the additional inclusion of wortmannin reduced uptake to a value indistinguishable from that observed in the presence of hypoxia alone (p > 0.05) (Fig. 4).
      Figure thumbnail gr4
      Figure 4:The effect of wortmannin on hypoxia and hypoxia plus insulin stimulation of 2-DG uptake in muscle. Isolated rat epitrochlearis muscles were treated with or without 1 μM wortmannin for 30 min, followed by incubation in an atmosphere of 5% CO2, 95% N2 for 60 min (n = 10). In hypoxia plus insulin stimulation experiments (n = 5), insulin was added to 300 nM 30 min after the start of incubation in 5% CO2, 95% N2. 2-DG uptake rate was then measured as described in the text and . Data are presented as the mean ± S.E. Wortmannin had no effect on hypoxia-stimulated hexose transport (p = 0.52).

       The Accumulation of PtdIns(3,4)P2and PtdIns(3,4,5)P3in Skeletal Muscle in Response to Insulin and Hypoxia

      Insulin stimulated the accumulation of PtdIns(3,4)P2 and PtdIns(
      • Nesher R.
      • Karl I.E.
      • Kipnis D.M.
      ,
      • Henriksen E.J.
      • Sleeper M.D.
      • Zierath J.R.
      • Holloszy J.O.
      ,
      • Cartee G.D.
      • Briggs-Tung C.
      • Holloszy J.O.
      ) P3 in epitrochlearis muscle, as assayed by the incorporation of [P]phosphate into lipids (Fig. 5). Hypoxia was without effect on the labeling of phosphatidylinosotides phosphorylated at the D-3 position. Wortmannin completely blocked the increase in PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in response to insulin (Fig. 6). Wortmannin did not affect the pattern of labeled phospholipids in muscles untreated with insulin or exposed to hypoxia (Data not shown).
      Figure thumbnail gr5
      Figure 5:The effect of insulin and hypoxia on the production of phosphatidylinositol lipids in muscle. Isolated rat epitrochlearis muscles were incubated in phosphate-free KHB containing [P]orthophosphate for a total of 4.5 h. When present, insulin (dottedline) was added to 300 nM at 30 min before the end of the incubation. Hypoxic conditions (solid line) were induced 60 min before the end of the incubation by changing the gassing mixture from 5% CO2, 95% O2 to 5% CO2, 95% N2. The lipids were extracted and analyzed as described under “Materials and Methods.” Shown are the disintegrations/min eluting coincident with the PtdIns(3,4)P2 standard at about 57 min (panelA) and the PtdIns(3,4,5)P3 standard at about 88.5 min (panelB). Each tracing was the result of four muscles.
      Figure thumbnail gr6
      Figure 6:The effect of wortmannin on the production of phosphatidylinositol lipids in muscle. Rat epitrochlearis muscle was incubated (dottedline) or not (solidline) in the presence of 1 μM wortmannin for 30 min, followed by exposure to insulin for an additional 30 min. Lipids were extracted and analyzed as in . PanelA, PtdIns(3,4)P2; panelB, PtdIns(3,4,5)P3. Each tracing was the result of four muscles; note that the data for insulin treatment in the absence of wortmannin represents the same as that shown in .

      DISCUSSION

      In this series of experiments, we have used sensitivity to the fungal metabolite wortmannin to define two distinct signaling pathways leading to increased hexose uptake into rat skeletal muscle. We believe that the ability of wortmannin to inhibit accelerated transport in response to insulin but not contraction or hypoxia reflects the obligatory role of PI 3-kinase in the insulin-dependent pathway but not that activated by the latter stimuli. Several results support this notion. Insulin, but not hypoxia, stimulated the in vivo accumulation of the products of PI 3-kinase, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Fig. 5). This is in contrast to the results of a previous study, in which no labeled PtdIns(3,4)P2 and PtdIns(3,4,5)P3 was detected (
      • Chen K.S.
      • Friel J.C.
      • Ruderman N.B.
      ). The reason for the difference in PtdIns labeling is unclear, but increased efficiency of labeling in the current series of experiments is most likely. The inhibition of insulin-activated hexose uptake by 1 μM wortmannin correlated with suppression of the accumulation of 3′-phosphatidylinositol lipids (Fig. 6). Moreover, wortmannin, which did not affect the phospholipid pattern of hypoxic muscle, also was completely without effect on the stimulation of hexose uptake in response to hypoxia or contraction. It should be emphasized, however, that whereas the accumulation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 probably reflects in vivo PI 3-kinase activity, these experiments do not address whether the lipids actually represent the critical signaling molecules. It has been reported that the catalytic subunit of the PI 3-kinase is also capable of acting as a serine protein kinase(
      • Carpenter C.L.
      • Auger K.R.
      • Duckworth B.C.
      • Hou W.M.
      • Schaffhausen B.
      • Cantley L.C.
      ,
      • Dhand R.
      • Hiles I.
      • Panayotou G.
      • Roche S.
      • Fry M.J.
      • Gout I.
      • Totty N.F.
      • Truong O.
      • Vicendo P.
      • Yonezawa K.
      • Kasuga M.
      • Courtneidge S.A.
      • Waterfield M.D.
      ), and it is likely that wortmannin inhibits this activity in parallel in these experiments.
      Several recently published studies have been interpreted as indicative of the presence of multiple independent pathways for the physiological activation of glucose transport in muscle(
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ,
      • Nesher R.
      • Karl I.E.
      • Kipnis D.M.
      ,
      • Douen A.G.
      • Ramlal T.
      • Rastogi S.
      • Bilan P.J.
      • Cartee G.D.
      • Vranic M.
      • Holloszy J.O.
      • Klip A.
      ). One of the strongest arguments has been based on the observation that a maximal stimulation of hexose uptake by insulin can be further increased by contraction or hypoxia, whereas maximal activations by hypoxia and contraction are completely non-additive(
      • Cartee G.D.
      • Douen A.G.
      • Ramlal T.
      • Klip A.
      • Holloszy J.O.
      ). Efforts to show differential sensitivities of insulin and contraction-stimulated hexose transport to calcium channel blockers have not been successful(
      • Henriksen E.J.
      • Sleeper M.D.
      • Zierath J.R.
      • Holloszy J.O.
      ,
      • Cartee G.D.
      • Briggs-Tung C.
      • Holloszy J.O.
      ). In experiments relying on the subcellular fractionation of muscle, Douen et al.(
      • Douen A.G.
      • Ramlal T.
      • Rastogi S.
      • Bilan P.J.
      • Cartee G.D.
      • Vranic M.
      • Holloszy J.O.
      • Klip A.
      ) showed that in rat hindlimb the amount of GLUT4 on plasma membrane increased significantly and the intracellular membrane GLUT4 decreased correspondingly after insulin stimulation; while the plasma membrane GLUT4 increased after exercise, there was no corresponding decrease in the recovered intracellular membrane pool of GLUT4. Based on these results, they concluded there are two distinct cytoplasmic populations of GLUT4, and, by inference, the biochemical signals initiating translocation of these pools must be different(
      • Douen A.G.
      • Ramlal T.
      • Rastogi S.
      • Bilan P.J.
      • Cartee G.D.
      • Vranic M.
      • Holloszy J.O.
      • Klip A.
      ). Our results provide the first direct evidence that there are at least two pathways to activate hexose transport in skeletal muscle, since wortmannin obliterates the response to insulin while leaving the effects of contraction and hypoxia unaffected. The possibility that wortmannin acts at the insulin receptor or IRS-1 to inhibit insulin stimulation has been excluded(
      • Okada T.
      • Kawano Y.
      • Sabakibara T.
      • Hazeki O.
      • Ui M.
      ).
      The potential importance of PI 3-kinase as a mediator of insulin-stimulated hexose uptake has been suggested from studies in several different model systems. For example, there have been several demonstrations of an inverse correlation between insulin resistance and the activation of PI 3-kinase. In the hyperinsulinemic ob/ob diabetic mouse model, the amount of PI 3-kinase activity associated with the phosphorylated IRS-1 is decreased in muscle and liver(
      • Folli F.
      • Saad M.J.A.
      • Backer J.M.
      • Kahn C.R.
      ); insulin-stimulated PI 3-kinase activity is decreased in muscle from mice made obese and insulin-resistant by injections of gold thioglucose (
      • Heydrick S.J.
      • Jullien D.
      • Gautier N.
      • Tanti J.F.
      • Giorgetti S.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ); and long term treatment with dexamethasone also reduces the PI 3-kinase-associated IRS-1 in rat muscle(
      • Saad M.J.A.
      • Folli F.
      • Kahn J.A.
      • Kahn C.R.
      ). PI 3-kinase has been implicated more directly in studies on adipocytes using inhibitors of the lipid kinase. In isolated rat adipocytes, wortmannin blocks both the stimulation of hexose uptake and the inhibition of lipolysis by insulin(
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ,
      • Okada T.
      • Kawano Y.
      • Sabakibara T.
      • Hazeki O.
      • Ui M.
      ). Similar results have been obtained in 3T3 L1 adipocytes when another PI 3-kinase inhibitor, LY294002, was used(
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ). However, these studies have failed to distinguish whether a reduction in PtdIns(3,4)P2 and PtdIns(3,4,5)P3 affects the insulin-signaling process or more directly interferes with translocation of GLUT4 glucose transporters. There are now abundant data implicating a role for PI 3-kinase in vesicular transport events. In the human Hep G2 cell line, platelet-derived growth factor receptors lacking the high affinity binding sites for PI 3-kinase could not be internalized after platelet-derived growth factor stimulation(
      • Joly M.
      • Kazlauskas A.
      • Fay F.S.
      • Corvera S.
      ). Even more strikingly, the yeast PI 3-kinase homologue VPS34p plays a critical role in delivering newly synthesized proteins to the vacuoles (
      • Schu P.V.
      • Takegawa K.
      • Fry M.J.
      • Stack J.H.
      • Waterfield M.D.
      • Emr S.D.
      ). Exposure of mammalian fibroblasts to wortmannin alters the steady-state distribution of transferrin receptors in the absence of exposure of cells to mitogens.
      Y. Shibasaki and M. J. Birnbaum, unpublished observations.
      Thus, the ability of wortmannin to inhibit insulin-stimulated but not contraction-activated hexose uptake in skeletal muscle argues strongly that the site of action is specific to the insulin-signaling system and not the apparatus responsible for movement of transporters to the cell surface. This interpretation, of course, relies on the fact that both contraction and insulin stimulate hexose uptake predominantly via translocation of GLUT4 transporters, which, as described above, has been concluded from sub-cellular fractionation experiments.
      In summary, our results support the view that PI 3-kinase is necessary for insulin stimulation of hexose uptake but is not involved in contraction and hypoxia stimulation of hexose uptake.

      Acknowledgements

      We thank Glen A. Seidner for technical assistance, Dr. Lewis C. Cantley for helpful discussions and suggestions, and Diane C. Fingar and Sharon F. Hausdorff for reading the manuscript and discussions.

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