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Differential Effects of Phosphatidylinositol 3-Kinase Inhibition on Intracellular Signals Regulating GLUT4 Translocation and Glucose Transport*

Open AccessPublished:December 07, 2001DOI:https://doi.org/10.1074/jbc.M109093200
      Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC50 of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC50 = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC50 = 8 and 10 μm, respectively). The sensitivity of insulin-stimulated activation of PKCζ/λ, Akt1, Akt2, and Akt3 to wortmannin (IC50 = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC50of 7 nm. Insulin-dependent p38α and p38β MAPK activities were also markedly reduced by wortmannin (IC50 = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Δp85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKCζ/λ, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.
      PI
      phosphatidylinositol
      MAPK
      mitogen-activated protein kinase
      PKC
      protein kinase C
      PBS
      phosphate-buffered saline
      IRS
      insulin receptor substrate
      It has been known for 20 years that insulin causes recruitment of glucose transporters to the surface of muscle and fat cells (
      • Douen A.G.
      • Ramlal T.
      • Rastogi S.
      • Bilan P.J.
      • Cartee G.D.
      • Vranic M.
      • Holloszy J.O.
      • Klip A.
      ). However, numerous studies have concluded that the increase in the cell surface content of the muscle/fat-specific GLUT4 does not correlate quantitatively with the degree of stimulation of glucose uptake. This disparity between the magnitude of GLUT4 translocation and the stimulation of glucose uptake has been observed in mature skeletal muscle (
      • Hansen P.A.
      • Wang W.
      • Marshall B.A.
      • Holloszy J.O.
      • Mueckler M.
      ,
      • Goodyear L.J.
      • Hirshman M.F.
      • Smith R.J.
      • Horton E.S.
      ,
      • Winder W.W.
      • Hardie D.G.
      ,
      • King P.A.
      • Horton E.D.
      • Hirshman M.F.
      • Horton E.S.
      ), primary adipocytes (
      • Joost H.G.
      • Weber T.M.
      • Cushman S.W.
      ,
      • Clark A.E.
      • Holman G.D.
      • Kozka I.J.
      ,
      • Malide D.
      • Ramm G.
      • Cushman S.W.
      • Slot J.W.
      ), and muscle and fat cell lines (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ). Recently, we developed a muscle cell line overexpressing GLUT4 fused to a Myc epitope that becomes exposed at the cell surface, allowing for the detection of GLUT4 translocation in intact cells (
      • Wang Q.
      • Khayat Z.
      • Kishi K.
      • Ebina Y.
      • Klip A.
      ,
      • Ueyama A.
      • Yaworsky K.L.
      • Wang Q.
      • Ebina Y.
      • Klip A.
      ). Using this system, we reported that GLUT4 translocation precedes the stimulation of glucose uptake by at least 2 min (
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ). In addition, we have identified conditions in which insulin-dependent stimulation of glucose uptake can be reduced in the face of intact GLUT4 translocation (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Somwar R.
      • Koterski S.
      • Stashko M.
      • Sciotti R.
      • Djuric S.
      • Berg C.
      • Kramer D.
      • Ramlal T.
      • Trevillyan J.
      • Rondinone C.
      ). These conditions include diverse inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Somwar R.
      • Koterski S.
      • Stashko M.
      • Sciotti R.
      • Djuric S.
      • Berg C.
      • Kramer D.
      • Ramlal T.
      • Trevillyan J.
      • Rondinone C.
      ), low temperature (
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ), and leptin (
      • Sweeney G.
      • Keen J.
      • Somwar R.
      • Konrad D.
      • Klip A.
      ). Conversely, other studies have shown that glucose uptake can be augmented in 3T3-L1 adipocytes by insulin, while GLUT4 translocation is completely inhibited (
      • Van Epps-Fung M.
      • Gupta K.
      • Hardy R.W.
      • Wells A.
      ). Similarly, protein synthesis inhibitors elevate glucose uptake in 3T3-L1 adipocytes without any significant gain in cell surface GLUT4 content (
      • Harrison S.A.
      • Clancy B.M.
      • Pessino A.
      • Czech M.P.
      ,
      • Clancy B.M.
      • Harrison S.A.
      • Buxton J.M.
      • Czech M.P.
      ). Taken together, these studies have suggested that at least two events culminate in the stimulation of glucose uptake: translocation and activation of GLUT4. We have further proposed that p38 MAPK may be an integral component of the signaling pathway regulating GLUT4 activity (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ,
      • Somwar R.
      • Koterski S.
      • Stashko M.
      • Sciotti R.
      • Djuric S.
      • Berg C.
      • Kramer D.
      • Ramlal T.
      • Trevillyan J.
      • Rondinone C.
      ,
      • Somwar R.
      • Perreault M.
      • Kapur S.
      • Taha C.
      • Sweeney G.
      • Ramlal T.
      • Kim D.Y.
      • Keen J.
      • Cote C.H.
      • Klip A.
      • Marette A.
      ).
      It is well established that the activity of the lipid kinase phosphatidylinositol (PI)1kinase is necessary for GLUT4 translocation to the plasma membrane in muscle and fat tissues and cells, based on the use of pharmacological inhibitors (
      • Lund S.
      • Pryor P.R.
      • Ostergaard S.
      • Schmitz O.
      • Pedersen O.
      • Holman G.D.
      ,
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Tsakiridis T.
      • McDowell H.E.
      • Walker T.
      • Downes C.P.
      • Hundal H.S.
      • Vranic M.
      • Klip A.
      ,
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ,
      • Clarke J.F.
      • Young P.W.
      • Yonezawa K.
      • Kasuga M.
      • Holman G.D.
      ), expression of dominant-negative mutants of type 1 PI 3-kinase (
      • Wang Q.
      • Somwar R.
      • Bilan P.J.
      • Liu Z.
      • Jin J.
      • Woodgett J.R.
      • Klip A.
      ,
      • Cong L.N.
      • Chen H.
      • Li Y.
      • Zhou L.
      • McGibbon M.A.
      • Taylor S.I.
      • Quon M.J.
      ,
      • Sakaue H.
      • Ogawa W.
      • Takata M.
      • Kuroda S.
      • Kotani K.
      • Matsumoto M.
      • Sakaue M.
      • Nishio S.
      • Ueno H.
      • Kasuga M.
      ), or microinjection of PI 3-kinase-neutralizing antibodies (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ). Two downstream effectors of PI 3-kinase, the serine/threonine kinases Akt and atypical protein kinase C, appear to relay the signals required for GLUT4 translocation (
      • Hill M.M.
      • Clark S.F.
      • Tucker D.F.
      • Birnbaum M.J.
      • James D.E.
      • Macaulay S.L.
      ,
      • Bandyopadhyay G.
      • Kanoh Y.
      • Sajan M.P.
      • Standaert M.L.
      • Farese R.V.
      ,
      • Kotani K.
      • Ogawa W.
      • Matsumoto M.
      • Kitamura T.
      • Sakaue H.
      • Hino Y.
      • Miyake K.
      • Sano W.
      • Akimoto K.
      • Ohno S.
      • Kasuga M.
      ).
      It was recently demonstrated that glucose uptake in 3T3-L1 adipocytes was reduced by concentrations of wortmannin that do not affect GLUT4 translocation (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ). These results raise the question of where the signals regulating GLUT4 translocation and activation bifurcate. Answering this question was the objective of the present study. Using L6 myotubes expressing GLUT4myc, we determined the sensitivity of glucose uptake, GLUT4 translocation, and the signals thought to regulate these two parameters to wortmannin and LY294002. Strikingly, we found that very low concentrations of wortmannin, but not LY294002 or dominant negative PI 3-kinase (Δp85), prevented p38α and p38β MAPK activation by insulin. The stimulation of glucose uptake was also exquisitely susceptible to inhibition by wortmannin. In contrast, inhibition of Akt or PKCζ/λ activity by LY294002 and/or wortmannin correlated more closely with GLUT4 translocation.

      DISCUSSION

      Several studies have shown that increasing the amount of GLUT4 at the cell surface of skeletal muscle (
      • Hansen P.A.
      • Wang W.
      • Marshall B.A.
      • Holloszy J.O.
      • Mueckler M.
      ), rat adipocytes (
      • Clark A.E.
      • Holman G.D.
      • Kozka I.J.
      ), L6 muscle cells (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ), and 3T3-L1 adipocytes (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ) is not sufficient to elicit maximum stimulation of glucose uptake. In addition, it has been demonstrated that GLUT4 translocation precedes the stimulation of glucose uptake in L6 muscle cells (
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ) and rat adipocytes (
      • Clark A.E.
      • Holman G.D.
      • Kozka I.J.
      ). A view that emerged from these studies is that two events culminate in full stimulation of glucose uptake: (a) translocation of GLUT4 to the cell surface and (b) increase in GLUT4 activity.

      Differential Sensitivity of Glucose Uptake and GLUT4 Translocation to Wortmannin

      The differential sensitivity to wortmannin of the stimulation of glucose uptake and GLUT4 translocation by insulin was the first evidence for a dual input resulting in glucose uptake in this study. Glucose uptake was inhibited by wortmannin with an IC50 of 3 nm. In contrast, the drug prevented the arrival of GLUT4 at the cell surface with an IC50 of 43 nm. The assay used to determine the amount of GLUT4 on the cell surface is based on quantitative immunological detection of the exofacial Myc epitope on the GLUT4 molecule (i.e. it detects transporters that are fully inserted in the plasma membrane and exposed to the extracellular milieu). Glucose transport, therefore, displayed a higher sensitivity to wortmannin than GLUT4 translocation, by 1 order of magnitude. A similar observation was made in 3T3-L1 adipocytes (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ), where the IC50 of the drug was 6 nm for glucose uptake and 80 nm for GLUT4 translocation. In the latter study, GLUT4 translocation was detected by exposure of another exofacial epitope using fluorescence microscopy (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ). Collectively, these results suggest that two different wortmannin targets might regulate these processes. It is unlikely that the inhibition of glucose uptake by wortmannin is due to a direct interaction of wortmannin with GLUT4, since the drug had no effect on the stimulation of glucose uptake by dinitrophenol in L6 myotubes (
      • Tsakiridis T.
      • Vranic M.
      • Klip A.
      ) or by contraction of rat skeletal muscle (
      • Lund S.
      • Pryor P.R.
      • Ostergaard S.
      • Schmitz O.
      • Pedersen O.
      • Holman G.D.
      ,
      • Yeh J.I.
      • Gulve E.A.
      • Rameh L.
      • Birnbaum M.J.
      ) at concentrations as high as 1 μm. In addition, wortmannin does not affect the stimulation of glucose uptake caused by expression of constitutively active Akt mutants in L6 myotubes and 3T3-L1 adipocytes (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ,
      • Ueki K.
      • Yamamoto-Honda R.
      • Kaburagi Y.
      • Yamauchi T.
      • Tobe K.
      • Burgering B.M.
      • Coffer P.J.
      • Komuro I.
      • Akanuma Y.
      • Yazaki Y.
      • Kadowaki T.
      ,
      • Hajduch E.
      • Alessi D.R.
      • Hemmings B.A.
      • Hundal H.S.
      ). It is easy to envisage that concentrations of wortmannin that inhibit GLUT4 translocation (>25 nm) will prevent stimulation of glucose uptake in response to insulin. However, the fact that 60% of the insulin response of glucose uptake is inhibited at concentrations that do not affect GLUT4 availability at the cell surface and that wortmannin does not affect GLUT4 per se suggest the existence of a different signal targeted by the drug that leads to activation of the translocated transporters.
      To begin to examine whether the differential sensitivity to wortmannin of GLUT4 translocation and putative activation of GLUT4 is due to different PI 3-kinase inputs (e.g. through different PI 3-kinase isoforms or PI 3-kinase products), we compared the sensitivity of both phenomena to another chemically unrelated inhibitor of PI 3-kinase, LY294002. Interestingly, glucose uptake and GLUT4 translocation were inhibited with similar potency by LY294002. The simplest interpretation is that the reduction in glucose uptake observed in the presence of LY294002 is due mainly to a reduction in cell surface GLUT4 levels. Intriguingly, LY294002 reduced the stimulation of glucose uptake by only 70% when GLUT4 translocation was fully blocked. In contrast, in the presence of wortmannin, the stimulation of glucose uptake was completely abrogated when cell surface GLUT4 was reduced to basal levels. These observations raise the possibility that GLUT4 activity may be regulated by either a PI 3-kinase that is highly sensitive to wortmannin but not to LY294002 or by another wortmannin-sensitive target that is not a PI 3-kinase.
      When the sensitivity to wortmannin was measured in vitro, PI 3-kinases associated with IRS-1 or phosphotyrosine-containing proteins were inhibited with IC50 values of 0.3 and 0.5 nm, respectively. These values are clearly lower than even those determined for inhibition of glucose uptake in intact cells, consistent with the view that, in vivo, higher concentrations of wortmannin may be needed to inhibit PI 3-kinases. It has recently been suggested that this difference may be due to the high concentration of intracellular ATP that competes with wortmannin for binding to PI 3-kinase (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ). We were unable to determine the IC50 for inhibition of PI 3-kinase by wortmannin in intact cells, because the drug does not remain bound to the enzyme following isolation of the IRS-1 or phosphotyrosine immunoprecipitates. Therefore, it is difficult to draw any conclusion about the participation of PI 3-kinases in GLUT4 activation or GLUT4 traffic based solely on the sensitivity to wortmannin of PI 3-kinase measuredin vitro.

      Inhibition of the Signaling Pathway Regulating GLUT4 Activation and the Stimulation of Glucose Uptake by Wortmannin but Not LY294002 or Δp85

      We recently demonstrated that several structurally different inhibitors of p38 MAPK reduced glucose uptake but not GLUT4 translocation (
      • Sweeney G.
      • Somwar R.
      • Ramlal T.
      • Volchuk A.
      • Ueyama A.
      • Klip A.
      ,
      • Somwar R.
      • Kim D.Y.
      • Sweeney G.
      • Huang C.
      • Niu W.
      • Lador C.
      • Ramlal T.
      • Klip A.
      ) in both L6 myotubes and 3T3-L1 adipocytes. This situation is highly reminiscent of the observations reported in the present study at low concentrations of wortmannin. In addition, in our hands expression of a dominant negative p38 MAPK mutant in 3T3-L1 adipocytes reduced glucose uptake (

      Somwar, R., Niu, W., Kim, D., Rhandawa, V., Ramlal, T., Sweeney, G., and Klip, A. (2001) in VIIIth International Symposium on Insulin Receptors and Insulin Action, Geneva, Switzerland

      ). Together, these studies suggested that GLUT4 activity may be regulated by a p38 MAPK-dependent pathway. Accordingly, we report here that the stimulation of p38 MAPK activity by insulin also displayed high sensitivity to inhibition by wortmannin. Insulin-stimulated p38 MAPK phosphorylation and activation of p38α and p38β isoforms were inhibited by wortmannin with IC50 values of 7, 6, and 2 nm, respectively. It is unlikely that wortmannin interacts directly with p38 MAPK, since wortmannin prevented insulin-induced phosphorylation of p38 MAPK, a step that is dependent on an upstream kinase. In addition, treatment of intact cells with wortmannin had no effect on the activation of p38 MAPK by anisomycin or mannitol (data not shown). Furthermore, it was recently reported that wortmannin did not inhibit recombinant p38 MAPK in in vitro kinase assays (
      • Davies S.P.
      • Reddy H.
      • Caivano M.
      • Cohen P.
      ). Our results support the view that insulin engages a wortmannin-sensitive target to activate p38 MAPK, leading to enhanced GLUT4 activity and maximum stimulation of glucose uptake. The identity of this high affinity wortmannin target remains to be determined.
      Contrary to the exquisite sensitivity of the activation of p38 MAPK by insulin to wortmannin, this response was largely unaffected by concentrations of LY294002. Only concentrations of LY294002 higher than those required for inhibition of GLUT4 translocation affected p38 MAPK phosphorylation. This result is consistent with the observation, described above, that there was no detectable reduction in GLUT4 activity by LY294002 independently of GLUT4 translocation. Moreover, expression of Δp85 to inhibit type IA PI 3-kinases did not reduce p38 MAPK phosphorylation, akin to the results obtained with LY294002. Under similar conditions, Δp85 completely prevented the arrival of GLUT4 at the cell surface in response to insulin. These results support the notion that insulin activates p38 MAPK through a mechanism that involves a highly sensitive wortmannin target that is not a type IA PI 3-kinase. In agreement with this conclusion, it was reported that wortmannin inhibits bombesin-stimulated cytosolic phospholipase A2 activity in Swiss 3T3 cells with an IC50 of 2 nm (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.
      ). This inhibition of enzyme activity was not due to inhibition of PI 3-kinase and could not be accounted for by direct inhibition of cytosolic phospholipase A2 by wortmannin (
      • Cross M.J.
      • Stewart A.
      • Hodgkin M.N.
      • Kerr D.J.
      • Wakelam M.J.
      ). These studies support the existence of a highly sensitive wortmannin target.

      Signaling Pathway Regulating GLUT4 Translocation

      In contrast to the emerging knowledge about the signals regulating GLUT4 activity, much more is known about the signals that meditate insulin-stimulated GLUT4 translocation. Many studies have shown that PI 3-kinase plays an important role in mediating this effect. The numerous experimental approaches showing a need for PI 3-kinase include the use of the pharmacological inhibitors wortmannin (
      • Tsakiridis T.
      • McDowell H.E.
      • Walker T.
      • Downes C.P.
      • Hundal H.S.
      • Vranic M.
      • Klip A.
      ,
      • Okada T.
      • Kawano Y.
      • Sakakibara T.
      • Hazeki O.
      • Ui M.
      ) and LY294002 (
      • Cheatham B.
      • Vlahos C.J.
      • Cheatham L.
      • Wang L.
      • Blenis J.
      • Kahn C.R.
      ) and expression of both inhibitory (
      • Wang Q.
      • Somwar R.
      • Bilan P.J.
      • Liu Z.
      • Jin J.
      • Woodgett J.R.
      • Klip A.
      ,
      • Kotani K.
      • Carozzi A.J.
      • Sakaue H.
      • Hara K.
      • Robinson L.J.
      • Clark S.F.
      • Yonezawa K.
      • James D.E.
      • Kasuga M.
      ) and constitutively active (
      • Tanti J.F.
      • Gremeaux T.
      • Grillo S.
      • Calleja V.
      • Klippel A.
      • Williams L.T.
      • Van Obberghen E.
      • Le Marchand-Brustel Y.
      ,
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ) mutant constructs of PI 3-kinase. More recently, microinjection of an antibody to the p110 catalytic subunit of PI 3-kinase (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ) or of peptides encompassing the Src homology 2 domain of p85 (
      • Haruta T.
      • Morris A.J.
      • Vollenweider P.
      • Nelson J.G.
      • Rose D.W.
      • Mueckler M.
      • Olefsky J.M.
      ) resulted in a reduction in insulin-stimulated GLUT4 translocation. Microinjection of antibody to the 3′ lipid phosphatase PTEN increased basal and insulin-stimulated GLUT4 translocation, whereas overexpression of PTEN reduced basal and insulin-stimulated GLUT4 translocation and glucose uptake (
      • Nakashima N.
      • Sharma P.M.
      • Imamura T.
      • Bookstein R.
      • Olefsky J.M.
      ). We have shown that GLUT4 translocation (
      • Sweeney G.
      • Somwar R.
      • Foster L.
      • Jiang T.
      • Neilsen P.O.
      • Prestwich G.D.
      • Klip A.
      ), but not glucose uptake (
      • Sweeney G.
      • Somwar R.
      • Foster L.
      • Jiang T.
      • Neilsen P.O.
      • Prestwich G.D.
      • Klip A.
      ,
      • Jiang T.
      • Sweeney G.
      • Rudolf M.T.
      • Klip A.
      • Traynor-Kaplan A.
      • Tsien R.Y.
      ), is stimulated by the introduction of phosphatidylinositol 3,4,5-trisphosphate into 3T3-L1 adipocytes and L6 muscle cells (
      • Jiang T.
      • Sweeney G.
      • Rudolf M.T.
      • Klip A.
      • Traynor-Kaplan A.
      • Tsien R.Y.
      ). This finding supports the notion that at least this PI 3-kinase product may not be involved in regulating GLUT4 activity. Alternatively, it is possible that GLUT4 translocation requires a lower level of phosphatidylinositol 3,4,5-trisphosphate than is required for the stimulation of glucose uptake. Such a scenario would explain why a higher concentration of wortmannin is needed to fully inhibit GLUT4 translocation. However, this remains a weak possibility, because we did not observe a differential sensitivity to LY294002 of glucose uptake and GLUT4 translocation, in agreement with results in 3T3-L1 adipocytes (
      • Hausdorff S.F.
      • Fingar D.C.
      • Morioka K.
      • Garza L.A.
      • Whiteman E.L.
      • Summers S.A.
      • Birnbaum M.J.
      ). Strengthening this argument would require measuring the endogenous levels of PI 3-kinase products under the different conditions and the subcellular location of these lipids. PI 3-kinase products are thought to mediate GLUT4 translocation by activating Akt and/or atypical PKC isoforms (
      • Bandyopadhyay G.
      • Kanoh Y.
      • Sajan M.P.
      • Standaert M.L.
      • Farese R.V.
      ,
      • Bandyopadhyay G.
      • Standaert M.L.
      • Galloway L.
      • Moscat J.
      • Farese R.V.
      ). In the present study, Akt isoforms and PKCζ/λ activities were inhibited by wortmannin with IC50 values that were similar and correlated closely with inhibition of GLUT4 translocation.
      In summary, our results suggest a segregation of signaling events leading to GLUT4 translocation and GLUT4 activation. We demonstrate here a differential sensitivity of insulin-stimulated GLUT4 translocation and glucose uptake to wortmannin. The lower sensitivity of GLUT4 translocation correlates with the sensitivity of insulin-induced Akt and PKCζ/λ activity to inhibition by wortmannin. In contrast, the high sensitivity of glucose uptake to inhibition by wortmannin correlates with inhibition of p38 MAPK. Collectively, these results suggest that a cellular target of insulin with a high affinity for wortmannin regulates the activation of p38 MAPK and GLUT4. We also suggest that such target is unlikely to be a type IA PI 3-kinase, since LY294002 and Δp85 did not inhibit insulin-stimulated p38 MAPK phosphorylation.

      ACKNOWLEDGEMENT

      We thank Dr. P. J. Bilan for helpful suggestions and critical reading of the manuscript.

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