An Inhibitor of p38 Mitogen-activated Protein Kinase Prevents Insulin-stimulated Glucose Transport but Not Glucose Transporter Translocation in 3T3-L1 Adipocytes and L6 Myotubes

Abstract

The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 μm). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane. The precise mechanisms underlying insulin-stimulated glucose transport still require investigation. Here we assessed the effect of SB203580, an inhibitor of the p38 MAP kinase family, on insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 myotubes. We found that SB203580, but not its inactive analogue (SB202474), prevented insulin-stimulated glucose transport in both cell types with an IC50 similar to that for inhibition of p38 MAP kinase (0.6 μm). Basal glucose uptake was not affected. Moreover, SB203580 added only during the transport assay did not inhibit basal or insulin-stimulated transport. SB203580 did not inhibit insulin-stimulated translocation of the glucose transporters GLUT1 or GLUT4 in 3T3-L1 adipocytes as assessed by immunoblotting of subcellular fractions or by immunofluorescence of membrane lawns. L6 muscle cells expressing GLUT4 tagged on an extracellular domain with a Myc epitope (GLUT4myc) were used to assess the functional insertion of GLUT4 into the plasma membrane. SB203580 did not affect the insulin-induced gain in GLUT4myc exposure at the cell surface but largely reduced the stimulation of glucose uptake. SB203580 had no effect on insulin-dependent insulin receptor substrate-1 phosphorylation, association of the p85 subunit of phosphatidylinositol 3-kinase with insulin receptor substrate-1, nor on phosphatidylinositol 3-kinase, Akt1, Akt2, or Akt3 activities in 3T3-L1 adipocytes. In conclusion, in the presence of SB203580, insulin caused normal translocation and cell surface membrane insertion of glucose transporters without stimulating glucose transport. We propose that insulin stimulates two independent signals contributing to stimulation of glucose transport: phosphatidylinositol 3-kinase leads to glucose transporter translocation and a pathway involving p38 MAP kinase leads to activation of the recruited glucose transporter at the membrane. phosphatidylinositol 3-kinase insulin receptor substrate-1 protein kinase B mitogen-activated protein kinase MAP kinase kinase activating transcription factor-2 interleukin-1 phosphate-buffered saline polyacrylamide gel electrophoresis phenylmethylsulfonyl fluoride The phenomenon of insulin-stimulated glucose transporter (GLUT) translocation from an intracellular location to the plasma membrane has been demonstrated in several fat and muscle cell systems since its initial report in 1980 (1Cushman S.W. Wardzala L.J. J. 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SB203580 also glucose transport in 3T3-L1 adipocytes L.F. M. J. S.A. J. 1997; PubMed Scopus Google Scholar), with an IC50 of In the we assessed the effect of SB203580 and its inactive on insulin-stimulated glucose transport. We report that SB203580 uptake of or in muscle and fat cells without with translocation of effect was not due to of SB203580 to GLUTs nor to a effect on signaling The that SB203580 the of insulin to the intrinsic activity of glucose transporter recruited to the plasma membrane. cell and were from 3T3-L1 cells were a from of L6 cells with GLUT4 were by Y. of insulin was from and protein were from and glucose transporter was from to and p38 MAP kinase and to Myc were from and and were from and were from and were from was from and and were from was from (PI) was from were from was from was from SB203580 and were from electrophoresis and immunoblotting were from were of the 3T3-L1 cells were in in with and and in an of at 3T3-L1 were into adipocytes as A. Liu L. Klip A. Mol. Biol. Cell. 1996; 7: PubMed Scopus Google Scholar). L6 cells from a of the L6 muscle cells and L6 cells GLUT4 tagged with a Myc epitope were and into as Y. Klip A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar). transport cells were with and in and in as with were the in for of cell and in for with insulin or SB203580 as were in of and Cell were for at to cell and was added to for at by of of protein for The were with and in of for and by MAP kinase was added to the by to and protein was by the of p38 MAP kinase and and of their kinase activity was in a similar to that for R. S. C. Klip A. J. 1998; Google Scholar). both cells were with and MAP kinase to a of protein and protein was added to of protein from cell and were also to a of protein and protein and added to of were with and with were by with the for were and with of and and with of kinase and The were for at with of of of as in p38 MAP kinase and as in the of the was and for with of and with for were and to L6 were of for with and glucose 3T3-L1 adipocytes were of by in for uptake were as R. Ramlal T. Klip A. 1998; Full Text PDF PubMed Scopus Google Scholar). and with cell were with saline and any was were for in saline and for L6 and for in the of The was by with uptake was in the presence of Cell was by the cells with by protein was by the PubMed Scopus Google Scholar). uptake was in a similar with the was added to saline and uptake to for a which uptake is to be cell were with in saline with 3T3-L1 on in were with SB203580 or insulin as in the membrane were as L.J. S. J. J. Cell Biol. 1992; PubMed Scopus Google Scholar) with the the cells were on and in was added in of were added to and the was and a to cell The were in and with in for on by in was with for by with at The were by a in in at with for at and in was added for with with and the with were a with a were gain was was as E. A. R. L. Liu Klip A. J. 1993; PubMed Google Scholar). were in and and the at for The was at to the plasma the from was at for to the plasma were by on a and and at in a for The at the was in and at for to plasma fractions were in to a of and at The of GLUT4 to the cell surface was by an assay K. Y. Klip A. FEBS Lett. 1998; PubMed Scopus Google Scholar) as L6 GLUT4myc cells as in the for were with with in for at and the was by with in at for The cells were with and in at for at was added into the at a of and for at The cells were extensively with at the cells were extensively and of and in was added to well for at The was by of of The was and the was at of of SB203580 to assay during of uptake in 3T3-L1 uptake in L6 muscle adipocytes and L6 were with or without insulin for to of uptake as uptake was in the presence or of SB203580 during the assay the of one of was in in a of SB203580 on transport and GLUT4 translocation in L6 GLUT4myc of SB203580 on of SB203580 on GLUT4myc translocation to plasma with GLUT4 were with or without SB203580 for by insulin for in the were uptake or Myc epitope exposure at the cell surface were as to basal and represent of which was in in a 3T3-L1 adipocytes and L6 were with or without insulin for to of uptake as uptake was in the presence or of SB203580 during the assay the of one of was in L6 with GLUT4 were with or without SB203580 for by insulin for in the were uptake or Myc epitope exposure at the cell surface were as to basal and represent of which was in 3T3-L1 adipocytes were with for was and of and association of the p85 subunit of PI 3-kinase was as R. Ramlal T. Klip A. 1998; Full Text PDF PubMed Scopus Google Scholar). were by and To the of the was with and protein by the to as The of the was with and protein to To PI 3-kinase cell were the as for and PI 3-kinase activity was on as T. T. M. Klip A. Endocrinology. 1995; 136: PubMed Google Scholar). the of PI 3-kinase with to phosphatidylinositol to phosphatidylinositol was by of by and of on the were a was or of as in the of p38 MAP kinase on and stimulated of p38 MAP kinase in 3T3-L1 adipocytes by by with MAP kinase We a similar in L6 T. C. S. Klip A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). SB203580, which with the domain of p38 MAP kinase S. S. S. S.A. M.J. J.L. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus (535) Google Scholar), did not affect the basal or insulin-stimulated of p38 in kinase assay was used to the of p38 MAP kinase to one of its caused an in and effect was by SB203580 a of the effect of SB203580 on the uptake of into 3T3-L1 adipocytes. The was to cells for to of with the for caused an in glucose uptake of protein with a of of SB203580 from to prevented stimulation by insulin in a The IC50 for effect was to be the of SB203580 had no effect on basal uptake in B that the effect of SB203580 on insulin-stimulated transport was also in L6 myotubes. In the IC50 was and SB203580 had no effect on basal glucose uptake. the effect of SB203580 with that of the similar on insulin-stimulated and transport in 3T3-L1 adipocytes. is to inhibit p38 MAP kinase and in is to be a inactive analogue of SB203580 S. M.J. S.W. J.L. PubMed Scopus Google Scholar). from in SB203580 insulin-stimulated uptake by had no effect on the of insulin to uptake. SB203580 nor had any effect on basal transport To whether the effect of SB203580 is on glucose transport activity and not on of the we its on uptake of the glucose B that SB203580 prevented the stimulation of uptake by insulin to a similar as prevented stimulation of uptake. The inactive analogue had no effect on insulin-stimulated uptake. In the cells were for with SB203580 insulin In to whether SB203580 has on the transport cells were with or without insulin and SB203580 was added only during the of the glucose uptake SB203580 had no effect on basal or insulin-stimulated uptake in 3T3-L1 adipocytes or L6 were used to assess the effect of SB203580 on insulin-induced glucose transporter translocation in 3T3-L1 adipocytes. the content of plasma glucose transporters was by of plasma membrane and immunofluorescence of GLUT1 and an in the plasma membrane content of both GLUT1 and GLUT4 in to which was by of cells with of the for GLUT4 on membrane insulin SB203580, The of GLUT1 on membrane were as insulin SB203580, There was also no effect of SB203580 on GLUT1 and GLUT4 basal not the of effect of SB203580 on glucose transporter with the plasma membrane in the of inhibition of glucose transport was not we also the insulin-stimulated glucose transporter translocation by subcellular to the effect of SB203580 on both the and the plasma of membrane protein from subcellular fractions from cells with or without SB203580 and with or without insulin were by and immunoblotting The the in plasma membrane that SB203580 not the translocation of GLUT1 or GLUT4 to the plasma membrane from intracellular It was in the presence of SB203580, GLUT4 might be but not with the plasma and that membrane and subcellular not be to two To we the effect of SB203580 on the insulin-dependent of GLUT4 tagged with a Myc epitope that is GLUT4 is in the plasma membrane. one to not only the GLUT4 translocation to the cell surface but also the functional insertion of transporter In L6 expressing GLUT4 we that SB203580 prevented the insulin-dependent stimulation of uptake by The exposure of GLUT4 on the cell was not by SB203580 the that SB203580 insulin-dependent glucose transport but not glucose transporter at the cell in the insulin signaling pathway for stimulation of glucose transport is of to activate PI pathway is required for translocation of GLUTs but is not can also the intrinsic activity of We the effect of SB203580 on the of insulin to in to gain on its of leading to of stimulation of glucose uptake by was from 3T3-L1 cell with and without insulin or SB203580, by to with that the insulin-induced of was not by The of p85 with was increased by insulin and effect was also not prevented by SB203580 We the effect of SB203580 on the stimulation of PI 3-kinase activity by insulin an in kinase assay to the of PI 3-kinase with in cell to into B that insulin PI 3-kinase activity and that SB203580 had no effect on insulin-stimulated or basal PI 3-kinase activity in is by of PI 3-kinase and several a for in insulin-stimulated glucose transport in 3T3-L1 adipocytes and L6 M.R. Martinez C. Liu H. El Jack A.K. Birnbaum M.J. Pilch P.F. J. Biol. Chem. 1998; 273: 7201-7204Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, E. 1998; PubMed Scopus (296) Google Scholar). To the effect of SB203580 on stimulation of and Akt3 by insulin were with and in activity as We found that insulin stimulated in 3T3-L1 adipocytes and stimulation was not by SB203580 SB203580 also had no effect on basal Akt1, Akt2, or Akt3 of stimuli the of protein leading to activation of p38 MAP the being as and as well as A. S. R. 1997; PubMed Scopus Google Scholar). p38 MAP kinase is by on and two MAP kinase and been as the that and activate p38 MAP kinase R. J. A. P. J. 1996; PubMed Scopus (112) Google Scholar). were found to be to activate the has been to activate both and been as of A. S. R. 1997; PubMed Scopus Google Scholar). substrates been to for p38 MAP MAP protein and A. S. R. 1997; PubMed Scopus Google Scholar). regulation of p38 MAP kinase and to be in a of p38 MAP kinase been and Y. H. M. L. J. L. J. J. Biol. Chem. 1997; 272: PubMed Scopus Google Scholar). and and by SB203580 A. FEBS Lett. 1998; PubMed Scopus Google Scholar). In to muscle also the Y. J. 1996; PubMed Scopus Google Scholar). The found in 3T3-L1 cells not been of and the and of p38 MAP kinase by insulin is an signaling phenomenon and of p38 MAP kinase by to in a It was in cells that insulin-induced and activation of p38 MAP kinase W. S. M. 1998; PubMed Scopus Google Scholar) and insulin stimulation of p38 MAP kinase was also demonstrated in cells C. 1997; PubMed Scopus Google Scholar). insulin of p38 and p38 activity in the is a J.L. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). activation of p38 MAP kinase by insulin in muscle A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), yet in a no stimulation of p38 MAP kinase was L.J. J. 1996; 271: Google Scholar). is on regulation of p38 MAP kinase activity by insulin in that insulin stimulated p38 MAP kinase activity in 3T3-L1 and adipocytes K. 1996; Scholar). In the we and that p38 activation by insulin is by We that SB203580, a cell inhibitor of p38 MAP the stimulation of glucose transport by of GLUT1 and GLUT4 to the cell surface is an of the stimulation of glucose transport by insulin in 3T3-L1 adipocytes. still remains whether of the plasma membrane with GLUTs can account for insulin-stimulated glucose uptake (4Zierler K. Diabetologia. 1998; 41: 724-730Crossref PubMed Scopus (23) Google Scholar), and one that to only 30% of the gain in glucose transport. We two with 3T3-L1 adipocytes of plasma membrane by immunofluorescence or immunoblotting of subcellular to the inhibition of insulin-stimulated glucose transport by SB203580 be by a of glucose transporters at the plasma membrane. we that in the presence of SB203580, insulin was to normal increases in the of GLUT1 and GLUT4 at the plasma membrane without a stimulation of glucose transport. the a to increased glucose transporter at the cell surface from increased glucose transport with both of is that that glucose transporters into the plasma membrane is not that in glucose can be but from the membrane S.A. J.M. Czech M.P. J. Biol. Chem. Full Text PDF PubMed Google Scholar, H. S. S.W. G.D. J. 1992; PubMed Scopus Google Scholar). to the glucose transporters were into the plasma membrane we the L6 muscle cells GLUT4 that is tagged with a Myc epitope on the surface K. Y. Klip A. FEBS Lett. 1998; PubMed Scopus Google Scholar). cells can be used to the of GLUT4 into the plasma membrane by exposure of the Myc epitope on the surface of insulin-induced translocation and of GLUT4myc into the plasma membrane was not by the with 3T3-L1 adipocytes or cells that insertion of glucose transporters into the plasma membrane in to insulin is not to account for the increased of glucose the membrane. or which is prevented by SB203580 is required to the cell surface transporters into functional insertion into the plasma membrane. that the effect of SB203580 was to inhibit insulin-stimulated glucose transport by we that insulin-stimulated glucose transport require a of increased GLUT translocation and increased intrinsic activity of The also that insulin and p38 MAP kinase in 3T3-L1 adipocytes. SB203580 insulin-stimulated kinase activity but had no effect on insulin-stimulated phosphorylation, did not with the signaling pathway involved in activation of p38 MAP kinase by on its to SB203580, a for p38 has been in the stimulation of glucose transport by in 3T3-L1 adipocytes and in cells A. P. J. 1995; PubMed Scopus Google Scholar, L.F. M. J. S.A. J. 1997; PubMed Scopus Google Scholar). did not a of glucose transporters to the cell surface and was proposed to the intrinsic activity of GLUT1 glucose transporters that at the cell surface L.F. M. J. S.A. J. 1997; PubMed Scopus Google Scholar). stimulates glucose transport into adipocytes by the functional activity of in a Y. S. H. Y. S.W. T. J. 1998; PubMed Scopus Google Scholar). There is also for activation of glucose transporters as of the by which insulin stimulates glucose transport in fat cells M. Harel C. Burvin R. Karnieli E. Endocrinology. 1995; 136: 3292-3298Crossref PubMed Scopus (20) Google Scholar, H. T. K. T. H. Y. T. J. M. Y. Y. J. 1997; 272: Google Scholar). of activation has been by the that and insulin a of glucose transporters to the cell has been to the for activation of transporters in the of increased of transporters on the cell due to in the of insulin-stimulated PI 3-kinase activity and glucose uptake in 3T3-L1 adipocytes has been that an pathway is for insulin-stimulated glucose transport H. T. K. T. H. Y. T. J. M. Y. Y. J. 1997; 272: Google Scholar). Moreover, we that of PI 3-kinase the insulin stimulation of glucose the inhibition of insulin caused by T. Klip A. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). also that signals to PI 3-kinase for the stimulation of glucose transport in 3T3-L1 adipocytes. In the we evidence to and that insulin stimulates both translocation and intrinsic activity of with the that the PI 3-kinase pathway is involved in GLUT We also signaling pathway involving p38 MAP which in increased intrinsic activity of required for stimulation of glucose transport. is that SB203580 to the recruited glucose their a was for the which insulin-stimulated glucose transport in a to its stimulation of A. Ramlal T. Bilan P.J. J. 255: PubMed Scopus Google Scholar). was found to affect glucose transport by a with GLUT4 without GLUT1 Mol. Pharmacol. Google Scholar). In the effect of SB203580 not be to from inhibition of glucose transporters since insulin-stimulated glucose transport was not by the presence of SB203580 during the transport that SB203580 not with glucose transporters. the that SB203580 insulin-dependent glucose transport by with an intracellular is by the the IC50 of SB203580 for the inhibition of glucose transport in 3T3-L1 adipocytes was to that for inhibition of p38 MAP kinase (0.6 and an inactive analogue of SB203580 was without effect on insulin-stimulated glucose transport in 3T3-L1 adipocytes. p38 MAP kinase in the insulin pathway leading to the stimulation of glucose transport. of glucose into cells is by its to glucose for uptake been that SB203580 of glucose To we assessed the of SB203580 on insulin-stimulated transport of the but similar to with that inhibition of glucose transport by SB203580 is at the of transport and not at the of The of effect of SB203580 on insulin-stimulated translocation of GLUT1 and GLUT4 to the cell surface that inhibitor not affect the signaling required for the of insulin-induced or activation of several of the insulin signaling pathway that be for insulin-dependent GLUT translocation were not by of was as was association of the subunit of PI 3-kinase with of PI 3-kinase and Akt1, Akt2, and Akt3 by insulin were also by The of SB203580 to inhibit insulin-stimulated activation of of also to M. P. P. J. 1996; 15: PubMed Scopus Google Scholar), p38 is not in the signaling pathway leading to the activation of in to In conclusion, we that the inhibitor of p38 MAP SB203580, can insulin-stimulated glucose uptake in 3T3-L1 adipocytes and L6 muscle effect is not due to a effect on insulin signaling pathways nor on glucose transporters. In the of SB203580 not at the of glucose with the translocation of glucose transporters is not the for the effect of We propose that glucose transporters their translocation to the plasma membrane and that activation is prevented by SB203580, by its to inhibit insulin-dependent activation of p38 MAP We Y. of for GLUT4myc into L6 muscle cells and of for the 3T3-L1 also to P. Bilan for on