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Opposite Translational Control of GLUT1 and GLUT4 Glucose Transporter mRNAs in Response to Insulin

ROLE OF MAMMALIAN TARGET OF RAPAMYCIN, PROTEIN KINASE B, AND PHOSPHATIDYLINOSITOL 3-KINASE IN GLUT1 mRNA TRANSLATION*
Open AccessPublished:November 12, 1999DOI:https://doi.org/10.1074/jbc.274.46.33085
      Prolonged exposure of 3T3-L1 adipocytes to insulin increases GLUT1 protein content while diminishing GLUT4. These changes arise in part from changes in mRNA transcription. Here we examined whether there are also specific effects of insulin on GLUT1 and GLUT4 mRNA translation. Insulin enhanced association of GLUT1 mRNA with polyribosomes and decreased association with monosomes, suggesting increased translation. Conversely, insulin arrested the majority of GLUT4 transcripts in monosomes. Insulin inactivates the translational suppressor eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) through the mammalian target of rapamycin (mTOR). Hence, we examined the effect of rapamycin on GLUT1 mRNA translation and protein expression. Rapamycin abrogated the insulin-mediated increase in GLUT1 protein synthesis through partial inhibition of GLUT1 mRNA translation and partial inhibition of the rise in GLUT1 mRNA. 4E-BP1 inhibited GLUT1 mRNA translationin vitro. Because phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB), in concert with mTOR, inactivate 4E-BP1, we explored their role in GLUT1 protein expression. Cotransfection of cytomegalovirus promoter-driven, hemagglutinin epitope-taggedGLUT1 with dominant inhibitory mutants of PI3K or PKB inhibited the insulin-elicited increase in hemagglutinin-tagged GLUT1 protein. These results unravel the opposite effects of insulin on GLUT1 and GLUT4 mRNA translation. Increased GLUT1 mRNA translation appears to occur via the PI3K/PKB/mTOR/4E-BP1 cascade.
      eIF-4
      eukaryotic initiation factor 4
      4E-BP
      eIF-4E-binding protein
      mTOR
      mammalian target of rapamycin
      PI3K
      phosphatidylinositol 3-kinase
      PKB
      protein kinase B
      HA
      hemagglutinin
      GST
      glutathione S-transferase
      GAPDH
      glyceraldehyde-3-phosphate dehydrogenase
      UTR
      untranslated region
      MEK
      mitogen-activated protein kinase/extracellular signal-regulated kinase kinase
      Glucose transport into most tissues occurs through the action of members of a family of facilitative diffusion glucose transport proteins designated GLUT1–5 (
      • Gould G.W.
      • Holman G.D.
      ). GLUT1 is ubiquitously distributed and has been proposed to act as a constitutive transport protein (
      • Gould G.W.
      • Holman G.D.
      ). In contrast, the GLUT4 isoform is expressed almost exclusively in adipose cells and skeletal muscles, tissues responsible for the major portion of insulin stimulation of glucose transport after a meal (
      • Birnbaum M.J.
      ,
      • James D.E.
      • Strube M.
      • Mueckler M.
      ). An acute insulin challenge results in an increase in glucose uptake via the translocation of GLUT proteins, mainly GLUT4, from internal stores to the plasma membrane (
      • Birnbaum M.J.
      ,
      • Cushman S.W.
      • Wardzala L.J.
      ,
      • James D.E.
      • Brown R.
      • Navarro J.
      • Pilch P.F.
      ,
      • Guma A.
      • Zierath J.R.
      • Wallberg-Henriksson H.
      • Klip A.
      ).
      In addition to its acute effect on the redistribution of glucose transporters, insulin also exerts long-term regulation of glucose transporter concentration. Prolonged exposure to insulin, a condition that occurs in type II diabetes, which is characterized by insulin resistance and compensatory hyperinsulinemia, results in an increase in GLUT1 protein levels (
      • Ciaraldi T.P.
      • Abrams L.
      • Nikoulina S.
      • Mudaliar S.
      • Henry R.R.
      ). In cells in culture, prolonged exposure to insulin also increases GLUT1 protein content and reduces GLUT4 protein (
      • Sargeant R.J.
      • Paquet M.R.
      ,
      • Koivisto U.M.
      • Martinez-Valdez H.
      • Bilan P.J.
      • Burdett E.
      • Ramlal T.
      • Klip A.
      ,
      • Walker P.S.
      • Ramlal T.
      • Sarabia V.
      • Koivisto U.M.
      • Bilan P.J.
      • Pessin J.E.
      • Klip A.
      ,
      • Garcia de Herreros A.
      • Birnbaum M.J.
      ). In 3T3-L1 fibroblasts and adipocytes, the elevation in GLUT1 protein expression is due in part to an elevation in GLUT1 mRNA transcription (
      • Garcia de Herreros A.
      • Birnbaum M.J.
      ) and to a rise in the GLUT1 mRNA half-life (
      • Maher F.
      • Harrison L.C.
      ). Conversely, chronic insulin treatment of 3T3-L1 adipocytes decreases GLUT4 protein levels as a result of a reduction in mRNA levels (
      • Flores-Riveros J.R.
      • McLenithan J.C.
      • Ezaki O.
      • Lane M.D.
      ) and a decrease in the half-life of GLUT4 protein (
      • Sargeant R.J.
      • Paquet M.R.
      ). Therefore, the pathways involved in GLUT1 and GLUT4 gene expression are complex, and insulin appears to exert both transcriptional and post-transcriptional regulation. It remains unknown, however, whether the hormone can also regulate the expression of these two transporters at the level of their mRNA translation.
      Translational control usually occurs at the rate-limiting step of initiation. Eukaryotic cellular mRNAs (except organellar) contain a cap structure (m7GpppX, where X is any nucleotide) at their 5′ termini, and initiation involves recognition of this structure by the mRNA cap-binding protein eIF-4E1 (
      • Sonenberg N.
      • Gingras A.-C.
      ). eIF-4E, together with eIF-4A (an RNA helicase) and eIF-4G (a bridge between eIF-4E and eIF-4A), forms the eIF-4F initiation complex (
      • Tahara S.M.
      • Morgan M.A.
      • Shatkin A.L.
      ,
      • Edery I.
      • Humbelin M.
      • Darveau A.
      • Lee K.A.
      • Milburn S.
      • Hershey J.W.
      • Trachsel H.
      • Sonenberg N.
      ). eIF-4E activity is regulated through the formation of complexes with inhibitory eIF-4E-binding proteins (
      • Sonenberg N.
      • Gingras A.-C.
      ). In mammals, the eIF-4E-binding proteins (4E-BPs) compose a family of three members termed 4E-BP1 (
      • Pause A.
      • Belsham G.J.
      • Gingras A.-C.
      • Donze O.
      • Lin T.-A.
      • Lawrence Jr., J.C.
      • Sonenberg N.
      ), 4E-BP2 (
      • Lin T.-A.
      • Lawrence Jr., J.C.
      ), and 4E-BP3 (
      • Poulin F.
      • Gingras A.-C.
      • Olsen H.
      • Chevalier S.
      • Sonenberg N.
      ). 4E-BPs compete with eIF-4G for interaction with eIF-4E, thereby inhibiting cap-dependent translation (
      • Sonenberg N.
      • Gingras A.-C.
      ). Whether this mechanism affects equally all mRNAs has not been determined.
      In response to insulin, 4E-BP1 (also termed PHAS-I (phosphorylated heat- andacid-stable protein I)) becomes hyperphosphorylated, leading to its dissociation from eIF-4E to relieve translational inhibition. This phenomenon has been demonstrated in rat adipose tissue (
      • Pause A.
      • Belsham G.J.
      • Gingras A.-C.
      • Donze O.
      • Lin T.-A.
      • Lawrence Jr., J.C.
      • Sonenberg N.
      ,
      • Lin T.-A.
      • Kong X.
      • Haystead T.A.J.
      • Pause A.
      • Belsham G.
      • Sonenberg N.
      • Lawrence Jr., J.C.
      ), 3T3-L1 adipocytes (
      • Lin T.-A.
      • Lawrence Jr., J.C.
      ), rat skeletal muscle (
      • Kimball S.R.
      • Jurasinski C.V.
      • Lawrence Jr., J.C.
      • Jefferson L.S.
      ), and L6 myoblasts (
      • Kimball S.R.
      • Horetsky R.L.
      • Jefferson L.S.
      ). The phosphorylation of 4E-BP1 by insulin is inhibited by rapamycin (
      • Beretta L.
      • Gingras A.-C.
      • Svitkin Y.V.
      • Hall M.N.
      • Sonenberg N.
      ), a drug that forms a complex with the immunophilin FKBP12 to inhibit the kinase mammalian target of rapamycin (mTOR). mTOR phosphorylates 4E-BP1 both in vitro andin vivo (
      • Brunn G.J.
      • Hudson C.C.
      • Sekulic A.
      • Williams J.M.
      • Hosoi H.
      • Houghton P.J.
      • Lawrence Jr., J.C.
      • Abraham R.T.
      ). Subsequent studies have demonstrated that phosphatidylinositol 3-kinase (PI3K) and its downstream effector, protein kinase B (PKB; also known as Akt), are critical intermediates in the signal transduction pathway leading from the insulin receptor to the activation of mTOR and phosphorylation of 4E-BP1 (
      • Dufner A.
      • Andjelkovic M.
      • Burgering B.M.T.
      • Hemmings B.A.
      • Thomas G.
      ,
      • Gingras A.-C.
      • Kennedy S.G.
      • O'Leary M.A.
      • Sonenberg N.
      • Hay N.
      ,
      • Scott P.H.
      • Brunn G.J.
      • Kohn A.D.
      • Roth R.A.
      • Lawrence Jr., J.C.
      ).
      In addition to stimulating overall protein synthesis, insulin preferentially regulates the biosynthesis of certain proteins over and above its general anabolic effects on protein synthesis and proteolysis. A large number of these specific effects on protein synthesis are dependent upon continued mRNA synthesis (
      • Levenson R.M.
      • Nairn A.C.
      • Blackshear P.J.
      ), whereas few others occur without changes in mRNA levels (
      • Levenson R.M.
      • Nairn A.C.
      • Blackshear P.J.
      ,
      • Manzella J.M.
      • Rychlik W.
      • Rhoads R.E.
      • Hershey J.W.B.
      • Blackshear P.J.
      ,
      • Davis B.B.
      • Magge S.
      • Mucenski C.G.
      • Drake R.L.
      ). In this study, we show that insulin specifically up-regulates GLUT1 mRNA translation, in contrast to GLUT4 mRNA and the glyceraldehyde-3-phosphate dehydrogenase housekeeping gene. Furthermore, the increase in GLUT1 mRNA translation appears to occur via the PI3K/PKB/mTOR/4E-BP1 pathway.

      Acknowledgments

      We thank Dr. Phillip Pekala and Dr. Bin Zhou for valuable advice on polysome profiles.

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