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Prolonged Insulin Stimulation Down-regulates GLUT4 through Oxidative Stress-mediated Retromer Inhibition by a Protein Kinase CK2-dependent Mechanism in 3T3-L1 Adipocytes*

  • Jinhui Ma
    Affiliations
    Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
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  • Yuko Nakagawa
    Affiliations
    Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
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  • Itaru Kojima
    Affiliations
    Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
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  • Hiroshi Shibata
    Correspondence
    To whom correspondence should be addressed: Dept. of Cell Biology, Inst. for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi 371-8512, Japan. Tel.: 81-27-220-8836; Fax: 81-27-220-8893
    Affiliations
    Department of Cell Biology, Institute for Molecular and Cellular Regulation, Gunma University, Maebashi 371-8512, Japan
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  • Author Footnotes
    * This work was supported by the Global Centers of Excellence Program “Signal Transduction in the Regulatory System and Its Disorders” and Grant-in-aid 23591295 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H. S.). Parts of this study were presented in abstract form at the 69th Scientific Sessions of the American Diabetes Association, New Orleans, LA, June 5–9, 2009.
Open AccessPublished:January 03, 2014DOI:https://doi.org/10.1074/jbc.M113.533240
      Although insulin acutely stimulates glucose uptake by promotion of GLUT4 translocation from intracellular compartments to the plasma membrane in adipocytes and muscles, long term insulin stimulation causes GLUT4 depletion that is particularly prominent in the insulin-responsive GLUT4 storage compartment. This effect is caused mainly by accelerated lysosomal degradation of GLUT4, although the mechanism is not fully defined. Here we show that insulin acutely induced dissociation of retromer components from the low density microsomal membranes of 3T3-L1 adipocytes that was accompanied by disruption of the interaction of Vps35 with sortilin. This insulin effect was dependent on the activity of protein kinase CK2 but not phosphatidylinositol 3-kinase or extracellular signal-regulated kinase 1/2. Knockdown of Vps26 decreased GLUT4 to a level comparable with that with insulin stimulation for 4 h. Vps35 with a mutation in the CK2 phosphorylation motif (Vps35-S7A) was resistant to insulin-induced dissociation from the low density microsomal membrane, and its overexpression attenuated GLUT4 down-regulation with insulin. Furthermore, insulin-generated hydrogen peroxide was an upstream mediator of the insulin action on retromer and GLUT4. These results suggested that insulin-generated oxidative stress switches the GLUT4 sorting direction to lysosomes through inhibition of the retromer function in a CK2-dependent manner.

      Introduction

      Although insulin acutely stimulates glucose uptake by promotion of translocation of the insulin-regulated glucose transporter GLUT4 from intracellular compartments to the plasma membrane in adipocytes and muscles (
      • Cushman S.W.
      • Wardzala L.J.
      Potential mechanism of insulin action on glucose transport in the isolated rat adipose cell. Apparent translocation of intracellular transport systems to the plasma membrane.
      ,
      • Suzuki K.
      • Kono T.
      Evidence that insulin causes translocation of glucose transport activity to the plasma membrane from an intracellular storage site.
      ), long term insulin stimulation causes a depletion of GLUT4 protein (
      • Flores-Riveros J.R.
      • McLenithan J.C.
      • Ezaki O.
      • Lane M.D.
      Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover.
      • Sargeant R.J.
      • Pâquet M.R.
      Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes.
      • Thomson M.J.
      • Williams M.G.
      • Frost S.C.
      Development of insulin resistance in 3T3-L1 adipocytes.
      ), which is particularly prominent in the highly insulin-responsive GLUT4 storage compartment (GSC)
      The abbreviations used are:
      GSC
      GLUT4 storage compartment
      TGN
      trans-Golgi network
      SNX
      sorting nexin
      BAR
      Bin-Amphiphysin-Rvs
      LDM
      low density microsome
      TBB
      4,5,6,7-tetrabromobenzotriazole
      PDH
      pyruvate dehydrogenase
      PI
      phosphatidylinositol
      PMAC
      phosphate metal affinity chromatography
      PP
      protein phosphatase
      CK2β
      β subunit of CK2.
      (
      • Maier V.H.
      • Gould G.W.
      Long-term insulin treatment of 3T3-L1 adipocytes results in mis-targeting of GLUT4: implications for insulin-stimulated glucose transport.
      ,
      • Liu L.B.
      • Omata W.
      • Kojima I.
      • Shibata H.
      The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes.
      ), and is thus accompanied by unresponsiveness to insulin of the glucose transport system (
      • Thomson M.J.
      • Williams M.G.
      • Frost S.C.
      Development of insulin resistance in 3T3-L1 adipocytes.
      ). Although the precise mechanism of this downside insulin action is not fully understood, accelerated degradation of GLUT4 in the lysosomes may play a major role in its down-regulation with long term insulin stimulation because previous studies demonstrated that GLUT4 protein turnover is accelerated about 3-fold with insulin in 3T3-L1 adipocytes (
      • Sargeant R.J.
      • Pâquet M.R.
      Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes.
      ), and lysosome inhibitors effectively prevent insulin-induced GLUT4 depletion (
      • Liu L.B.
      • Omata W.
      • Kojima I.
      • Shibata H.
      The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes.
      ). Although insulin also inhibits transcription of GLUT4 (
      • Flores-Riveros J.R.
      • McLenithan J.C.
      • Ezaki O.
      • Lane M.D.
      Insulin down-regulates expression of the insulin-responsive glucose transporter (GLUT4) gene: effects on transcription and mRNA turnover.
      ,
      • Thomson M.J.
      • Williams M.G.
      • Frost S.C.
      Development of insulin resistance in 3T3-L1 adipocytes.
      ), this may contribute less to the insulin-evoked GLUT4 depletion especially in the early few hours of insulin stimulation because the turnover of GLUT4 protein is much slower (with half-lives of 50 and 15.4 h in the absence and presence of insulin, respectively) (
      • Sargeant R.J.
      • Pâquet M.R.
      Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes.
      ,
      • Liu L.B.
      • Omata W.
      • Kojima I.
      • Shibata H.
      The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes.
      ) than the rate of GLUT4 loss with insulin stimulation (
      • Thomson M.J.
      • Williams M.G.
      • Frost S.C.
      Development of insulin resistance in 3T3-L1 adipocytes.
      ). These observations suggested that insulin down-regulates GLUT4 mainly by promotion of its sorting toward the lysosomes.
      Previous kinetic analyses have shown that insulin stimulation elicits entry of a significant number of GLUT4 from the relatively static insulin-responsive intracellular pool(s) into the endocytic recycling pathway (
      • Slot J.W.
      • Geuze H.J.
      • Gigengack S.
      • Lienhard G.E.
      • James D.E.
      Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat.
      • Shibata H.
      • Suzuki Y.
      • Omata W.
      • Tanaka S.
      • Kojima I.
      Dissection of GLUT4 recycling pathway into exocytosis and endocytosis in rat adipocytes. Evidence that GTP-binding proteins are involved in both processes.
      • Govers R.
      • Coster A.C.
      • James D.E.
      Insulin increases cell surface GLUT4 levels by dose dependently discharging GLUT4 into a cell surface recycling pathway.
      ). Although this shift in the subcellular traffic route of GLUT4 leads to a substantial increase in the number of GLUT4 at the cell surface (i.e. apparent “translocation”), the appearance of GLUT4 in the early endosomes, a major sorting center in the endocytic pathway, may possibly be relevant to promotion of its sorting to the lysosomes. Membrane proteins recycling from the endosome to the plasma membrane (e.g. the transferrin receptor and the LDL receptor) or those recycling between the endosome and the trans-Golgi network (TGN) (e.g. the mannose 6-phosphate receptor) would be delivered to the lysosomes when the sorting machinery on the early endosome is crippled (
      • Arighi C.N.
      • Hartnell L.M.
      • Aguilar R.C.
      • Haft C.R.
      • Bonifacino J.S.
      Role of the mammalian retromer in sorting of the cation-independent mannose 6-phosphate receptor.
      ), whereas non-recycling proteins (e.g. the EGF receptor) are transported all the way from the endosomes to the lysosomes for degradation after endocytosis. Thus, one possible mechanism for insulin-induced GLUT4 down-regulation would be that, under insulin stimulation, the endosomal GLUT4 sorting machinery may be functionally impaired, and GLUT4 not retrieved from the endosome-to-lysosome degradative flow would be transported to lysosomes with a consequent depletion of the transporter.
      In the present study, to test this possibility, we investigated the role for the retromer complex in GLUT4 trafficking in 3T3-L1 adipocytes. Retromer is a key component of the endosomal protein sorting machinery that mediates the endosome-to-TGN retrieval of diverse functional transmembrane proteins (cargos) such as the cation-independent mannose 6-phosphate receptor, the Wnt-binding protein Wntless, and the sortilin family proteins (for reviews, see Refs.
      • Bonifacino J.S.
      • Hurley J.H.
      Retromer.
      • McGough I.J.
      • Cullen P.J.
      Recent advances in retromer biology.
      • Seaman M.N.
      The retromer complex—endosomal protein recycling and beyond.
      • Cullen P.J.
      • Korswagen H.C.
      Sorting nexins provide diversity for retromer-dependent trafficking events.
      ). The prototypical mammalian retromer (sorting nexin (SNX)-BAR retromer) is a heteropentameric complex comprising two functionally separate subcomplexes: the cargo-selective subcomplex composed of Vps26, Vps29, and Vps35 that recruits cargos via an association between Vps35 and a sorting motif within the cytoplasmic tail of cargo and the SNX-BAR dimer subcomplex composed of SNX1 and SNX2 or of SNX5 and SNX6 that drives membrane curvature by binding to the early endosomal phosphatidylinositol (PI) 3-phosphate resulting in the formation of membrane tubules.
      We show herein that retromer components were present mainly in the low density microsomal (LDM) membranes of 3T3-L1 adipocytes and that retromer played a pivotal role in the regulation of GLUT4 protein level. Notably, insulin negatively regulated retromer function by disrupting its interaction with the membranes, which may be a possible mechanism of insulin-promoted GLUT4 sorting to the degradative pathway. Furthermore, the signaling mechanism of this insulin action was exceptionally unique in that it depends on insulin-generated oxidative stress, particularly hydrogen peroxide, as well as on the activity of protein kinase CK2 (formerly known as casein kinase 2) but not PI 3-kinase or extracellular signal-regulated kinase 1/2 (Erk1/2). Thus, this study revealed a unique oxidative stress-mediated insulin signal cascade that regulates the fate of GLUT4 by interfering with the retromer function.

      DISCUSSION

      In the present study, we studied the mechanism of GLUT4 down-regulation by prolonged insulin stimulation and have provided evidence that retromer plays a pivotal role in the regulation of GLUT4 amount and is a critical functional component in this downside effect of insulin. This was supported by the following observations. First, manipulation of the retromer function by knockdown of Vps26 reduced GLUT4 to the level induced by 4-h insulin stimulation, whereas overexpression of Vps26 caused an increase in GLUT4. Second, insulin acutely interfered with the membrane interaction of retromer, and this was accompanied by disruption of the Vps35 interaction with its cargo protein sortilin. Thus, retromer plays an indispensable role in the maintenance of GLUT4 amount, and its function is negatively regulated by insulin. Given that retromer mediates the retrograde transport of cargo proteins from endosome to TGN, thus retrieving them from the endosome-to-lysosome degradative flow, the insulin interference with the retromer function may cause switching of the GLUT4 traffic route toward the lysosomes with consequent acceleration of GLUT4 degradation. This model is consistent with previous observations that insulin accelerates GLUT4 turnover (
      • Sargeant R.J.
      • Pâquet M.R.
      Effect of insulin on the rates of synthesis and degradation of GLUT1 and GLUT4 glucose transporters in 3T3-L1 adipocytes.
      ) and that prolonged insulin stimulation reduces GLUT4 by a de novo protein synthesis-independent mechanism (
      • Rudich A.
      • Tirosh A.
      • Potashnik R.
      • Hemi R.
      • Kanety H.
      • Bashan N.
      Prolonged oxidative stress impairs insulin-induced GLUT4 translocation in 3T3-L1 adipocytes.
      ). On the other hand, because TGN is the most likely compartment for the generation of insulin-sensitive GLUT4 pool(s) (
      • Bogan J.S.
      • Kandror K.V.
      Biogenesis and regulation of insulin-responsive vesicles containing GLUT4.
      ,
      • Kandror K.V.
      • Pilch P.F.
      The sugar is sIRVed: sorting Glut4 and its fellow travelers.
      ), retromer-mediated retrograde transport of GLUT4 may be critical for the generation of an insulin-sensitive pool (
      • Hatakeyama H.
      • Kanzaki M.
      Molecular basis of insulin-responsive GLUT4 trafficking systems revealed by single molecule imaging.
      ), and insulin-elicited switching of GLUT4 sorting at the endosomes may partly explain the inverse correlation between GLUT4 turnover and its targeting to GSC or biogenesis of the insulin-responsive GLUT4 vesicles (
      • Liu L.B.
      • Omata W.
      • Kojima I.
      • Shibata H.
      The SUMO conjugating enzyme Ubc9 is a regulator of GLUT4 turnover and targeting to the insulin-responsive storage compartment in 3T3-L1 adipocytes.
      ,
      • Shi J.
      • Kandror K.V.
      Sortilin is essential and sufficient for the formation of Glut4 storage vesicles in 3T3-L1 adipocytes.
      ). A recent proteomic analysis of GLUT4 vesicles (
      • Jedrychowski M.P.
      • Gartner C.A.
      • Gygi S.P.
      • Zhou L.
      • Herz J.
      • Kandror K.V.
      • Pilch P.F.
      Proteomic analysis of GLUT4 storage vesicles reveals LRP1 to be an important vesicle component and target of insulin signaling.
      ) has shown that the insulin-responsive GLUT4 vesicles contain retromer components such as Vps35 and Vps26 as well as retromer cargo proteins including the mannose 6-phosphate receptor, sortilin, and sortilin-related LDL receptor relative 11 (SorLA). Because the latter two proteins contain the luminal Vps10p domain that can serve as the binding site for GLUT4 (
      • Shi J.
      • Kandror K.V.
      The luminal Vps10p domain of sortilin plays the predominant role in targeting to insulin-responsive Glut4-containing vesicles.
      ), they are possible candidates for mediating retromer-dependent sorting of GLUT4.
      Another important finding in the present study is that protein kinase CK2, but not PI 3-kinase or Erk1/2, plays a crucial role in the insulin signaling cascade to retromer inhibition as well as GLUT4 down-regulation. This was demonstrated by using pharmacological inhibitors and RNAi-based knockdown of CK2. In addition, by using the Vps35-S7A mutant, we showed that CK2-mediated phosphorylation of Vps35 is a possible mechanism for the insulin-induced disruption of the Vps35 interaction with the LDM membrane. Furthermore, by real time monitoring of the HyPer fluorescence, we showed that insulin stimulates rapid and sustained generation of H2O2, which is a possible candidate for the upstream mediator in the CK2 regulation of Vps35, although the mechanism of insulin regulation of CK2 activity is unclear at present. The basal activity of CK2 is very high, and there has been no known intracellular signal molecule that activates CK2 (
      • Litchfield D.W.
      Protein kinase CK2: structure, regulation and role in cellular decisions of life and death.
      ). In this regard, our data indicated that a microcystin-LR-sensitive protein phosphatase (i.e. PP1 or PP2A) antagonized the Vps35 dissociation from the LDM membrane. Thus, it is possible that H2O2 may facilitate CK2-mediated Vps35 phosphorylation by inhibiting a putative redox-sensitive serine/threonine phosphatase(s) such as PP2A (
      • Guy G.R.
      • Cairns J.
      • Ng S.B.
      • Tan Y.H.
      Inactivation of a redox-sensitive protein phosphatase during the early events of tumor necrosis factor/interleukin-1 signal transduction.
      ,
      • Rao R.K.
      • Clayton L.W.
      Regulation of protein phosphatase 2A by hydrogen peroxide and glutathionylation.
      ) that may dephosphorylate Vps35. Alternatively, because H2O2 inactivates redox-sensitive protein tyrosine phosphatases such as PTP1B (
      • Denu J.M.
      • Tanner K.G.
      Specific and reversible inactivation of protein tyrosine phosphatases by hydrogen peroxide: evidence for a sulfenic acid intermediate and implications for redox regulation.
      ,
      • Lee S.R.
      • Kwon K.S.
      • Kim S.R.
      • Rhee S.G.
      Reversible inactivation of protein-tyrosine phosphatase 1B in A431 cells stimulated with epidermal growth factor.
      ), it is also possible that H2O2 may inactivate PP2A through phosphorylation of its catalytic subunit at tyrosine 307, which negatively regulates the PP2A activity (
      • Chen J.
      • Martin B.L.
      • Brautigan D.L.
      Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation.
      ). Also, we cannot exclude the possibility that H2O2 may regulate CK2 activity through subcellular localization or interaction with other regulatory molecules.
      Altogether, we propose a working model deduced from the present study for insulin-induced switching of GLUT4 traffic route (Fig. 7). In the model, insulin stimulates H2O2 generation via activation of the NADPH oxidase (possibly Nox4) by a yet undefined mechanism. The generated H2O2 promotes CK2-mediated Vps35 phosphorylation by inactivating a microcystin-LR-sensitive serine/threonine phosphatase, causing disruption of the retromer interaction with cargo proteins such as sortilin, thus switching GLUT4 sorting to lysosomes. Apparently, a few missing links are present in this model among which are (a) the postreceptor mechanism of insulin activation of NADPH oxidase, (b) the mechanism of H2O2 regulation of CK2 activity, and (c) the entity of the cargo protein that connects GLUT4 and retromer in the endosome-to-TGN retrograde transport. Further work will be necessary to elucidate these points.
      Figure thumbnail gr7
      FIGURE 7A model for insulin-induced switching of GLUT4 traffic route from “recycling” to “degradation.” Insulin receptor tyrosine kinase activation stimulates Nox4-mediated H2O2 generation, which may increase the apparent activity of CK2 by inhibition of counteracting redox-sensitive phosphatase(s). CK2-induced Vps35 phosphorylation may dissociate retromer from the LDM membrane, leading to switching of GLUT4 sorting at endosomes from “recycling to TGN” to “lysosomal degradation.” This may cause shortening of the GLUT4 half-life and depletion of GLUT4 in GSC.
      Finally, although the present study provides a novel insight into the mechanism of the downside action of insulin, it is unclear whether retromer plays any physiological role in the insulin action(s) such as GLUT4 translocation. In this regard, we unexpectedly found the cargo-selective components (Vps35, Vps26, and Vps29) in the GSC fraction without the SNX-BAR components (SNX1 and SNX2), which were mainly present in the endosome/TGN fraction (Fig. 2B). The reason for this discrepancy is unknown, but it may reflect the absence of PI 3-phosphate or the presence of as yet unidentified SNX protein(s) in the GSC fraction. In light of recent studies showing that non-prototypical retromer is involved in the endosome-to-plasma membrane traffic of G-protein-coupled receptors (
      • Seaman M.N.
      The retromer complex—endosomal protein recycling and beyond.
      ,
      • Cullen P.J.
      • Korswagen H.C.
      Sorting nexins provide diversity for retromer-dependent trafficking events.
      ), it would be intriguing to investigate the role of GSC-associated retromer components in the physiological action of insulin on subcellular trafficking of GLUT4.

      Acknowledgments

      We thank Howard Green (Harvard University) for providing 3T3-L1 cells and Juan S. Bonifacino (National Institutes of Health) for the Vps26-myc-pcDNA3.1 expression plasmid.

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