The Glucose Transporter 4-regulating Protein TUG Is Essential for Highly Insulin-responsive Glucose Uptake in 3T3-L1 Adipocytes

Abstract

Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake. Insulin stimulates glucose uptake in fat and muscle by redistributing GLUT4 glucose transporters from intracellular membranes to the cell surface. We previously proposed that, in 3T3-L1 adipocytes, TUG retains GLUT4 within unstimulated cells and insulin mobilizes this retained GLUT4 by stimulating its dissociation from TUG. Yet the relative importance of this action in the overall control of glucose uptake remains uncertain. Here we report that transient, small interfering RNA-mediated depletion of TUG causes GLUT4 translocation and enhances glucose uptake in unstimulated 3T3-L1 adipocytes, similar to insulin. Stable TUG depletion or expression of a dominant negative fragment likewise stimulates GLUT4 redistribution and glucose uptake, and insulin causes a 2-fold further increase. Microscopy shows that TUG governs the accumulation of GLUT4 in perinuclear membranes distinct from endosomes and indicates that it is this pool of GLUT4 that is mobilized by TUG disruption. Interestingly, in addition to translocating GLUT4 and enhancing glucose uptake, TUG disruption appears to accelerate the degradation of GLUT4 in lysosomes. Finally, we find that TUG binds directly and specifically to a large intracellular loop in GLUT4. Together, these findings demonstrate that TUG is required to retain GLUT4 intracellularly in 3T3-L1 adipocytes in the absence of insulin and further implicate the insulin-stimulated dissociation of TUG and GLUT4 as an important action by which insulin stimulates glucose uptake. Insulin controls glucose utilization by adipose and muscle tissues by modulating the subcellular distribution of GLUT4 2The abbreviations used are: GLUT4, glucose transporter 4; TUG, tether, containing a UBX domain, for GLUT4; TfnR, transferrin receptor; GSV, GLUT4 storage vesicle; siRNA, small interfering RNA; shRNA, short hairpin RNA; GFP, green fluorescent protein; LM, light microsome; HM, heavy microsome; PM, plasma membrane; GST, glutathione S-transferase; SNARE, soluble NSF attachment receptor; TM, transmembrane segment; Cter, C-terminal. glucose transporters (1Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 267-277Crossref PubMed Scopus (936) Google Scholar, 2Watson R.T. Kanzaki M. Pessin J.E. Endocr. Rev. 2004; 25: 177-204Crossref PubMed Scopus (357) Google Scholar). In unstimulated cells, GLUT4 is retained in intracellular membranes, and its predominant exclusion from the plasma membrane minimizes glucose uptake from the extracellular space. Within minutes of insulin stimulation, GLUT4 is translocated so that the number of transporters in the plasma membrane is increased and glucose uptake is accelerated. This insulin action is defective in muscle and adipose from individuals with type 2 diabetes and other insulin-resistant states (3Shulman G.I. J. Clin. Invest. 2000; 106: 171-176Crossref PubMed Scopus (2189) Google Scholar, 4Abel E.D. Shepherd P.R. Kahn B.B. Le Roith D. Taylor S.I. Olefsky J.M. Diabetes Mellitus: A Fundamental and Clinical Text. 3rd Ed. Lippincott-Raven, Philadelphia2003: 917-938Google Scholar, 5Maianu L. Keller S.R. Garvey W.T. J Clin. Endocrinol. Metab. 2001; 86: 5450-5456Crossref PubMed Scopus (77) Google Scholar). The molecular mechanisms governing GLUT4 distribution and its regulation by insulin remain poorly characterized. To redistribute GLUT4, insulin acts primarily on the exocytic arm of the GLUT4 recycling pathway. The signaling proteins involved include Akt/protein kinase and its the to to intracellular of GLUT4 in unstimulated cells M. J. M. M. James D.E. J. Biol. PubMed Scopus Google Scholar, L. Metab. PubMed Scopus Google Scholar). of GLUT4 by the that unstimulated cells, the used 3T3-L1 cell of intracellular GLUT4. include GLUT4 in the and poorly membranes as the or storage Insulin stimulates which a 2-fold in cell GLUT4 and other proteins as the transferrin The insulin-stimulated in cell GLUT4, and in other recycling is to of membranes that of and other J. Biol. 2002; PubMed Scopus Google Scholar, J. Biol. 2002; PubMed Scopus Google Scholar). that by from the with endosomes R.T. M. L. Kanzaki M. Pessin J.E. J. 2004; PubMed Scopus Google Scholar, Mol. Endocrinol. PubMed Scopus Google Scholar, Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar). In adipocytes, distinct from containing Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). The of these is cell and J. Cell. PubMed Scopus Google Scholar). retained intracellularly in the absence of and these membranes mobilized by insulin stimulation, remains uncertain. We proposed that in unstimulated cells, GLUT4 that is in to intracellular to its to the plasma membrane PubMed Scopus Google Scholar). to this insulin this GLUT4 to accelerate its to the cell and the plasma We TUG as a which acts in with other proteins to retain GLUT4 within cells in the absence of insulin. this TUG and GLUT4 a in unstimulated 3T3-L1 adipocytes, and on intracellular membranes that as light PubMed Scopus Google Scholar). by The is insulin acts to the dissociation of TUG and GLUT4 PubMed Scopus Google Scholar). of TUG the of the GLUT4 and the number of PubMed Scopus Google Scholar). expression of a dominant negative TUG fragment the of the pool and the number of Together, these that TUG is of an governing the of GLUT4 in within unstimulated 3T3-L1 Here we depletion of TUG to further its in GLUT4 and glucose uptake, 3T3-L1 that TUG is required for intracellular of GLUT4 in perinuclear membranes within unstimulated cells, with the The of the we that the an important of insulin action to glucose uptake in 3T3-L1 and adipocytes as that the used for and containing insulin Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). 3T3-L1 adipocytes as previously M. PubMed Scopus Google Scholar). In 3T3-L1 cells a with or the TUG fragment or TUG previously Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). of 3T3-L1 cells with a containing a TUG shRNA, the used from 2004; PubMed Scopus Google Scholar). as and of a Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. J. PubMed Google Scholar). To TUG the cells, a TUG containing in the the and cells by cell of a PubMed Scopus Google Scholar, D. 2000; PubMed Scopus Google Scholar). from and the TUG TUG and and and the its with a the for TUG in the to TUG previously PubMed Scopus Google Scholar). to the insulin from and to GLUT4 a from and to a as 86: PubMed Scopus Google or from to from from from from and from and as previously Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). previously and light and heavy in containing as Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). in or and a in and and to membranes a and on as previously Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). in the and a as that or used and PubMed Scopus Google Scholar). 3T3-L1 adipocytes cell or cells with in cells 2 or insulin for so that the of and uptake to for or for and in A of used for a so that used to for in the number of cells in uptake in the of and from from other in or and a and containing the large intracellular loop of GLUT4 or in a a of and proteins and control in the or its cells and in from the soluble To of or proteins in of the and the of by in and TUG in the and the to the TUG containing or and with a or TUG proteins with GST, or in The the with and proteins in proteins by and or the with to demonstrate of or adipocytes a GLUT4 on and for as previously Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar). To with on cells to intracellular cells and for used for with on a a and for an of with a of and of or and and in by to the of from with and with for The used to TUG and GLUT4 and GLUT4 and and and GLUT4 and GLUT4 and with and to control expression of in with a of in an in the of on GLUT4 3T3-L1 adipocytes in containing with or as J. Cell. PubMed Scopus Google Scholar). for of and in The of as and of by and To the of TUG in the control of glucose uptake, we 3T3-L1 adipocytes with that TUG In cells, and in with on TUG In the that TUG the by of insulin the and specifically TUG in 3T3-L1 The of TUG depletion on glucose uptake in 3T3-L1 adipocytes is in In control or cells, insulin a in glucose uptake. In cells with of the TUG glucose uptake and further insulin In the of the and it glucose uptake this We that depletion of TUG glucose uptake, similar to in unstimulated 3T3-L1 To the glucose uptake we TUG is to increased GLUT4 or these proteins on of cell in GLUT4 increased in 3T3-L1 adipocytes with TUG with control GLUT4 the that TUG GLUT4 and as further on the glucose uptake we to increased GLUT4 or with the that TUG depletion GLUT4 to the plasma membrane to glucose uptake in cells with insulin. To further the of TUG depletion on GLUT4 subcellular we of 3T3-L1 adipocytes a and GLUT4 Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar). in cells a control and with insulin GLUT4 to intracellular membranes, and the perinuclear distribution in these unstimulated insulin GLUT4 in perinuclear and plasma membrane and of that GLUT4 the plasma membrane In cells in which TUG by of GLUT4 the cell in the absence of insulin that this GLUT4 the plasma The GLUT4 in the control cells, which a the overall distinct from that in unstimulated control insulin of the TUG cells, GLUT4 the cell and a the that transient, TUG depletion of GLUT4 the plasma membrane in unstimulated 3T3-L1 To we 3T3-L1 cells in which TUG by expression of a This the as TUG We a of 3T3-L1 by cells adipose as by accumulation and expression of GLUT4, with 3T3-L1 that TUG by in adipocytes containing the To further control for the of TUG the cells with a containing a of TUG, to cells by and adipose shows that the TUG in that that of TUG in 3T3-L1 of GLUT4 similar or in cells, and increased in cells, that GLUT4 by TUG large on from and 3T3-L1 adipocytes that TUG GLUT4 as further We the of TUG depletion on glucose uptake. shows that unstimulated 3T3-L1 adipocytes containing the increased glucose uptake with control This increased glucose uptake to that by insulin of the control Insulin further glucose uptake in cells of TUG, by of the increased uptake. The glucose uptake in the cells to increased or GLUT4 which similar or in cells with control cells, as of TUG the on glucose uptake, that it from TUG depletion and a of the glucose uptake in cells similar to that in control cells the increased GLUT4 in cells This the of TUG to retain GLUT4 within cells, it from the plasma membrane PubMed Scopus Google Scholar). Finally, it that the in glucose uptake in cells in cells the of a of insulin to glucose uptake in the and depletion of TUG in increased glucose uptake in unstimulated 3T3-L1 adipocytes, and in this to increased of GLUT4 or To the of the TUG on glucose transporter we subcellular of and insulin-stimulated shRNA, and TUG 3T3-L1 in insulin increased GLUT4 in plasma membranes of control cells, and a of GLUT4 in light In cells, GLUT4 in plasma membranes, and in light in and cells GLUT4 in light and by this in control cells and to In the TUG cells, of TUG GLUT4 in light and insulin-stimulated GLUT4 to the plasma membrane these to TUG depletion and of the by in GLUT4 from plasma membranes of TUG of the to that, in control cells, insulin of of light and to the plasma membrane with Olefsky J.M. PubMed Scopus Google Scholar). TUG depletion to plasma membranes in 3T3-L1 This by of TUG and to TUG it the on GLUT4 and Insulin a further in plasma membrane in cells and to in light depletion of TUG by a a large on GLUT4 and a on in 3T3-L1 The redistribution of GLUT4 of light and to the plasma membrane in the absence of insulin. 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PubMed Scopus Google Scholar). to dominant negative fragment GLUT4 3T3-L1 adipocytes containing this fragment with for of in we that in unstimulated to insulin stimulation, cells in and in increased GLUT4 in control cells, and this with this This with the of GLUT4 in unstimulated 3T3-L1 adipocytes, as previously J. PubMed Scopus Google Scholar). In cells containing increased GLUT4 and this as as its addition that large on TUG TUG is primarily by the it is and these the that expression GLUT4 degradation in lysosomes. To further GLUT4 degradation is by TUG and by TUG similar with of control cells, cells, and TUG shows that, in control cells, addition a in GLUT4 with previously of GLUT4 J. Cell. PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). 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The that TUG is an of GLUT4 and of glucose uptake in 3T3-L1 In unstimulated cells, TUG required to GLUT4 glucose transporters in intracellular of TUG by or by a dominant negative to GLUT4 redistribution of light TUG and GLUT4 in unstimulated control The GLUT4 to the cell and glucose uptake. Insulin to glucose uptake in cells with disruption of TUG, this of the large on glucose uptake. that insulin stimulates the dissociation of TUG and GLUT4 and that this GLUT4 of light Mol. Cell. Biol. 2001; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). it that the insulin-stimulated dissociation of TUG and GLUT4 for a large of the overall action of insulin to glucose uptake. The which TUG acts to control GLUT4 distribution remains poorly with a in GLUT4 to or in intracellularly in the absence of or in of these PubMed Scopus Google Scholar). The the that TUG in GLUT4 or other in the recycling pathway. on these the of GLUT4 that translocated by insulin with the of GLUT4 that from TUG. increased by TUG and by TUG TUG with GLUT4 in light membranes that distinct from by TUG binds with to GLUT4 to This is similar to that for translocation Olefsky J.M. PubMed Scopus Google Scholar). the to for is that TUG in of GLUT4 and controls the accumulation of a pool of in unstimulated The this for TUG action the in the of a in which TUG an intracellular GLUT4 GLUT4 translocation to the cell and insulin or to and Insulin enhances the of from membranes by and this important for insulin action J. Biol. 2002; PubMed Scopus Google Scholar). In unstimulated cells, GLUT4 in these is retained and in a perinuclear in 3T3-L1 A action of insulin is to this pool of GLUT4. The by which GLUT4 is retained within unstimulated cells is is that TUG and other proteins GLUT4 in to intracellular PubMed Scopus Google Scholar). proteins to which TUG GLUT4 include and M. Mol. Biol. Cell. PubMed Scopus Google Scholar, D. Pessin J.E. Mol. Biol. Cell. PubMed Scopus Google Scholar). proteins to the TUG a distinct from that which binds GLUT4 PubMed Scopus Google Scholar). A is that, in unstimulated cells, TUG an intracellular in which and or membranes (1Bryant N.J. Govers R. James D.E. Nat. Rev. Mol. Cell. Biol. 2002; 3: 267-277Crossref PubMed Scopus (936) Google Scholar, Mol. Endocrinol. PubMed Scopus Google Scholar, Mol. Biol. Cell. 2004; PubMed Scopus Google Scholar, M. N.J. Mol. Biol. Cell. PubMed Google Scholar). Insulin these a plasma membrane recycling by the PubMed Scopus Google Scholar, R. James D.E. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar). A is that, in the absence of TUG the of GLUT4 with which insulin addition the transporters the plasma membrane J. J. PubMed Scopus Google Scholar, J. J. Olefsky J.M. Mol. Cell. Biol. PubMed Scopus Google Scholar). and that TUG disruption and insulin that similar in TUG the intracellular of by of the dissociation of TUG and GLUT4 an important action by which insulin these to the plasma We the mobilized to the cell by TUG disruption to mobilized by insulin that TUG primarily as a to GLUT4 to in unstimulated cells in which TUG is GLUT4 to the cell in the membranes from which endosomes or The that TUG is a is with the that TUG directly intracellular of GLUT4, by of the mechanisms The of GLUT4 and the intracellular of these that In addition to an intracellular to insulin acts the plasma membrane to accelerate the of containing GLUT4 J. Metab. PubMed Scopus Google Scholar). insulin to of that the cell M. M. J. Biol. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, Govers R. James D.E. Mol. Endocrinol. PubMed Google Scholar). of TUG directly on mechanisms the plasma TUG is in this PubMed Scopus Google Scholar). by the intracellular of GLUT4, TUG disruption is to the of the plasma membrane of unstimulated The that this is to GLUT4 in the plasma membrane and to glucose uptake. the of the plasma membrane is of which to a large by of intracellular GLUT4 The of insulin to accelerate the of the plasma membrane for other in 3T3-L1 adipocytes in which TUG is by or insulin a 2-fold further to glucose uptake. This transient, depletion of TUG, is in this The of insulin in or cells from of containing GLUT4 the plasma of insulin on GLUT4 to this 2-fold a is that TUG disruption in a of plasma membrane with that in insulin control for this it that this from an accumulation of that the plasma The of a the plasma In of the insulin of control cells of in the plasma so that a is by The that expression and TUG depletion with to GLUT4 and glucose uptake, in 3T3-L1 The fragment is to an intracellular to which TUG GLUT4 in unstimulated cells and to TUG from this with the of GLUT4 to as that expression the of a containing TUG and GLUT4 PubMed Scopus Google Scholar). depletion of TUG the of this that TUG depletion GLUT4 the plasma similar to and expression of GLUT4 the plasma the is in GLUT4 is in unstimulated cells and cells containing Finally, TUG depletion and expression glucose uptake. expression and TUG depletion on GLUT4 distribution and glucose uptake, that the dominant negative of is to a In the of glucose uptake is in 3T3-L1 adipocytes in which TUG is in control cells in which TUG is In GLUT4 is by the of insulin and TUG or as with the of insulin findings with the that insulin acts to and to of GLUT4 the plasma membrane and that TUG is required the previously that insulin to the pool of GLUT4 in 3T3-L1 adipocytes R. James D.E. Mol. Cell. Biol. 2004; PubMed Scopus Google Scholar, Govers R. James D.E. 2004; PubMed Scopus Google Scholar). remains a of the to a of the of that is to the plasma to retained by the action of TUG and other insulin stimulates the dissociation of of the in 3T3-L1 adipocytes and the of these PubMed Scopus Google Scholar). The retain the that mobilized by insulin. TUG disruption an that of it the pool of and of this The that TUG depletion TUG by and indicates disruption of by expression PubMed Scopus Google Scholar). with the that the of GLUT4 in of unstimulated cells, that membranes with control cells or cells TUG it that retained by TUG disruption by insulin and that a of GLUT4 is for the plasma The that TUG GLUT4 as as its transient, TUG the increased glucose uptake GLUT4 of GLUT4 and is with the that GLUT4 from or increased and the the that by the of GLUT4 to the plasma TUG the of GLUT4 to and its proteins to GLUT4 and and J. Cell. PubMed Scopus Google Scholar, L. 2000; PubMed Scopus Google Scholar). GLUT4 in it is this from GLUT4 or degradation S.R. J. Biol. 2002; PubMed Scopus Google Scholar). The GLUT4 that remains is that GLUT4 that a is by and in other an on in cells in 3T3-L1 adipocytes, a of GLUT4 in in 3T3-L1 Finally, it for that GLUT4 degradation is by insulin J. PubMed Scopus Google Scholar, J. Google Scholar). the that TUG disruption of the of insulin on GLUT4 and glucose uptake the of insulin to accelerate GLUT4 The demonstrate that TUG binds directly and specifically to the large intracellular loop of GLUT4. This in GLUT4 R.T. Pessin J.E. J. Biol. 2004; PubMed Scopus Google Scholar). The of TUG that binds this loop in to important for the of TUG to retain GLUT4 intracellularly in cells PubMed Scopus Google Scholar). In the that TUG and GLUT4 in a as This a of TUG and or of TUG required for intracellular of GLUT4 in cells, and for of TUG and GLUT4 in to this the GLUT4 which is in insulin The that TUG disruption a small to redistribute to the plasma This is with GLUT4 is so in 3T3-L1 adipocytes, the on distribution to the increased glucose uptake we that insulin translocation to the plasma membrane of 3T3-L1 adipocytes, this is that of GLUT4 Olefsky J.M. PubMed Scopus Google Scholar). is that the in we of TUG and the this is the of TUG and the GLUT4 the and a large of TUG in GLUT4 distribution a in distribution in 3T3-L1 is other proteins in a or this to retain GLUT4 within unstimulated it is insulin acts on the to dissociation of TUG and GLUT4 and to GLUT4. Finally, it is TUG is in insulin-resistant states as type 2 the demonstrate that TUG is an important of GLUT4 and glucose uptake and that this a in insulin We and of for expression for the for and and for and on the This used the for and the Diabetes of