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.
Highlights
Glucose transporters [1, 2]
The data presented here show that TUG is an essential regulator of GLUT4 targeting and of glucose uptake in 3T3-L1 adipocytes
Disruption of TUG function, by RNAi or by a dominant negative fragment, led to marked GLUT4 redistribution out of light microsomes, where both TUG and insulin-responsive GLUT4 reside in unstimulated control cells
Summary
Glucose transporters [1, 2]. In unstimulated cells, GLUT4 is retained in intracellular membranes, and its predominant exclusion from the plasma membrane minimizes glucose uptake from the extracellular space. Regulation of GLUT4 Glucose Transporters by TUG the absence of insulin Supporting this model, TUG and GLUT4 form a complex in unstimulated 3T3-L1 adipocytes, and colocalize on TfnR-negative intracellular membranes that sediment as light microsomes [15]. Expression of a dominant negative TUG UBX-Cter fragment decreases the size of the insulin-responsive pool and decreases the number of TUG-GLUT4 complexes Together, these data suggested that TUG is part of an insulin-responsive mechanism governing the sequestration of GLUT4 in GSVs within unstimulated 3T3-L1 cells. We use RNAi-mediated depletion of TUG to further characterize its role in GLUT4 trafficking and glucose uptake, using 3T3-L1 adipocytes These data show that TUG is required for intracellular retention of GLUT4 in nonendosomal, perinuclear membranes within unstimulated cells, consistent with the model described above. The magnitude of the effects we observe suggests that the TUG-GLUT4 complex may be an important site of insulin action to regulate glucose uptake in 3T3-L1 adipocytes
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