Abstract
The majority of GLUT4 is sequestered in unique intracellular vesicles in the absence of insulin. Upon insulin stimulation GLUT4 vesicles translocate to, and fuse with, the plasma membrane. To determine the effect of GLUT4 content on the distribution and subcellular trafficking of GLUT4 and other vesicle proteins, adipocytes of adipose-specific, GLUT4-deficient (aP2-GLUT4-/-) mice and adipose-specific, GLUT4-overexpressing (aP2-GLUT4-Tg) mice were studied. GLUT4 amount was reduced by 80-95% in aP2-GLUT4-/- adipocytes and increased approximately 10-fold in aP2-GLUT4-Tg adipocytes compared with controls. Insulin-responsive aminopeptidase (IRAP) protein amount was decreased 35% in aP2-GLUT4-/- adipocytes and increased 45% in aP2-GLUT4-Tg adipocytes. VAMP2 protein was also decreased by 60% in aP2-GLUT4-/- adipocytes and increased 2-fold in aP2-GLUT4-Tg adipocytes. IRAP and VAMP2 mRNA levels were unaffected in aP2-GLUT4-Tg, suggesting that overexpression of GLUT4 affects IRAP and VAMP2 protein stability. The amount and subcellular distribution of syntaxin4, SNAP23, Munc-18c, and GLUT1 were unchanged in either aP2-GLUT4-/- or aP2-GLUT4-Tg adipocytes, but transferrin receptor was partially redistributed to the plasma membrane in aP2-GLUT4-Tg adipocytes. Immunogold electron microscopy revealed that overexpression of GLUT4 in adipocytes increased the number of GLUT4 molecules per vesicle nearly 2-fold and the number of GLUT4 and IRAP-containing vesicles per cell 3-fold. In addition, the proportion of cellular GLUT4 and IRAP at the plasma membrane in unstimulated aP2-GLUT4-Tg adipocytes was increased 4- and 2-fold, respectively, suggesting that sequestration of GLUT4 and IRAP is saturable. Our results show that GLUT4 overexpression or deficiency affects the amount of other GLUT4-vesicle proteins including IRAP and VAMP2 and that GLUT4 sequestration is saturable.
Highlights
The number of GLUT4 and Insulin-responsive aminopeptidase (IRAP)-containing vesicles per cell 3-fold
The residual glucose transport is probably due to the presence of GLUT1 and to GLUT4 remaining in a small subset of adipocytes that escaped Cre recombinase-mediated deletion
Intracellular Sequestration of GLUT4 Is Saturable in Adipocytes—A major finding of this study is that GLUT4 overexpression in adipocytes causes subcellular redistribution of GLUT4
Summary
TfR, transferrin receptor; IRAP, insulin-responsive aminopeptidase; SNARE, soluble NSF attachment protein receptors; LDM, low density microsomal membranes; PM, plasma membranes; -COP, -coatomer protein; WT, wild type. We hypothesized that altering adipocyte GLUT4 content may alter the protein composition, number, and/or sequestration of GLUT4-containing vesicles but not affect plasma membrane proteins mediating GLUT4 trafficking or other proteins trafficking through endosomes in response to insulin. Levels of plasma membrane proteins syntaxin, SNAP23, and Munc-18c, which mediate GLUT4-vesicle docking and fusion with plasma membranes, are unchanged. GLUT4 overexpression results in an increased proportion of GLUT4 at the plasma membrane in the absence of insulin, supporting the notion that GLUT4 sequestration is saturable. This could occur at the level of retention, endocytosis, and/or recycling of GLUT4 from the plasma membrane to intracellular stores. Our results indicate that GLUT4 is a major determinant of GLUT4 vesicle net biogenesis and sequestration in vivo
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