Glucose uptake in PC12 cells: GLUT3 vesicle trafficking and fusion as revealed with a novel GLUT3-GFP fusion protein

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Abstract
The distribution of glucose transporters at the cell surface has a major impact on cellular glucose uptake. In muscle cells and adipocytes, this distribution is under the control of insulin; however, neuronal glucose uptake is not acutely regulated by insulin. Factors that affect the translocation of the neuronal glucose transporter isoform GLUT3 vesicles to and their fusion with the plasma membrane are not well understood. We report that GLUT3 in PC12 cells colocalizes with SNARE complex proteins SNAP-25 and syntaxin 1, suggesting that fusion of GLUT3-containing vesicles with the plasma membrane is mediated by these proteins. In addition, it seems that GLUT3 vesicle fusion is regulated, as depolarization increases GLUT3 insertion into the plasma membrane. To study the dynamics of GLUT3 vesicle trafficking, we have created a GLUT3-GFP fusion protein that is easily expressed in PC12 cells. Trafficking of GLUT3-GFP seems normal, as 1). its distribution is similar to endogenous GLUT3, 2). GLUT3-GFP containing vesicles fuse with the plasma membrane evidenced by labeling of the fusion protein with an antibody directed against the exofacial epitope of GLUT3, and 3). glucose uptake is similar to PC12 cells not transfected with GLUT3 fusion protein. These studies are the first to examine GLUT3 trafficking and fusion in PC12 cells, and establish a model system to study regulation of the neuronal glucose transporter.