PACSIN family members regulate intracellular vesicle trafficking via their ability to regulate cytoskeletal rearrangement. These processes are known to be involved in trafficking of GLUT1 and GLUT4 in adipocytes. In this study, PACSIN3 was observed to be the only PACSIN isoform that increases in expression during 3T3-L1 adipocyte differentiation. Overexpression of PACSIN3 in 3T3-L1 adipocytes caused an elevation of glucose uptake. Subcellular fractionation revealed that PACSIN3 overexpression elevated GLUT1 plasma membrane localization without effecting GLUT4 distribution. In agreement with this result, examination of GLUT excifacial presentation at the cell surface by photoaffinity labeling revealed significantly increased GLUTI, but not GLUT4, after overexpression of PACSIN3. These results establish a role for PACSIN3 in regulating glucose uptake in adipocytes via its preferential participation in GLUTI trafficking. They are consistent with the proposal, which is supported by a recent study, that GLUTI, but not GLUT4, is predominantly endocytosed via the coated pit pathway in unstimulated 3T3-L1 adipocytes. (c) 2007 Elsevier Inc. All rights reserved.