title: GLUT8 subcellular localization and absence of translocation to the plasma membrane in PC12 cells and hippocampal neurons.
abstract: GLUT8 is a high-affinity glucose transporter present mostly in testes and a subset of brain neurons. At the cellular level, it is found in a poorly defined intracellular compartment in which it is retained by an N-terminal dileucine motif. Here we assessed GLUT8 colocalization with markers for different cellular compartments and searched for signals, which could trigger its cell surface expression. We showed that when expressed in PC12 cells, GLUT8 was located in a perinuclear compartment in which it showed partial colocalization with markers for the endoplasmic reticulum but not with markers for the trans-Golgi network, early endosomes, lysosomes, and synaptic-like vesicles. To evaluate its presence at the plasma membrane, we generated a recombinant adenovirus for the expression of GLUT8 containing an extracellular myc epitope. Cell surface expression was evaluated by immunofluorescence microscopy of transduced PC12 cells or primary hippocampal neurons exposed to different stimuli. Those included substances inducing depolarization, activation of protein kinase A and C, activation or inhibition of tyrosine kinase-linked signaling pathways, glucose deprivation, AMP-activated protein kinase stimulation, and osmotic shock. None of these stimuli-induced GLUT8 cell surface translocation. Furthermore, when GLUT8myc was cotransduced with a dominant-negative form of dynamin or GLUT8myc-expressing PC-12 cells or neurons were incubated with an anti-myc antibody, no evidence for constitutive recycling of the transporter through the cell surface could be obtained. Thus, in cells normally expressing it, GLUT8 was associated with a specific intracellular compartment in which it may play an as-yet-uncharacterized role.
authors.affiliation: Department of Physiology, University of Lausanne, 27 rue du Bugnon, 1005 Lausanne, Switzerland.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Widmer
authors.forename: Marc
authors.initials: M
authors.lastname: Uldry
authors.forename: Bernard
authors.initials: B
authors.lastname: Thorens
mesh_terms: D055372:AMP-Activated Protein Kinases; D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology; D004594:Electrophysiology; D004789:Enzyme Activation; D005947:Glucose / Q000172:deficiency; D051246:Glucose Transport Proteins, Facilitative / Q000235:genetics / Q000378:metabolism*; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008856:Microscopy, Fluorescence; D009097:Multienzyme Complexes / Q000378:metabolism; D009474:Neurons / Q000378:metabolism* / Q000648:ultrastructure; D009997:Osmotic Pressure; D016716:PC12 Cells / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D051246:Glucose Transport Proteins, Facilitative; C489427:MYC protein, human; D009097:Multienzyme Complexes; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; C498629:Slc2a8 protein, rat; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; D005947:Glucose