title: Real-time imaging of sodium glucose transporter (SGLT1) trafficking and activity in single cells.
abstract: The processes controlling targeting of glucose transporters to apical and basolateral membranes of polarized cells are complex and not-well understood. We have engineered SGLT1 and GLUT4 constructs linked to fluorescent proteins to highlight the differences in transporter expression and trafficking, in real time, in different cell types. Activity was assessed in parallel using a FRET glucose sensor. In COS cells and HEK cells, SGLT1 was distributed between the plasma membrane and intracellular compartments, but there was little expression in CHO cells. Trafficking was investigated using the lysosome inhibitors NH4Cl (10 mmol/L) and chloroquine (150μmol/L) and the proteasome inhibitors MG-262 (1μmol/L) and lactacystin (5μmol/L). Lysosome inhibitors caused SGLT1 accumulation into intracellular bodies, whereas proteasome inhibitors induced SGLT1 accumulation in the plasma membrane, even in CHO cells. Our data suggest that a fraction of SGLT1 is rapidly degraded by lysosomes and never reached the plasma membrane; another fraction reaches the membrane and is subsequently degraded by lysosomes following internalization. The latter process is regulated by the ubiquitin/proteasome pathway, acting at a late stage of the lysosomal pathway. Using the cholesterol inhibitor MβCD (3 mmol/L), a dominant negative dynamin (K44A) and caveolin, we showed that SGLT1 internalization is lipid raft-mediated, but caveolin-independent. In contrast, GLUT4 internalization is dynamin-dependent, but cholesterol-independent. The physiological relevance of these data is discussed in terms of differential membrane compartmentalization of the transporters and expression under stress conditions.
authors.affiliation: Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, California.
authors.forename: Chiara
authors.initials: C
authors.lastname: Ghezzi
authors.affiliation: Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, California.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Calmettes
authors.affiliation: Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, California.
authors.forename: Pauline
authors.initials: P
authors.lastname: Morand
authors.affiliation: Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, California bribalet@mednet.ucla.edu.
authors.forename: Bernard
authors.initials: B
authors.lastname: Ribalet
authors.affiliation: Departments of Physiology and Medicine and the Cardiovascular Research Laboratory, David Geffen School of Medicine at UCLA, Los Angeles, California.
authors.forename: Scott
authors.initials: S
authors.lastname: John
mesh_terms: D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D016764:Cell Polarity; D002522:Chlorocebus aethiops; D003412:Cricetulus; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins; D008247:Lysosomes / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D021381:Protein Transport; D059748:Proteolysis; D051269:Sodium-Glucose Transporter 1 / Q000378:metabolism*
chemical_list: D051275:Glucose Transporter Type 4; D008164:Luminescent Proteins; C495692:SLC2A4 protein, human; C495674:SLC5A1 protein, human; D051269:Sodium-Glucose Transporter 1; D046988:Proteasome Endopeptidase Complex
keywords: Endocytosis; SGLT1; lysosome; proteasome