Insulin and isoproterenol have opposing roles in the maintenance of cytosol pH and optimal fusion of GLUT4 vesicles with the plasma membrane.
Insulin treatment of rat adipocytes increases both cytoplasmic alkalinity and glucose transport activity. Both processes are blocked by the phosphatidylinositol 3-kinase inhibitor wortmannin. Isoproterenol pre-treatment reverses the alkalinizing effects of insulin and leads to attenuation of insulin-stimulated glucose transport activity and exposure of GLUT4 to photolabeling reagents at the cell surface. These effects of isoproterenol are mimicked by acid loading and are reversed by cell-alkalinizing conditions. However, neither isoproterenol nor acid loading alters the total level of GLUT4 at the plasma membrane as revealed by Western blotting of plasma membrane fractions or immunodetection of GLUT4 in plasma membrane lawns. GLUT4 is therefore occluded from participation in glucose transport catalysis by a pH-sensitive process. To examine the kinetics of trafficking that lead to these changes in cell surface GLUT4 occlusion, we have utilized a new biotinylated photolabel, GP15. This reagent has a 70-atom spacer between the biotin and the photolabeling diazirine group, and this allows quenching of the surface signal of biotinylated GLUT4 by extracellular avidin. The rates of GLUT4 internalization are only slightly altered by isoproterenol or acidification, mainly due to reduced recycling over long internalization times. By contrast, insulin stimulation of GLUT4 exocytosis is slowed by isoproterenol or acidification pre-treatments. Biphasic time courses are evident, with an initial burst of exposure at the cell surface followed by a slow phase. It is hypothesized that the burst kinetics are a consequence of a two-phase fusion reaction that is rapid in the presence of insulin but slowed by cytosol acidification.
Yang
Jing
J
Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, United Kingdom.
Hodel
Alois
A
Holman
Geoffrey D
GD
D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D021961:Cell Membrane Structures / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005947:Glucose / Q000378:metabolism; D051275:Glucose Transporter Type 4; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000494:pharmacology*; D007545:Isoproterenol / Q000494:pharmacology*; D007700:Kinetics; D008297:Male; D018613:Microscopy, Confocal; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D051381:Rats; D017208:Rats, Wistar
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; C495694:Slc2a4 protein, rat; D005947:Glucose; D007545:Isoproterenol