Depletion of intracellular potassium (K+) induced a 4-fold increase in basal and 1 μM phorbol-12-myristate-13-acetate (PMA)-stimulated 3-O- methylglucose transport in rat adipose cells. K+ depletion had no effect on the maximum insulin (0.7 μM)-stimulated transport rate but enhanced the sensitivity to insulin 3-fold (EC50 = 0.05 versus 0.15 nM) by a mechanism that did not result from changes in the insulin receptor binding, autophosphorylation, or tyrosine kinase activity. Western blotting analysis revealed that K+ depletion induced a 2.2-fold increase in GLUT4 in plasma membranes from basal cells, enhanced the PMA-stimulated GLUT4 translocation by 4-fold, and increased the 5-fold insulin-stimulated GLUT4 translocation by 15%, indicating the presence of an inactive GLUT4 intermediate. The time course for insulin's stimulation of transport activity was accelerated by K+ depletion (t( 1/2 ) = 3 versus 1.5 min). Conversely, the reversal of transport activity, on removal of insulin, was delayed (t( 1/2 ) = 11 versus 22 min). The corresponding t( 1/2 ) values for the loss of GLUT4 were 22 min in control cells and 40 min in K+-depleted cells, again indicating the existence of an inactive intermediate. Photolabeling intact cells with the impermeant, exofacial photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D- mannos-4-yloxy)-2-propylamine in the continuous presence of insulin revealed that K+ depletion had no effect on the GLUT4 externalization rate but halved the rate of internalization. K+ depletion elicited entirely analogous effects on the recycling of insulin-like growth factor II/mannose 6-phosphate receptor, strongly supporting the involvement of a coated pit mechanism in the recycling of GLUT4 transporters. An inactive conformation of GLUT4 has been detected in plasma membranes from insulin-stimulated cells, which is enhanced by K+ depletion, suggesting a limitation in the adipose cells' capacity to express active GLUT4 transporters.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|State||Published - 1993|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology