Abstract
A novel method was developed to measure relative amounts of the GLUT4 glucose transporter on the surface of intact fat cells and to monitor the action of insulin on cell surface glucose transporters as they internalize into intracellular membranes. The method takes advantage of two predicted trypsin cleavage sites in the major exofacial loop of this transporter protein. Treatment of cyanide-poisoned rat adipocytes with 1 mg/ml trypsin at 37 degrees C for 30 min produced an immunoreactive GLUT4 protein species in subsequently isolated plasma membranes that migrated with higher mobility (apparent M(r) = 35,000) than native GLUT4 (apparent M(r) = 46,000) on SDS-polyacrylamide gel electrophoresis. This proteolyzed GLUT4 protein was absent in the intracellular low density microsomes. Insulin treatment of adipocytes for 20 min prior to sequential additions of cyanide and trypsin caused a 16-fold increase in the proteolytically cleaved GLUT4 species. Incubation of fresh fat cells with trypsin caused a rapid and progressive appearance of the proteolyzed GLUT4 species in the intracellular low density membranes as well as plasma membranes. After 5 min of trypsinization, 66% of the total cleaved GLUT4 in these cells had moved into the low density membranes. Insulin treatment markedly decreased the internalized cleaved GLUT4 to 20% of the total. These data indicate the following: 1) trypsinization of the GLUT4 transporter protein on intact fat cells is a convenient means to monitor the extent of transporter recruitment to the plasma membrane by insulin, as well as to estimate GLUT4 internalization rates; and 2) the action of insulin on glucose transporter redistribution to the cell surface is associated with a marked inhibition of the fraction of cell surface GLUT4 transporters internalized per unit time.
Highlights
A novel method was developed to measure relative proteins from intracellular stores to the plasma membrane amounts of the GLUT4 glucose transporter on the sur- where they can catalyzesugar uptake [12,13,14]
Like cells to mature muscle or fat cells correlates with the de nouo expression of GLUT4 protein in the latter
This effect has face of intact fat cells and to monitor the action of been observed by quantifying GLUTl and GLUT4 protein insulin on cell surface glucose transporters as they content in isolated plasma membranes derived from control internalize into intracellularmembranes
Summary
Like cells to mature muscle or fat cells correlates with the de nouo expression of GLUT4 protein in the latter. A novel method was developed to measure relative proteins from intracellular stores to the plasma membrane amounts of the GLUT4 glucose transporter on the sur- where they can catalyzesugar uptake [12,13,14]. This effect has face of intact fat cells and to monitor the action of been observed by quantifying GLUTl and GLUT4 protein insulin on cell surface glucose transporters as they content in isolated plasma membranes derived from control internalize into intracellularmembranes. These data indicate the following: 1) trypsinization of the GLUT4 transporter protein on intactfat cells is a convenient means to monitor the extentof transporter recruitment to thpelasma membrane by insulin, as well
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