Abstract
To examine the role of clathrin-coated vesicle endocytosis in insulin receptor signaling and GLUT4 trafficking, we used recombinant adenovirus to express a dominant interfering mutant of dynamin (K44A/dynamin) in 3T3L1 adipocytes. Functional expression of K44A/dynamin, as measured by inhibition of transferrin receptor internalization, did not affect insulin-stimulated insulin receptor autophosphorylation, Shc tyrosine phosphorylation, or mitogen-activated protein kinase activation. Although the tyrosine phosphorylation of insulin receptor substrate-1 was slightly reduced, correlating with a 25% decrease in insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity, insulin-stimulated Akt kinase activation was unaffected. In contrast, expression of K44A/dynamin resulted in the cell-surface accumulation of GLUT4 under basal conditions and an inhibition of GLUT4 endocytosis without affecting insulin-stimulated GLUT4 exocytosis. These data demonstrate that disruption of clathrin-mediated endocytosis does not significantly perturb insulin receptor signal transduction pathways. Furthermore, K44A/dynamin expression causes an accumulation of GLUT4 at the cell surface, suggesting that GLUT4 vesicles exist in at least two distinct intracellular compartments, one that undergoes continuous recycling and a second that is responsive to insulin.
Highlights
The insulin-responsive glucose transporter, GLUT4,1 is expressed at high levels in adipose tissue, skeletal muscle, and cardiac myocytes [1,2,3,4]
Concurrent with the insulin-dependent increase in GLUT4 vesicle exocytosis, insulin reduces the rate of GLUT4 endocytosis up to 3-fold (8 –10)
Dynamin Expression Is Increased during 3T3L1 Adipocyte Differentiation—Differentiated 3T3L1 adipocytes represent an excellent model system to study GLUT4 trafficking since they share many of the characteristics of primary adipocytes, including movement of GLUT4 from intracellular stores to the plasma membrane in response to insulin [36, 37]
Summary
The insulin-responsive glucose transporter, GLUT4,1 is expressed at high levels in adipose tissue, skeletal muscle, and cardiac myocytes [1,2,3,4]. Increased expression of K44A/ dynamin results in an inhibition of transferrin receptor internalization and functions in a dominant interfering manner to reduce coated vesicle endocytosis in 3T3L1 adipocytes. Under these conditions, uninfected, LacZ-, and K44A/dynamin-expressing cells exhibited similar patterns of Akt activation in response to increasing doses or times of insulin stimulation.
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