Kinetic analysis of glucose transporter trafficking in fibroblasts and adipocytes

Abstract

Insulin regulates hexose uptake by the redistribution of glucose transport proteins from intracellular compartments to the cell surface. We have submitted the trafficking of GLUT1, GLUT4, and GLUT1/GLUT4 chimeras to a mathematical analysis in the context of different models. Our data suggest that a model with one intracellular and one cell surface compartment can describe the glucose transporter-trafficking kinetics in fibroblasts. Moreover, the difference in cellular distribution between GLUT1 and GLUT4 overexpressed in fibroblasts is best explained by a slower rate of movement of GLUT4 to the plasma membrane. In 3T3-L1 adipocytes, glucose transporter-trafficking kinetics is adequately described by a three-pool model which includes flow of transporters from the endosomal compartment to cell surface. The kinetic roles of previously identified motifs in GLUT4 trafficking were defined in proposed fibroblast and adipocyte glucose transporter-trafficking models. The C-terminus is important in reducing the exocytosis rate from the endosomal compartment to the cell surface in both fibroblasts and adipocytes, and the N-terminus behaves similarly in adipocytes. The C-terminus has an additional signal(s) that allows GLUT4 to be sequestered more efficiently into the insulin responsive vesicle compartment. Mutation of the dileucine motif in the C-terminus significantly reduces the endocytosis of GLUT4 in both fibroblasts and adipocytes, but these amino acids appear not to be primarily responsible for the different kinetics of wild-type GLUT1 and GLUT4.

Insulin-induced Recruitment of Glucose Transporter 4 (GLUT4) and GLUT1 in Isolated Rat Cardiac Myocytes

“…Our finding that insulin depletes GLUT1 and SCAMP 39 from GLUT4 pool 2 is consistent with the model proposed by Yeh et al (50), according to which GLUT1 would recycle from the endosomal compartment to the cell surface in adipocytes. Furthermore, GLUT4 pool 1 might be analogous to the insulinrecruitable compartment proposed by others on the basis of kinetic (48,50) and immunoadsorption (53) experiments. In keeping with this, we found that GLUT4 was markedly depleted from the 1F8-agarose immunoprecipitates after insulin treatment (Fig.…”

“…First, mathematical analysis of kinetic data of GLUT4 endocytosis and exocytosis obtained with the photoaffinity reagent ATB-BMPA (6,35) predicts that GLUT4 localizes to at least two distinct intracellular compartments in fat cells (48). Second, data of subcellular trafficking of both GLUT4 and GLUT1 and chimeric transporters in adipocytes (49) are best explained by a model postulating two intracellular pools (50). According to the predictions of this model, GLUT4 is internalized into an endosomal compartment and then sorted into an insulin-recruitable compartment; in contrast, GLUT1 is endocytosed into the endosomal compartment and recycles from this compartment to the cell surface (50).…”

“…Second, data of subcellular trafficking of both GLUT4 and GLUT1 and chimeric transporters in adipocytes (49) are best explained by a model postulating two intracellular pools (50). According to the predictions of this model, GLUT4 is internalized into an endosomal compartment and then sorted into an insulin-recruitable compartment; in contrast, GLUT1 is endocytosed into the endosomal compartment and recycles from this compartment to the cell surface (50). Further support to the idea that GLUT4 is present in separate intracellular compartments comes from very recent compartment ablation analysis; thus, ablation of the endosomal compartment in 3T3-L1 adipocytes reduces by 40% the amount of cellular GLUT4 (51).…”

Recycling of the Insulin-sensitive Glucose Transporter GLUT4

“…The 12 transmembrane domain protein displays, in its amino and carboxy-cytoplasmic tails, Phe 5 -Gln 6 -Gln 7 -Ile 8 -based (13) and Leu 489 -Leu 490 -based motifs (14) that when inactivated provoke its cellular redistribution. Both motifs have been involved in GLUT4 endocytosis, intracellular retention, and targeting in transfected 3T3-L1fibroblasts, COS-7 and Chinese hamster ovary cells (15)(16)(17)(18)(19)(20), myoblasts (21), and adipocytes (18,(22)(23)(24). Whether the dileucine motif mediates the trafficking of GLUT4 in adipocytes is, however, the subject of much debate (18,22,23).…”

“…Both motifs have been involved in GLUT4 endocytosis, intracellular retention, and targeting in transfected 3T3-L1fibroblasts, COS-7 and Chinese hamster ovary cells (15)(16)(17)(18)(19)(20), myoblasts (21), and adipocytes (18,(22)(23)(24). Whether the dileucine motif mediates the trafficking of GLUT4 in adipocytes is, however, the subject of much debate (18,22,23). In addition, the mechanisms that mediate the intracellular retention and recycling of GLUT4 to the intracellular store compartments remain essentially unknown.…”