Jeffrey S Elmendorf Introduction

In 1980, two groups simultaneously provided evidence of the existence of an intracellular pool of glucose transporters in rat adipocytes (1,2). We now know that facilitative glucose uptake occurs through a family of highly related integral membrane proteins that share significant sequence similarity. Of the established glucose transporter isoforms, GLUT-4 is highly expressed in adipose tissue and striated muscle (3). In the basal state, GLUT-4 cycles slowly between the plasma membrane and one or more intracellular compartments, with the vast majority of the transporter residing in vesicular compartments within the cell interior (4-6). Activation of the insulin receptor triggers a large increase in the rate of GLUT-4 vesicle exocytosis and a smaller decrease in the rate of internalization by endocytosis (7-10). The stimulation of exocytosis by insulin is probably the major step for GLUT-4 translocation because complete inhibition of GLUT-4 endocytosis only modestly increases plasma membrane-associated GLUT-4 protein without affecting the extent of insulin-stimulated GLUT-4 translocation (11-13). In contrast to GLUT-4, GLUT-1 is an intracellular and plasma membrane localized in the basal state and displays a modest insulin-stimulated redistribution to the plasma membrane. Thus, the overall insulin-dependent shift in the cellular dynamics of GLUT-4 vesicle trafficking results in a net increase of GLUT-4 on the cell surface, thereby increasing the rate of glucose uptake.

Abundant studies have used both primary (isolated from adipose tissue by collagenase digestion) and cultured (3T3-L1 adipocyte model system) fat cells to evaluate insulin action as it pertains to GLUT-4 translocation. This is because these cells can be fractionated into relatively pure membrane fractions. Herein we will focus on the use of the subcellular fractionation technique to analyze GLUT-4 cellular distribution in the 3T3-L1 adipocyte model system. Adipocytes at 10-12 d after withdrawal from differentiation media are routinely used for fractionation, and at this time of adipocyte development, the mRNA and protein levels of GLUT-4 (14) together with the glucose-transport response to insulin (15) have reached a steady state. Subcellular fractionation of 3T3-L1 adipocytes uses a procedure similar to that developed for the fractionation of isolated adipocytes (16,17). Four fractions obtained by differential centrifuga-tion include the plasma membrane (PM), low-density microsomes (LDMs), high-density microsomes (HDMs), and cytosol (CYT). The intracellular pool of GLUT-4 is localized in the LDM. Because of its migration in sucrose density gradients, compared with other subcellular compartments, this membrane fraction is conventionally called the LDM fraction. As well as being enriched with GLUT-4, this fraction contains endosomes and the Golgi apparatus. The HDM fraction is enriched in endoplasmic reticulum but a considerable cross-contamination with PM and LDM organelles is inevitable.

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