Differential sorting of two glucose transporters expressed in insulin-sensitive cells

Abstract

The insulin-regulatable glucose transporter (IRGT) is specifically expressed in muscle and fat cells and undergoes translocation from an intracellular compartment to the cell surface following acute insulin treatment. This study examined sorting differences between the IRGT and the homologous HepG2/erythrocyte/brain glucose transporter (HepG2 GT) when expressed together in insulin-responsive 3T3-L1 adipocytes. The ratio of the amount of transporter per unit protein in the plasma membrane fraction vs. the intracellular membrane fraction was 1:2 for the HepG2 GT and 1:30 for the IRGT. Insulin treatment increased the plasma membrane concentration of the IRGT by 10-fold and the HepG2 GT by 3.5-fold. This distribution was confirmed by confocal immunofluorescence microscopy. Differential sorting within intracellular organelles was evident by sucrose gradient analysis and immunoisolation of transporter vesicles and by double immunofluorescence labeling. We propose that differential sorting at an intracellular locus preferably withdraws the IRGT from a pathway which is in close communication with the plasma membrane, thus allowing the IRGT to regulate glucose entry into fat and muscle cells in a highly insulin-regulated fashion.

Compartment ablation analysis of the insulin-responsive glucose transporter (GLUT4) in 3T3-L1 adipocytes

“…Okadaic acid was from Moana BioTech, Hawaii. All other reagents were as described elsewhere [14,23].…”

“…Adipocytes were subjected to a differential centrifugation procedure as described previously [14]. Cells grown on 10-cm-diam.…”

“…Homogenates of adipocytes in HES buffer were centrifuged at 19 000 g for 20 min at 4 mC to pellet the plasma membrane fraction [14,27]. The supernatant from this step was then centrifuged at 150 000 g for 60 min at 4 mC to pellet the remaining membrane fraction.…”

“…All subsequent steps were performed at 4 mC. Plates were then rinsed with HES buffer, scraped with a rubber policeman into 6 ml of HES buffer and homogenized using a Yamato homogenizer [14]. The homogenate was centrifuged at 41 000 g for 20 min to remove plasma membranes, mitochondria, nuclei and high-density microsomes [14].…”

“…First, GLUT4 is virtually excluded from the cell surface in non-stimulated cells whereas this is not the case for other membrane proteins such as GLUT1 or the TfR [13,14]. Secondly, the externalization rate of GLUT4 is lower than that of other recycling proteins such as TfR in adipocytes [6,15] and insulin results in a much larger fold increase in GLUT4 at the surface compared with other endosomal proteins (reviewed in [2,9]).…”