Hexose Transport in Isolated Brown Fat Cells: A MODEL SYSTEM FOR INVESTIGATING INSULIN ACTION ON MEMBRANE TRANSPORT

Abstract

Abstract Direct measurement of 3-O-[3H]methyl-d-glucose uptake by brown fat cells with the use of a rapid filtration procedure was found to provide a suitable index of the d-glucose transport system activity. The rate of net 3-O-methylglucose influx across the fat cell membrane was 10 to 20 times greater than the uptake rate of l-[14C]glucose which enters the cells by simple diffusion. After equilibration the concentration of 3-O-methylglucose in the intracellular water space was the same as that in the medium, indicating that 3-O-methylglucose uptake occurs by facilitated diffusion. Detectable phosphorylation of this d-glucose analogue by brown fat cells under the conditions of these studies did not occur. 3-O-Methylglucose transport was consistently linear for 4 or more min at 23° and was several times greater at 37° and 23° than at 0°. Transport of 3-O-methylglucose was inhibited by d-glucose and by agents known to block d-glucose transport such as phlorizin and cytochalasin B. Both saturable and nonsaturable processes contributed to the initial velocity of 3-O-methylglucose uptake. The saturable component of net influx was estimated by subtraction of uptake rates monitored in the presence of cytochalasin B, which were used to approximate the nonsaturable component, from the initial velocities of 3-O-methylglucose uptake. The corrected values obtained followed simple Michaelis-Menten kinetics with an apparent Kt of about 4 mm. At high concentrations of the hexose, diffusion probably represented the major contribution to uptake since this portion of a Lineweaver-Burk plot of uncorrected values passed through the origin. Insulin markedly stimulated the initial rate of hexose transport, and this effect reflected an increase in apparent Vmax with no detectable difference in apparent Kt. Diamide, H2O2, vitamin K5, and spermine enhanced d-[1-14C]glucose conversion to 14CO2 to a greater extent than d-[6-14C]glucose oxidation, whereas in these experiments insulin increased oxidation of both sugars equally. Vitamin K5 and spermine had little effect on 3-O-methylglucose transport under conditions where insulin, diamide, and H2O2 significantly stimulated this process. The action of diamide and H2O2 to increase 3-O-methylglucose uptake was specific for the d-glucose transport system since, like insulin, they failed to alter l-glucose uptake. The data suggested that vitamin K5 and spermine stimulate fat cell glucose uptake and oxidation primarily by activating intra-cellular metabolic pathways while diamide and H2O2, in addition to an intracellular effect, also activate the d-glucose transport system. These results indicate that glucose uptake in brown fat cells is determined by both the activity of the membrane transport system and the rate of intra-cellular utilization and that alteration of either process can modulate glucose entry rates.