Uptake of glucose-3H into cultured HLM cells was measured. Equilibration of intracellular and extracellular pools occurred after 25 min. Glucose influx was determined subsequently by measuring the glucose-3H entering in precisely 1 min. Although saturation kinetics were demonstrated these were not of the simple Michaelis-Menten type. The Km of the glucose carrier system is probably about 60 mM glucose. Galactose did not compete with glucose. Insulin stimulated glucose flux without increasing the value of Vmax. The stimulation was fully demonstrable after 10 min, could be elicited at concentrations of 10-4 units/ml, and was absent 2–4 hr after removal. Increasing pH had little or no effect in stimulating glucose flux. Increasing osmotic pressure caused a marked increase and reduced the effect of insulin. Glucose influx was unaffected by anoxia. Glucose influx was increased and the effect of insulin abolished in the absence of K+. Glucose influx was increased by mercuric chloride, iodoacetate, and fluoride which abolished the effect of insulin. Dinitrophenol decreased the rate of glucose uptake but did not alter the effect of insulin. Phlorizin reduced the rate of glucose uptake and abolished the effect of insulin. ATP and AMP enhanced the rate of glucose uptake. These findings are discussed in relation to the mode of action of insulin.

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