Insulin and Epidermal Growth Factor Stimulate Glycolysis in Quiescent 3T3 Fibroblasts with no Changes in Key Glycolytic Enzyme Activities

Abstract

In order to study the effect of insulin and epidermal growth factor (EGF) on glycolysis in quiescent 3T3 fibroblasts and their mechanisms of action, l actic acid produced by cells and activities of key glycolytic enzymes in cell extracts were determined. Insulin increased lactic acid production; the maximal stimulation occurred at the concentrations above 250 ng/ml and the halfmaximal dose was 50ng/ml. This effect of insulin appeared as early as one hour, and lactic acid production in the presence of insulin linearly increased up to 4 h. The 24-h pretreatment with insulin exhibited no significant effect on the production by cells afterward incubated either with or without insulin. Lactic acid production decreased as the concentration of phloridzin increased. However, insulin stimulation of the production still remained in the presence of phloridzin. Parahydroxymercuribenzoate reduced production only by the equivalent of the increase due to insulin. EGF also increased lactic acid production ; this effect occurred at 1 ng/ml and was maximal at 100 ng/ml. The activities of hexokinase, phosphofructokinase and pyruvate kinase in quiescent cells were not increased by insulin, and the affinities for substrates of these enzymes remained unaltered. These findings suggest that glucose uptake is a rate-limiting step in glycolysis in quiescent 3T3 fibroblasts and that the stimulatory effect of insulin on glycolysis is mediated by enhanced glucose entry.