Stimulation of insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine‐dependent inhibition of phosphofructokinase 2 activation

Activation of glycolysis by insulin in cultured rat hepatocytes is preceeded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructase 2,6‐bisphosphate [Fru(2,6) P 2 ] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated.1 50 μM ATP completely prevented the 3–5‐fold insulin‐dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6) P 2 content 50 μM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6) P 2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin in cells with initial high Fru(2,6) P 2 content. 2 ATP exerted effects on its own; it decreased initially high Fru(2,6) P 2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half‐maximal effects on the Fru(2,6) P 2 level were obtained with about 25 μM ATP or 15 μM adenosine 5′[β,γ‐methylene]triphosphate. ADP and adenosine‐5‐(γ‐thio]triphosphate were as effective as ATP, whereas 100 μM adenosine 5′[α,β‐methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6) P 2 levels nor inhibited basal glycolysis. 3 Extracellular ATP (100 μM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6 P , citrate. ATP, ADP and AMP were increased by extracellular ATP, the phospho enol pyruvate content was decreased, Fru6 P and glycerol 3‐phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4 Both puromycin and ATP prevented the insulin‐dependent rise of the Fru(2,6) P 2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6) P 2 provoked by glucose addition; ATP also antagonized the glucose‐dependent increase. 5 100 μM ATP elevated the cAMP‐dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16‐fold within 5 min, whereas puromycin was without effect on either level.It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6) P 2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.