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

Abstract

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)P2] level. Extrdcelhlar addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated.1. 50 pM 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)P2 content. 50 pM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. I t did not antagonize the action of insulin in cells with initial high Fru(2,6)P2 content.2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 pM ATP or 15 pM adenosine S'[fl,y-methyleneltriphosphate. ADP and adenosine-5-[y-thio]triphosphate were as effective as ATP, whereas 100 pM adenosine 5'[a,fl-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis.3. Extracellular ATP (300 pM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P 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)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase.5. 100 pM 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)P2 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.Insulin as the principal hypoglycemic agent promotes disposal of external glucose in the liver in vivo [l]. For the study of its short-term effects on glucose metabolism, hepatocytes in primary culture have proved to be a suitable experimental tool. In cultured adult liver cells, insulin decreases glycogenolysis 12, 31 and increases glycolysis [4 -61. Acute stimulation of glycolysis by insulin proceeds only after a lag phase of 20 -

The diacylglycerol and protein kinase C pathways are not involved in insulin signalling in primary rat hepatocytes

“…At these higher concentrations, PLC strongly antagonized the action of insulin. As reported previously [42], ATP (> 10 lM) inhibited basal and insulinactivated glycolysis (results not shown).…”

Stimulation of glucose production from glycogen by glucagon, noradrenaline and non-degradable adenosine analogues is counteracted by adenosine and ATP in cultured rat hepatocytes

“…It is known that adenosine [37] and ATP [28] increase intracellular adenine nucleotide concentrations in isolated hepatocytes; it has also been demonstrated that adenosine decreases the cytosolic Pi concentration [38]. A tentative explanation for the inhibition of phosphorylase a described here could thus be a decrease in the availability of cytosolic Pi brought about by increased rephosphorylation of adenosine [39].…”

Purinergic signalling in the liver in health and disease

“…Stimulation of glycolysis by insulin in cultured hepatocytes is attenuated by ATP via inhibition of phosphofructokinase 2 [205]. The mammalian target of rapamycin (mTOR) pathway is one pathway utilised in hepatocytes to control glucose metabolism.…”