Volume 125, number 2 FEBS LETTERS March 1981 IS THE GLUCOSE-INDUCED STIMULATION OF GLYCOLYSIS IN PANCREATIC ISLETS ATTRIBUTABLE TO ACTIVATION OF PHOSPHOFRUCTOKINASE BY FRUCTOSE 2,6-BISPHOSPHATE? Willy J. MALAISSE, Francine MALAISSE-LAGAE, Abdullah SENER, Emile VAN SCHAFTINGEN and Henri-G&y HERS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO Laboratory of Experimental M edicine, Brussels Free University, Laboratory of Physiological Chemistry, Louvain University and International Institute of Cellular and Molecular Pathology, Brussels 1000, Belgium Received 2 February 1981 1. Introduction It is currently thought that the process through which glucose is identified in the pancreatic B-cell as a stimulus for both proinsulin biosynthesis and insulin release strictly depends on the integrity of glucose metabolism in the islet cells [ 1,2]. However, the mechanism by which the glycolytic and oxidative flux is increased in response to a rise in the extracellular concentration of glucose is not fully elucidated. In hepatocytes, the rate of glycolysis is regulated at the level of fructose 6-phosphate phosphorylation by a newly discovered hexose phosphate, fructose 2,6-bis- phosphate, which activates phosphofructokinase [3-S]. Here, we propose that a similar situation prevails in pancreatic islets. 2. Materials and methods For the assay of phosphofructokinase [3], groups of 1200-l 500 pancreatic islets each isolated from fed albino rats were homogenized in Potter-Elvehjem tubes with 0.65 ml Hepes-NaOH buffer (25 mM, pH 7.4) containing NaF (50 mM) and EGTA (7.5 mM). The homogenate was centrifuged (30 min, 30 000 X g) and the supernatant used in 0.1 ml samples/assay cuvette. The reaction mixture (1 .O ml) contained (final concentration): fructose 6-phosphate (0.1-5.0 mM); glucose 6-phosphate (glucose 6-phosphate/fructose 6-phosphate concentration ratio of 3 .O); ATP (1.5 mM); NADH (0.075 mM); AMP (0.1 mM); Hepes (50 mM, pH 7.0); NaF (5.0 mM); EGTA (0.75 mM); KC1 (100 mM); MgClz (5.0 mM); KH2P04 (5.0 mM); BlsevierjNorth-Holland Biomedical Press NH4C1 (1 .O mM); the auxiliary enzymes (phosphoglu- case-isomerate 1 U/ml, aldolase 0.5 U/ml, triose phos- phate isomerase 5 U/ml, and glycerophosphate dehydro- genase 0.5 U/ml); and, as required, fructose 2,6-bis- phosphate prepared from fructose 1,6-bisphosphate by an adaption of the method in [6]. The reaction was initiated by the addition of ATP and the fall in NADH concentration was recorded at room temperature by spectrophotometry at 340 nm. In a second series of experiments, paired groups of 600 islets each were incubated for 60 min at 37°C in the absence or presence of D-glucose (20 mM) in 2.0 ml bicarbonate-buffered medium equilibrated against a mixture of O2 (95%)-CO2 (5%) [7]. After incubation, the islets were briefly washed with a glucose free medi- um and homogenized. The homogenate was prepared and examined for its phosphofructokinase activity as described above. 3. Results Fig.1 illustrates the effect of fructose 2,6-bisphos- phate upon phosphofructokinase activity in islet homogenate. In the presence of 0.25 mM fructose 6-phosphate, the reaction velocity was increased in a dose-related fashion by fructose 2,6-bisphosphate (fig. 1, left). The increment in velocity attributable to fructose 2,6-bisphosphate reached 50% of its maximal value at -0.2 FM activator. The capacity of fructose 2,6-bisphosphate to increase the reaction velocity was much more marked at low than at high concentration of fructose 6-phosphate (fig.1, right). In the absence of fructose 2,6-bisphosphate, the curve relating the 217