Glucose is the major signal for insulin release from pancreatic islet beta cells, and its metabolism is an essential step in the propagation of that signal. We are testing the hypothesis that fructose 2,6- bisphosphate (Fru 2,6-P2) is involved in the signaling between glucose and the insulin gene, by modulating either the beta cell glycolytic pathway, or a specific insulin promoter binding protein. Intracellular Fru 2,6-P2 is the major determinant of glycolytic rate in most tissues, as the most potent activator of the glycolytic enzyme, 6-phosphofructo- 1-kinase. In cells capable of catalyzing the opposing pathway; gluconeogenesis, Fru 2,6-P2 is a competitive inhibitor of P2 are modulated by reciprocal changes in the two activities of the bifunctional enzyme, 6-phosphofructo-2-kinase-fructose-2,6-biphosphatase (6PF2K/Fru-2,6-P2ase). In liver, the major gluyconeogenic organ, an amino terminal cAMP dependent phosphorylation, inhibits the kinase and activates the bisphosphatase, resulting in Fru 2,6-P2 depletion, and the activation of gluconeogenesis. We have recently identified a novel gene of 6PF2K/Fru-2,6-P2ase, encoding at least 3 isozymes, that is expressed in brain cells and pancreatic beta islet cells, unique in the fact that they contain multiple putative phosphorylation sites at the carboxyl termini. In vitro phosphorylation of one of these sites, catalyzed by cAMP-dependent protein kinase, activates the kinase and inhibits the bisphospatase. Thus, in contrast to hepatic metabolism, where phosphorylation of 6PF2K/Fru-2,6-P2ase attenuates glycolytic rates, glycolysis in these tissues would be expected to be enhanced by phosphorylation of 6PF2K/Fru-2,6-P2ase. This is consistent with the enhanced response to glucose observed after hormonal treatment of islet cells, and may provide a molecular mechanism for the glucose/hormonal induced stimulation of insulin biosynthesis. With this in mind, we have just identified a nuclear Fru 2,6-P2-binding protein that may characterize the role of Fru 2,6-P2 in beta cell metabolism and insulin biosynthesis. Fru 2,6-P2 levels will be manipulated by over expression of mutant forms of 6PF2K/Fru-2,6-P2ase, that have only one of the two activities as well as by antisense methodology, to define its role in modulating glucose utilization/insulin release. Finally, we will elucidate the structure and function of the nuclear Fru-2,6-P2 binding protein and determine its involvement in the expression of islet cell genes.