HORMAL CONTROL OF HEPATIC GLUCONEOGENESIS

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The proposed research will concentrate on the mechanism of action of hormones on hepatic gluconeogenesis and glycolysis. The hypothesis which will be tested is as follows: Hormonal control of hepatic gluconeogenesis and glycolysis is mediated by cAMP-dependent and/or independent phosphorylation of several cytoplasmic enzymes involved in F6P/F1,6-P2 and PEP/Pyruvate substrate cycles and of the enzyme(s) involved in the synthesis and degradation of the recently discovered sugar diphosphate, fructose 2, 6-bisphosphate. Research will continue to focus on L-type pyruvate kinase, fructose 1, 6-bisphosphatase, 6-phosphofructo 1-kinase, and two recently discovered enzymes 6-phosphofructo 2-kinase and fructose 2,6-bisphosphatase. 6-Phosphofructo 2-kinase/fructose 2,6-bisphosphatase will be purified to homogeneity and their physical and kinetic properties studied. In vitro phosphorylation of these enzymes catalyzed by the cAMP-dependent protein kinase and perhaps by other kinases will be characterized. The effect of glucagon, insulin, catecholamines and various substrates on 32P incorporation into 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase in isolated hepatocytes will be investigated using immunological methods to isolate the enzymes. The phosphorylation state of the enzymes in intact cells will be correlated with enzyme activity measured in hepatocyte extracts and with cAMP levels. The regulation of activity of these enzymes will be studied in various dietary and hormonal states both in vivo and in isolated liver systems. We will also investigate the modulation of fructose 2, 6-bisphosphate levels in vivo and in isolated hepatocytes and assess the importance of this regulation vis a vis hepatic glycolysis and gluconeogenesis. In order to achieve this latter goal we will employ a conscious, intact dog model where endocrine pancreatic function can be controlled by virtue of a "pancreatic clamp" technique and substrate levels can be controlled by supplementation techniques. The interaction of fructose 2, 6-bisphosphate and other allosteric effectors with 6-phosphofructo 1-kinase, fructose 1,6-bisphosphatase, pyruvate kinase, and 6-phosphofructo 2-kinase will also be investigated. Finally, the effect of phosphorylation on the kinetic and physical properties of these enzymes will be assessed by a number of techniques.