The objective of this research is to investigate the effect of endotoxin administration on carbohydrate flux through two regulatory loci, phosphofructokinase and pyruvate kinase, in intact liver parenchymal cells and to elucidate the biochemical mechanism(s) by which endotoxin regulates carbohydrate flux at those sites. The first specific aim is to identify the biochemical mechanisms responsible for accumulation of hepatic Fru 2,6-P2 in response to endotoxin-administration in vivo and addition of endotoxin to perfused livers. The endotoxin-induced increase in Fru 2,6-P2 level, an important intracellular activator of phosphofructokinase and regulator of carbohydrate flux through the pathways of glycolysis and gluconeogenesis, have led to the focus on the hepatic """"""""bifunctional enzyme"""""""". Fru 2,6-P2 is synthesized and degraded by the unique """"""""bifunctional enzyme"""""""" catalyzing both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities. The ratio of these two activities is altered by changing the phosphorylation state of the enzyme by cAMP-dependent protein kinase and protein phosphatase activities. To determine the effect of endotoxin-treatment on mechanisms regulating the hepatic Fru 2,6-P2 level, liver homogenates will be used to measure the Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities, the extent of phosphorylation of the bifunctional enzyme protein and if there is an altered extent of phosphorylation the specific intracellular mechanisms involved would be identified. Since endotoxin rapidly affects expression of liver proteins, increasing some and decreasing others, the effect of endotoxin on expression of bifunctional enzyme protein by Western blot analysis and accumulation of mRNA by Northern blot analysis will be determined. The second specific aim is to measure the carbohydrate flux through pyruvate kinase using 13C NMR analysis in intact perfused livers or liver parenchymal cells as a function of time following endotoxin administration. The relative flux through pyruvate kinase will be determined from analysis of high resolution 13C NMR spectra and combined with independent enzymatic assays of specific metabolites for calculation of the absolute rates. Comparison of changes in the pyruvate kinase flux with altered concentrations of key liver metabolites as a function of time after endotoxin will be used to evaluate primary events and focus our studies on identification of intracellular mechanisms responsible. The results of these studies will provide insight into the biochemical mechanisms fundamental to the development of severe endotoxin-induced hypoglycemia and may aid development of therapeutic approaches in future investigations.
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