Coordinated Regulation of Hepatic Glucose Disposal

Abstract

The objective of these studies was to elucidate the molecular mechanisms that underlie the acute response of the fasting liver to refeeding. Our focus was on two major regulators of liver metabolism, glucokinase (GK) and 6-phosphofructo 2-kinase/Fructose 2,6-bisphosphatase (PFK-2/FBP-2). We detailed the binding of these enzymes with pull-down assays, fluorescence spectroscopy, and steady state kinetics. The results have confirmed that the enzymes bind. The formation of this protein:protein complex activates GK and produces a PFK-2/FBP-2 that has a higher kinase-to-bisphosphatase activity ratio (K/B) which will act to increase the level of fructose-2,6-bisphosphate (F-2,6-P2). Therefore, we conclude that the binding of GK and PFK-2/FBP-2 provides for a coordinated up-regulation of hepatic glucose disposal because it activates both the phosphorylation of glucose (GK) and the activation of the committing step to glycolysis (6-phosphofructo 1-kinase) via the biofactor F-2,6-P2. Our results have begun to reveal the molecular mechanisms by which the enzyme activities are modulated. Intrinsic protein fluorescence spectroscopy showed that binding of GK to PFK-2/FBP-2, or the FBP-2 domain, promotes the binding of glucose to GK, thus activating it. The mechanism for the increased K/B of PFK-2/FBP-2 is less clear, yet it appears to involve at least one other liver protein. On this basis we have developed a comprehensive model of how these enzymes respond to the extra-hepatic signals generated by meal consumption that is completely consistent with previously published reports concerning GK and PFK-2/FBP-2. Funding: VA Merit Award to DAO.