Regulation of Glucokinase in Pancreatic Beta Cells

Abstract

Glucokinase (GCK) is the main glucose sensor in pancreatic β cells and controls insulin secretion. Because of this, GCK is an important target for type 2 diabetes mellitus therapies. The mechanism of how GCK, a monomeric enzyme with a single binding site for glucose, exhibits cooperativity is still unknown although there are many proposed models. Our lab has shown that nitric oxide (NO) enhances GCK activity, but the underlying mechanism of activation and the relationship between GCK S-nitrosylation and cooperativity are unclear. To further investigate GCK regulation, we created a novel single-color GCK reporter. This Forster resonance energy transfer (FRET) biosensor allows for more precise and quantitative measurements than traditional two-color FRET reporters. Here we show that our single-color GCK sensor accurately measures changes in GCK conformation in response to increasing concentrations of glucose. 7.5mM glucose is representative of post-prandial blood glucose levels and also corresponds to half-maximal GCK activation. We show that 7.5mM also has the highest degree of variability further highlighting the need to elucidate the mechanism of GCK activation. Introducing known activating and inactivating GCK point mutations into our GCK FRET sensor provide evidence for two conformations of GCK. We measured distinct FRET efficiencies for the both the active and inactive states that converged on precise anisotropy values. Addition of the NO donor, SNAP, stimulates formation of the more active GCK conformation. Together, these findings provide support for a two state GCK model with NO facilitating formation of the more active GCK state. Learning how GCK is regulated is important for understanding the pathology of diabetes.