Nitric Oxide Activates β-Cell Glucokinase by Promoting Formation of the “Glucose-Activated” State

Abstract

The release of insulin from the pancreas is tightly controlled by glucokinase (GCK) activity that couples β-cell metabolism to changes in blood sugar. Despite having only a single glucose-binding site, GCK displays positive glucose cooperativity. Ex vivo structural studies have identified several potential protein conformations with varying levels of enzymatic activity, yet it is unclear how living cells regulate GCK cooperativity. To better understand the cellular regulation of GCK activation, we developed a homotransfer Förster resonance energy transfer (FRET) GCK biosensor and used polarization microscopy to eliminate fluorescence crosstalk from FRET quantification and improve the signal-to-noise ratio. This approach enhanced sensor contrast compared to that seen with the heterotransfer FRET GCK reporter and allowed observation of individual GCK states using an automated method to analyze FRET data at the pixel level. Mutations known to activate and inhibit GCK activity produced distinct anisotropy distributions, suggesting that at least two conformational states exist in living cells. A high glucose level activated the biosensor in a manner consistent with GCK's enzymology. Interestingly, glucose-free conditions did not affect GCK biosensor FRET, indicating that there is a single low-activity state, which is counter to proposed structural models of GCK cooperativity. Under low-glucose conditions, application of chemical NO donors efficiently shifted GCK to the more active conformation. Notably, GCK activation by mutation, a high glucose level, a pharmacological GCK activator, or S-nitrosylation all shared the same FRET distribution. These data suggest a simplified model for GCK activation in living cells, where post-translational modification of GCK by S-nitrosylation facilitates a single conformational transition that enhances GCK enzymatic activity.

Molecular and cellular regulation of human glucokinase

“…[133] As GLP-1 is known to potentiate GSIS at physiologically intermediate (8-11 mM) glucose concentrations [134], it seems likely that this insulinotropic effect is achieved, in part, by promoting S-nitrosylation of GCK. [133] A recent report also demonstrated that chemically-mediated S-nitrosylation activates GCK by promoting the highly active closed GCK conformation [135], much like α-type activating mutations [40], small molecule GKAs [15,16,117], and high glucose concentrations. Despite these results, the direct influence that granular association and S-nitrosylation have on GCK activity remain incompletely understood.…”

Insulin secretion: The nitric oxide controversy

“…Nitric oxide through S -nitrosylation of glucokinase (at cysteine-371) and syntaxin 4 (at cysteine-141) facilitates GSIS (Rizzo and Piston, 2003[ 105 ]; Wiseman et al, 2011[ 134 ]; Kruszelnicka, 2014[ 65 ]; Seckinger et al, 2018[ 113 ]). Using quantitative imaging of multicolor fluorescent proteins fused to glucokinase, it has been demonstrated that the dynamic association of glucokinase with secretory granules is modulated by NO (Rizzo and Piston, 2003[ 105 ]).…”

“…Indeed, insulin is found to stimulate NO production leading to S -nitrosylation of glucokinase in cultured β-cells (βTC3 cells); moreover, inhibition of NOS disrupts glucokinase association with secretory granules and glucokinase conformation (Rizzo and Piston, 2003[ 105 ]). It has been demonstrated that elevated glucose and S -nitrosylation, both induce the same high-activity glucokinase conformational state I in βTC3 cells (Seckinger et al, 2018[ 113 ]). Ultimately, attachment of a nuclear localization signal sequence to NOS in βTC3 cells drives glucokinase to the nucleus in addition to its normal cytoplasmic and granule targeting (Rizzo and Piston, 2003[ 105 ]).…”

Homotransfer FRET Reporters for Live Cell Imaging

“…Classic “molecular switch” type sensors, such as the Protein Kinase A activity reporters (AKARs) that contain both external sensing domains and internal effector-binding domains, have been converted to homotransfer reporters in four different colors [34]. Sensors that report changes in a full sequence protein, like myosin light chain kinase (MLCK) [34] or glucokinase [56], have also been successfully converted to FLAREs.…”