Regulation of Glucokinase by Intracellular Calcium Levels in Pancreatic β Cells

Abstract

Glucokinase (GCK) controls the rate of glucose metabolism in pancreatic β cells, and its activity is rate-limiting for insulin secretion. Posttranslational GCK activation can be stimulated through either G protein-coupled receptors or receptor tyrosine kinase signaling pathways, suggesting a common mechanism. Here we show that inhibiting Ca(2+) release from the endoplasmic reticulum (ER) decouples GCK activation from receptor stimulation. Furthermore, pharmacological release of ER Ca(2+) stimulates activation of a GCK optical biosensor and potentiates glucose metabolism, implicating rises in cytoplasmic Ca(2+) as a critical regulatory mechanism. To explore the potential for glucose-stimulated GCK activation, the GCK biosensor was optimized using circularly permuted mCerulean3 proteins. This new sensor sensitively reports activation in response to insulin, glucagon-like peptide 1, and agents that raise cAMP levels. Transient, glucose-stimulated GCK activation was observed in βTC3 and MIN6 cells. An ER-localized channelrhodopsin was used to manipulate the cytoplasmic Ca(2+) concentration in cells expressing the optimized FRET-GCK sensor. This permitted quantification of the relationship between cytoplasmic Ca(2+) concentrations and GCK activation. Half-maximal activation of the FRET-GCK sensor was estimated to occur at ∼400 nm Ca(2+). When expressed in islets, fluctuations in GCK activation were observed in response to glucose, and we estimated that posttranslational activation of GCK enhances glucose metabolism by ∼35%. These results suggest a mechanism for integrative control over GCK activation and, therefore, glucose metabolism and insulin secretion through regulation of cytoplasmic Ca(2+) levels.