title: 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.
authors.affiliation: From the University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Michele L
authors.initials: ML
authors.lastname: Markwardt
authors.affiliation: From the University of Maryland School of Medicine, Baltimore, Maryland 21201.
authors.forename: Kendra M
authors.initials: KM
authors.lastname: Seckinger
authors.affiliation: From the University of Maryland School of Medicine, Baltimore, Maryland 21201 mrizzo@som.umaryland.edu.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Rizzo
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D000242:Cyclic AMP / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000235:genetics / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000201:enzymology*; D008297:Male; D051379:Mice
chemical_list: D000242:Cyclic AMP; D005941:Glucokinase; D005947:Glucose; D002118:Calcium
keywords: calcium imaging; calcium intracellular release; glucokinase; nitric oxide synthase; optogenetics