Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.
We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.
Stubbs
M
M
Department of Diabetes and Metabolism, The Medical School, University of Newcastle, Newcastle upon Tyne, UK.
Aiston
S
S
Agius
L
L
D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D004072:Digitonin / Q000494:pharmacology; D005941:Glucokinase / Q000302:isolation & purification / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D006593:Hexokinase / Q000378:metabolism; D007158:Immunologic Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D009994:Osmolar Concentration; D010766:Phosphorylation; D013194:Staining and Labeling; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism*; D014018:Tissue Distribution
D007328:Insulin; D006593:Hexokinase; D005941:Glucokinase; D005947:Glucose; D004072:Digitonin