Novel insights into the regulation of the bound and diffusible glucokinase in MIN6 β-cells

Abstract

A stable MIN6 β-cell clone overexpressing glucokinase as an enhanced cyan fluorescent protein (ECFP) fusion construct was generated for analysis of glucokinase regulation in these glucose-responsive insulin-secreting cells. A higher glucokinase enzyme activity accompanied by an improved glucose-induced insulin secretion indicated the integration of ECFP-glucokinase into the functional pool of glucokinase protein in MIN6-ECFP-glucokinase cells. Fluorescence recovery after photobleaching experiments of MIN6-ECFP-glucokinase cells and photoactivation of a transiently transfected photoswitchable cyan fluorescent protein (PS-CFP)-glucokinase construct in MIN6 cells indicate a higher motility of the diffusible glucokinase fraction at high glucose concentrations. In agreement with previous studies, we observed significant binding of ECFPglucokinase to insulin secretory granules. Using fluorescence lifetime imaging, we obtained evidence for an association between glucokinase and -tubulin in MIN6-ECFP-glucokinase cells. Furthermore, immunohistochemistry and fluorescence resonance energy transfer analysis by acceptor photobleaching showed distinct association between endogenous glucokinase and α-tubulin as well as β-tubulin in MIN6 cells. Interestingly, glucokinase was also colocalized with kinesin, a motor protein involved in insulin secretory granule movement. Therefore, we suggest a role of a bound glucokinase protein fraction in the regulation of insulin granule movement along tubulin filaments. © 2007 by the American Diabetes Association.