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Novel Insights Into the Regulation of the Bound and Diffusible Glucokinase in MIN6 ß-Cells

From the Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany

Address correspondence and reprint requests to Dr. Simone Baltrusch, Institute of Clinical Biochemistry, Hannover Medical School, D-30623 Hannover, Germany. E-mail: baltrusch.simone{at}mh-hannover.de

Abbreviations: DMEM, Dulbecco's modified Eagle's medium; ECFP, enhanced cyan fluorescent protein; EYFP, enhanced yellow fluorescent protein; FRET, fluorescence resonance energy transfer; PS-CFP, photoswitchable cyan fluorescent protein

A stable MIN6 ß-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 ECFP-glucokinase 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 -tubulin as well as ß-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.

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