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Diabetes Publish Ahead of Print published online ahead of print February 7, 2007
DOI: 10.2337/db06-0894
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Original Research

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

Simone Baltrusch1, and Sigurd Lenzen1

1Institute of Clinical Biochemistry, Hannover Medical School, 30623 Hannover, Germany

Correspondence: baltrusch.simone{at}mh-hannover.de

A stable MIN6 beta cell clone overexpressing glucokinase as an 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-GK into the functional pool of glucokinase protein in MIN6-ECFP-GK cells. Fluorescence recovery after photobleaching experiments of MIN6-ECFP-GK cells and photoactivation of a transiently transfected PS-CFP-GK 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-GK to insulin secretory granules. Using fluorescence lifetime imaging, we obtained evidence for an association between glucokinase and alpha-tubulin in MIN6-ECFP-GK cells. Furthermore, immunohistochemistry and fluoresecence resonance energy transfer analysis by acceptor photobleaching showed distinct association between endogenous glucokinase and alpha-tubulin as well as beta-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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Copyright © 2007 by the American Diabetes Association.