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

Diabetes. 2007 May;56(5):1305-15. doi: 10.2337/db06-0894. Epub 2007 Feb 7.


A stable MIN6 beta-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 alpha-tubulin in MIN6-ECFP-glucokinase cells. Furthermore, immunohistochemistry and fluorescence 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Culture Techniques
  • Diffusion
  • Genes, Reporter
  • Glucokinase / genetics*
  • Glucokinase / metabolism*
  • Green Fluorescent Proteins / genetics
  • Insulin / physiology
  • Insulin-Secreting Cells / metabolism
  • Insulin-Secreting Cells / physiology*
  • Mice
  • Microscopy, Fluorescence
  • Recombinant Fusion Proteins / metabolism
  • Transfection
  • Tubulin / physiology


  • Cyan Fluorescent Protein
  • Insulin
  • Recombinant Fusion Proteins
  • Tubulin
  • Green Fluorescent Proteins
  • Glucokinase