Novel insights into the regulation of the bound and diffusible glucokinase in MIN6 beta-cells.
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.
Baltrusch
Simone
S
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany. baltrusch.simone@mh-hannover.de
Lenzen
Sigurd
S
D000818:Animals; D018929:Cell Culture Techniques; D004058:Diffusion; D017930:Genes, Reporter; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics; D007328:Insulin / Q000502:physiology; D050417:Insulin-Secreting Cells / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D014404:Tubulin / Q000502:physiology
C485182:Cyan Fluorescent Protein; D007328:Insulin; D011993:Recombinant Fusion Proteins; D014404:Tubulin; D049452:Green Fluorescent Proteins; D005941:Glucokinase