Quantitative image analysis reveals that phosphorylation of liver-type isozyme of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase does not affect nuclear translocation of glucokinase in rat primary hepatocytes.
We have developed a new quantification method to measure translocation of glucokinase between nucleus and cytoplasm in primary hepatocytes. The method is robust, reliable and sensitive with the use of a high content fluorescence microscope, which can analyse more than 20,000 hepatocytes under each experimental condition. Frequency distributions of the nuclear and cytoplasmic contents of glucokinase did not exhibit a Gaussian distribution. Moreover, the distributions have large standard deviation values compared with their average values. These results indicate that a large number of cells must be analysed for the accurate quantification. Glucose and sorbitol promoted the translocation of glucokinase from nucleus to cytoplasm. These results show good agreement with previous reports. However, glucagon did not affect the localization of glucokinase. Under the same conditions, liver-type isozyme of fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase (F6P2K), whose dephosphorylated form has been proposed as a cytoplasmic binding protein with glucokinase, was completely phosphorylated. These results indicate that the phosphorylation and dephosphorylation of F6P2K does not have any appreciable effect on the intracellular localization of glucokinase.
Watanabe
Fusao
F
Department of Chemistry, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka, Japan. watanabe@art.osaka-med.ac.jp
Furuya
Eisuke
E
D000818:Animals; D018929:Cell Culture Techniques; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005941:Glucokinase / Q000378:metabolism; D022781:Hepatocytes / Q000378:metabolism* / Q000648:ultrastructure; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D009687:Nuclear Proteins / Q000378:metabolism*; D025481:Phosphofructokinase-2 / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats
D007004:Hypoglycemic Agents; D009687:Nuclear Proteins; D020033:Protein Isoforms; D025481:Phosphofructokinase-2; D005941:Glucokinase