Glucokinase Is an Integral Component of the Insulin Granules in Glucose-Responsive Insulin Secretory Cells and Does Not Translocate During Glucose Stimulation

  1. Catherine Arden1,
  2. Andrew Harbottle1,
  3. Simone Baltrusch2,
  4. Markus Tiedge2 and
  5. Loranne Agius1
  1. 1School of Clinical Medical Sciences-Diabetes, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K
  2. 2Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany
  1. Address correspondence and reprint requests to Loranne Agius, School of Clinical Medical Sciences-Diabetes, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K. E-mail: loranne.agius{at}ncl.ac.uk

Abstract

The association of glucokinase with insulin secretory granules has been shown by cell microscopy techniques. We used MIN6 insulin-secretory cells and organelle fractionation to determine the effects of glucose on the subcellular distribution of glucokinase. After permeabilization with digitonin, 50% of total glucokinase remained bound intracellularly, while 30% was associated with the 13,000g particulate fraction. After density gradient fractionation of the organelles, immunoreactive glucokinase was distributed approximately equally between dense insulin granules and low-density organelles that cofractionate with mitochondria. Although MIN6 cells show glucose-responsive insulin secretion, glucokinase association with the granules and low-density organelles was not affected by glucose. Subfractionation of the insulin granule components by hypotonic lysis followed by sucrose gradient centrifugation showed that glucokinase colocalized with the granule membrane marker phogrin and not with insulin. PFK2 (6-phosphofructo-2-kinase-2/fructose-2,6-bisphosphatase)/FDPase-2, a glucokinase-binding protein, and glyceraldehyde phosphate dehydrogenase, which has been implicated in granule fusion, also colocalized with glucokinase after hypotonic lysis or detergent extaction of the granules. The results suggest that glucokinase is an integral component of the granule and does not translocate during glucose stimulation.

Footnotes

    • Accepted May 10, 2004.
    • Received January 8, 2004.
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