HEXOKINASE SHIFT TO MITOCHONDRIA IS ASSOCIATED WITH AN INCREASED SENSITIVITY TO GLUCOSE IN RAT PANCREATIC-ISLETS

When rat pancreatic islets are incubated in 5.5 or 16.7 mmol/l glucose for 3 h, an increased sensitivity is observed in islets pre-exposed t o high glucose, as indicated by a shift to the left of the glucose dos e-response curve (EC50 7.1 +/- 0.9 and 11.5 +/- 1.2 in high- and low-g lucose-exposed islets, respectively; n = 5, P < 0.05). To investigate the mechanism(s) responsible for this effect, we measured hexokinase a nd glucokinase activity both in the cytosolic fraction and in a mitoch ondrion-enriched fraction, since binding to the outer mitochondrial me mbrane has been reported to result in an increased enzyme activity. In islets cultured at 16.7 mmol/l glucose, the cytosolic hexokinase acti vity was similar to control islets, but mitochondrial enzyme activity was significantly increased (124 +/- 7 vs. 51 +/- 9 nmol.mu g(-1).90 m in(-1), P < 0.01). As a consequence, the cytosolic:mitochondrial fract ion ratio was altered in comparison with control islets. In contrast, glucokinase activity in the two groups of islets was similar in the cy tosolic fraction and undetectable in the mitochodrial fraction. Hexoki nase I quantitation by Western blot confirmed the enzyme translocation from the free cytosolic to the mitochondria-bound form in islets cult ured at 16.7 mmol/l glucose. Glucose-induced alterations were reversib le after Ih exposure to 5.5 mmol/l glucose. Moreover, in islets expose d to 16.7 mmol/l glucose, inhibition of hexokinase binding to mitochon dria by the addition of 20 nmol/l dicyclohexylcarbodiimide resulted in no increase of glucose sensitivity (EC50 10.9 +/- 0.4, n = 3, similar to that of control islets). These data indicate that after chronic ex posure to high glucose, the P-cen becomes more sensitive to glucose be fore eventually getting desensitized. This increased sensitivity is as sociated with (and may be due to) an increased hexokinase activity sec ondary to a subcellular shift of the enzyme from the free cytosolic to the mitochondria-bound, more active form.