Submitted on August 17, 2007
Accepted on January 16, 2008
Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of nuclear and cytoplasmic glucokinase receptors
Mohammed H Mukhtar1, Victoria A Payne1, Catherine Arden2, Andrew Harbottle3, Salmaan Khan4, Alex J. Lange5, and Loranne Agius6*
1 Institute of Cellular Medicine, Newcastle University, United Kingdom
2 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United States
3 Insitute of Cellular Medicine, Newcastle University, United Kingdom
4 Biochemistry, Molecular Biology & Biophysics, University of Minnesota, United States
5 Dept. of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota Medical School, Minneapolis,, Minnesota, United States
6 Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom
* To whom correspondence should be addressed. E-mail: loranne.agius{at}ncl.ac.uk.
The rate of glucose phosphorylation in hepatocytes is determined by the sub-cellular location of glucokinase and by its association with its regulatory protein (GKRP) in the nucleus. Elevated glucose concentrations and precursors of fructose 1-phosphate (e.g. sorbitol) cause dissociation of glucokinase from GKRP and translocation to the cytoplasm. In this study we investigated the counter-regulation of substrate-induced translocation by AICAR (5-aminoimidazole-4-carboxamide-1-
-D-ribofuranoside), which is metabolised by hepatocytes to an AMP analogue, and causes activation of AMP-activated protein kinase (AMPK) and depletion of ATP. During incubation of hepatocytes with 25 mM glucose, AICAR concentrations below 200 µM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 µM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR counteracted translocation induced by a GKA and partially counteracted translocation by sorbitol. However, AICAR did not block reversal of translocation (from cytoplasm to nucleus) after substrate withdrawal. Inhibition of glucose-induced translocation by AICAR was greater than inhibition by glucagon and was associated with phosphorylation of both the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32 and the nuclear glucokinase binding protein, GKRP. Expression of a kinase-active PFK2 variant lacking ser-32 partially reversed the inhibition of translocation by AICAR. Phosphorylation of GKRP by AMPK partially counteracted its inhibitory effect on glucokinase activity suggesting altered interaction of glucokinase and GKRP. In summary: mechanisms downstream of AMPK activation involving phosphorylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and GKRP are involved in the ATP-independent inhibition of glucose-induced glucokinase translocation by AICAR in hepatocytes.
