Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
The rate of glucose phosphorylation in hepatocytes is determined by the subcellular 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-beta-D-ribofuranoside), which is metabolized by hepatocytes to an AMP analog, 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 microM activated AMPK without depleting ATP and inhibited glucose phosphorylation and glucokinase translocation with half-maximal effect at 100-140 microM. Glucose phosphorylation and glucokinase translocation correlated inversely with AMPK activity. AICAR also counteracted translocation induced by a glucokinase activator and partially counteracted translocation by sorbitol. However, AICAR did not block the 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 GKRP and the cytoplasmic glucokinase binding protein, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK2) on ser-32. 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.
Mukhtar
Mohammed H
MH
Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
Payne
Victoria A
VA
Arden
Catherine
C
Harbottle
Andrew
A
Khan
Salmaan
S
Lange
Alex J
AJ
Agius
Loranne
L
D055372:AMP-Activated Protein Kinases; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000378:metabolism / Q000502:physiology*; D002478:Cells, Cultured; D004232:Diuretics / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D005941:Glucokinase / Q000235:genetics* / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D022781:Hepatocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007150:Immunohistochemistry; D008297:Male; D008687:Metformin / Q000494:pharmacology; D009097:Multienzyme Complexes / Q000502:physiology*; D025481:Phosphofructokinase-2 / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D021381:Protein Transport; D051381:Rats; D017208:Rats, Wistar; D012265:Ribonucleotides / Q000494:pharmacology; D013012:Sorbitol / Q000494:pharmacology
D002352:Carrier Proteins; D004232:Diuretics; D007004:Hypoglycemic Agents; D009097:Multienzyme Complexes; D012265:Ribonucleotides; C080906:glucokinase regulatory protein; D000620:Aminoimidazole Carboxamide; D013012:Sorbitol; D008687:Metformin; D025481:Phosphofructokinase-2; D005941:Glucokinase; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D005947:Glucose