Endogenous activation of glucokinase by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is glucose dependent.
Glucokinase (GK) plays a crucial role as glucose sensor in glucose-induced insulin secretion in pancreatic β-cells. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) acts as an endogenous GK activator. Therefore, the goal of this study was the analysis of GK-PFK-2/FBPase-2 complex formation and its effect on metabolic stimulus-secretion coupling in β-cells in dependence upon glucose. The interaction between GK and PFK-2/FBPase-2 was analyzed in insulin-secreting MIN6 cells with a new fluorescence-based mammalian two-hybrid system. In contrast to the commonly used mammalian two-hybrid systems that require sampling before detection, the system used allows monitoring of the effects of environmental changes on protein-protein interactions on the single-cell level. Increasing the glucose concentration in the cell culture medium from 3 to 10 and 25 mmol/liter amplified the interaction between the enzymes stepwise. Importantly, in line with these results, overexpression of PFK-2/FBPase-2 in MIN6 cells evoked only at 10 and 25 mmol/liter, an increase in insulin secretion. Furthermore, a PFK-2/FBPase-2 mutant with an abolished GK-binding motif neither showed a glucose-dependent GK binding nor was able to increase insulin secretion. The results obtained with the mammalian two-hybrid system could be confirmed by fluorescence resonance energy transfer experiments in COS cells. Furthermore, the established interaction between GK and the liver GRP served in all experiments as a control. Thus, this study clearly showed that binding and activation of GK by PFK-2/FBPase-2 in β-cells is promoted by glucose, resulting in an enhancement of insulin secretion at stimulatory glucose concentrations, without affecting basal insulin secretion.
Langer
Sara
S
Institute of Clinical Biochemistry, Hannover Medical School, Hannover, Germany.
Kaminski
Martin T
MT
Lenzen
Sigurd
S
Baltrusch
Simone
S
D000818:Animals; D019556:COS Cells; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D005941:Glucokinase / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism; D016296:Mutagenesis; D025481:Phosphofructokinase-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D011993:Recombinant Fusion Proteins; D025481:Phosphofructokinase-2; D005941:Glucokinase; D005947:Glucose