Endogenous activation of glucokinase by 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase is glucose dependent

Mol Endocrinol. 2010 Oct;24(10):1988-97. doi: 10.1210/me.2010-0115. Epub 2010 Aug 11.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • COS Cells
  • Cells, Cultured
  • Chlorocebus aethiops
  • Enzyme Activation
  • Glucokinase / genetics
  • Glucokinase / metabolism*
  • Glucose / metabolism*
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / metabolism
  • Mutagenesis
  • Phosphofructokinase-2 / genetics
  • Phosphofructokinase-2 / metabolism*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Two-Hybrid System Techniques

Substances

  • Recombinant Fusion Proteins
  • Phosphofructokinase-2
  • Glucokinase
  • Glucose