A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

Catherine Arden, Laura J. Hampson, Guo C. Huang, James A.M. Shaw, Ali Aldibbiat, Graham Holliman, Derek Manas, Salmaan Khan, Alex J. Lange, Loranne Agius

Research output: Contribution to journalArticle

28 Citations (Scopus)

Abstract

PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bis-phosphatase) catalyses the formation and degradation of fructose 2,6-P2 (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P2 in regulating glucose metabolism and insulin secretion in pancreatic β-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P2, in regulating β-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P2 were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P2 content, glucokinase activity and glucose-induced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P2 in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P2, in potentiating glucose-induced insulin secretion. Whereas downregulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P2 is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

Original languageEnglish (US)
Pages (from-to)41-51
Number of pages11
JournalBiochemical Journal
Volume411
Issue number1
DOIs
StatePublished - Apr 1 2008

Fingerprint

Fructose
Glucokinase
Insulin
Glucose
Protein Isoforms
Down-Regulation
Proteins
Gene encoding
Phosphotransferases
Small Interfering RNA
Phosphofructokinase-2
fructose 2,6-diphosphate
Phosphorylation
AMP-Activated Protein Kinases
Glycolysis
Transcription
Phosphoric Monoester Hydrolases
Metabolism
Reverse Transcription
Rats

Keywords

  • 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2)
  • Fructose 2,6-bisphosphate
  • Glucokinase
  • Insulin
  • Islets
  • β-cell

Cite this

Arden, C., Hampson, L. J., Huang, G. C., Shaw, J. A. M., Aldibbiat, A., Holliman, G., ... Agius, L. (2008). A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells. Biochemical Journal, 411(1), 41-51. https://doi.org/10.1042/BJ20070962

A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells. / Arden, Catherine; Hampson, Laura J.; Huang, Guo C.; Shaw, James A.M.; Aldibbiat, Ali; Holliman, Graham; Manas, Derek; Khan, Salmaan; Lange, Alex J.; Agius, Loranne.

In: Biochemical Journal, Vol. 411, No. 1, 01.04.2008, p. 41-51.

Research output: Contribution to journalArticle

Arden, C, Hampson, LJ, Huang, GC, Shaw, JAM, Aldibbiat, A, Holliman, G, Manas, D, Khan, S, Lange, AJ & Agius, L 2008, 'A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells', Biochemical Journal, vol. 411, no. 1, pp. 41-51. https://doi.org/10.1042/BJ20070962
Arden, Catherine ; Hampson, Laura J. ; Huang, Guo C. ; Shaw, James A.M. ; Aldibbiat, Ali ; Holliman, Graham ; Manas, Derek ; Khan, Salmaan ; Lange, Alex J. ; Agius, Loranne. / A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells. In: Biochemical Journal. 2008 ; Vol. 411, No. 1. pp. 41-51.
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T1 - A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic β-cells

AU - Arden, Catherine

AU - Hampson, Laura J.

AU - Huang, Guo C.

AU - Shaw, James A.M.

AU - Aldibbiat, Ali

AU - Holliman, Graham

AU - Manas, Derek

AU - Khan, Salmaan

AU - Lange, Alex J.

AU - Agius, Loranne

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N2 - PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bis-phosphatase) catalyses the formation and degradation of fructose 2,6-P2 (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P2 in regulating glucose metabolism and insulin secretion in pancreatic β-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P2, in regulating β-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P2 were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P2 content, glucokinase activity and glucose-induced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P2 in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P2, in potentiating glucose-induced insulin secretion. Whereas downregulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P2 is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

AB - PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bis-phosphatase) catalyses the formation and degradation of fructose 2,6-P2 (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P2 in regulating glucose metabolism and insulin secretion in pancreatic β-cells is unresolved. We down-regulated the endogenous isoforms of PFK-2/FBPase-2 with siRNA (small interfering RNA) and expressed KA (kinase active) and KD (kinase deficient) variants to distinguish between the role of PFK-2/FBPase-2 protein and the role of its product, fructose 2,6-P2, in regulating β-cell function. Human islets expressed the PFKFB2 (the gene encoding isoform 2 of the PFK2/FBPase2 protein) and PFKFB3 (the gene encoding isoform 3 of the PFK2/FBPase2 protein) isoforms and mouse islets expressed PFKFB2 at the mRNA level [RT-PCR (reverse transcription-PCR)]. Rat islets expressed PFKFB2 lacking the C-terminal phosphorylation sites. The glucose-responsive MIN6 and INS1E cell lines expressed PFKFB2 and PFKFB3. PFK-2 activity and the cell content of fructose 2,6-P2 were increased by elevated glucose concentration and during pharmacological activation of AMPK (AMP-activated protein kinase), which also increased insulin secretion. Partial down-regulation of endogenous PFKFB2 and PFKFB3 in INS1E by siRNA decreased PFK-2/FBPase-2 protein, fructose 2,6-P2 content, glucokinase activity and glucose-induced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P2 in the absence of down-regulation of PFK-2/FBPase-2 protein, using a KD PFK-2/FBPase-2 variant, resulted in sustained glycolysis and elevated glucose-induced insulin secretion, indicating an over-riding role of PFK-2/FBPase-2 protein, as distinct from its product fructose 2,6-P2, in potentiating glucose-induced insulin secretion. Whereas downregulation of PFK-2/FBPase-2 decreased glucokinase activity, overexpression of PFK-2/FBPase-2 only affected glucokinase distribution. It is concluded that PFK-2/FBPase-2 protein rather than its product fructose 2,6-P2 is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.

KW - 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2)

KW - Fructose 2,6-bisphosphate

KW - Glucokinase

KW - Insulin

KW - Islets

KW - β-cell

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