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 language | English (US) |
|---|---|
| Pages (from-to) | 41-51 |
| Number of pages | 11 |
| Journal | Biochemical Journal |
| Volume | 411 |
| Issue number | 1 |
| DOIs | |
| State | Published - Apr 1 2008 |
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Keywords
- 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2)
- Fructose 2,6-bisphosphate
- Glucokinase
- Insulin
- Islets
- β-cell
Cite this
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 journal › Article
}
TY - JOUR
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
PY - 2008/4/1
Y1 - 2008/4/1
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
UR - http://www.scopus.com/inward/record.url?scp=41649106843&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=41649106843&partnerID=8YFLogxK
U2 - 10.1042/BJ20070962
DO - 10.1042/BJ20070962
M3 - Article
C2 - 18039179
AN - SCOPUS:41649106843
VL - 411
SP - 41
EP - 51
JO - Biochemical Journal
JF - Biochemical Journal
SN - 0264-6021
IS - 1
ER -