A role for PFK-2/FBPase-2, as distinct from fructose 2,6-bisphosphate, in regulation of insulin secretion in pancreatic beta-cells.
PFK-2/FBPase-2 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase) catalyses the formation and degradation of fructose 2,6-P(2) (fructose 2,6-bisphosphate) and is also a glucokinase-binding protein. The role of fructose 2,6-P(2) in regulating glucose metabolism and insulin secretion in pancreatic beta-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-P(2), in regulating beta-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-P(2) 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-P(2) content, glucokinase activity and glucoseinduced insulin secretion. Selective down-regulation of glucose-induced fructose 2,6-P(2) 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-P(2), in potentiating glucose-induced insulin secretion. Whereas down-regulation 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-P(2) is the over-riding determinant of glucose-induced insulin secretion through regulation of glucokinase activity or subcellular targeting.
Arden
Catherine
C
Institute of Cellular Medicine, The Medical School, Leech Bld Level 4, Newcastle University, Newcastle upon Tyne NE2 4HH, UK.
Hampson
Laura J
LJ
Huang
Guo C
GC
Shaw
James A M
JA
Aldibbiat
Ali
A
Holliman
Graham
G
Manas
Derek
D
Khan
Salmaan
S
Lange
Alex J
AJ
Agius
Loranne
L
D000818:Animals; D015536:Down-Regulation; D005635:Fructosediphosphates; D005941:Glucokinase / Q000378:metabolism*; D006019:Glycolysis; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D007515:Islets of Langerhans / Q000166:cytology; D007527:Isoenzymes; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D025481:Phosphofructokinase-2 / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar
D005635:Fructosediphosphates; D007328:Insulin; D007527:Isoenzymes; D034741:RNA, Small Interfering; C027652:fructose 2,6-diphosphate; D025481:Phosphofructokinase-2; D005941:Glucokinase