TY - JOUR
T1 - Characterization of expression of phosphofructokinase isoforms in isolated rat pancreatic islets and purified beta cells and cloning and expression of the rat phosphofructokinase-A isoform
AU - Ma, Zhongmin
AU - Ramanadham, Sasanka
AU - Kempe, Kirsten
AU - Hu, Zhiqing
AU - Ladenson, Jack
AU - Turk, John
N1 - Funding Information:
We gratefully acknowledge the technical assistance of Alan Bohrer, Dr. Mary Mueller, and Bingbing Li. This research was supported by a grant from the National Institutes of Health (R37-DK-34388) and by the Washington University Diabetes Research and Training Center.
PY - 1996/8/14
Y1 - 1996/8/14
N2 - Phosphofructokinase (PFK) plays a key role in regulating glycolytic flux, and the mammalian enzyme is a tetramer. Three monomeric isoforms are encoded by separate genes, are differentially expressed in specific tissues, and are designated by tissues in which they are most abundant (A, muscle; B, liver; and C, brain). Glucose-induced insulin secretion from pancreatic islets requires glucose transport into islet p-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in β-cells is of interest because that isoform is thought to govern, glycolytic oscillations and to interact with a metabolically activated β-cell phospholipase A, enzyme. Using as probe a PCR product generated from rat islet RNA with primers designed from the human PFK-A sequence, we have cloned a full-length PFK-A cDNA from a rat islet cDNA library. The rat PFK-A deduced amino-acid sequence is 96% identical to that of human PFK-A, and all residues thought to participate in substrate or allosteric effector binding are conserved between the two sequences. The rat PFK-A amino-acid sequence is 69% and 68% identical to those for rat PFK-B and rat PFK-C, respectively, and differences in residues involved in binding of allosteric effectors were observed among the three isoforms. Rat PFK-A expressed as a glutathione-S-transferase fusion protein was recognized by antibodies raised against a peptide in the PFK-A sequence. Expression of PFK isoform mRNA species was examined by RT-PCR in rat islets, in purified populations of β-cells prepared by fluorescence-activated cell sorting (FAGS), and in RIN-m5F insulinoma cells, all of which expressed mRNA species for PFK-A, -B, and -C isoforms. PFK-A mRNA was expressed at much lower levels in an islet α-cell-enriched population. Interleukin-1 impairs islet glucose metabolism and insulin secretion and was found to induce a specific decline in islet expression of PFK-A mRNA. These findings establish the sequence of rat PFK-A, demonstrate that it is expressed in FAGS-purified islet β-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.
AB - Phosphofructokinase (PFK) plays a key role in regulating glycolytic flux, and the mammalian enzyme is a tetramer. Three monomeric isoforms are encoded by separate genes, are differentially expressed in specific tissues, and are designated by tissues in which they are most abundant (A, muscle; B, liver; and C, brain). Glucose-induced insulin secretion from pancreatic islets requires glucose transport into islet p-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in β-cells is of interest because that isoform is thought to govern, glycolytic oscillations and to interact with a metabolically activated β-cell phospholipase A, enzyme. Using as probe a PCR product generated from rat islet RNA with primers designed from the human PFK-A sequence, we have cloned a full-length PFK-A cDNA from a rat islet cDNA library. The rat PFK-A deduced amino-acid sequence is 96% identical to that of human PFK-A, and all residues thought to participate in substrate or allosteric effector binding are conserved between the two sequences. The rat PFK-A amino-acid sequence is 69% and 68% identical to those for rat PFK-B and rat PFK-C, respectively, and differences in residues involved in binding of allosteric effectors were observed among the three isoforms. Rat PFK-A expressed as a glutathione-S-transferase fusion protein was recognized by antibodies raised against a peptide in the PFK-A sequence. Expression of PFK isoform mRNA species was examined by RT-PCR in rat islets, in purified populations of β-cells prepared by fluorescence-activated cell sorting (FAGS), and in RIN-m5F insulinoma cells, all of which expressed mRNA species for PFK-A, -B, and -C isoforms. PFK-A mRNA was expressed at much lower levels in an islet α-cell-enriched population. Interleukin-1 impairs islet glucose metabolism and insulin secretion and was found to induce a specific decline in islet expression of PFK-A mRNA. These findings establish the sequence of rat PFK-A, demonstrate that it is expressed in FAGS-purified islet β-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.
KW - Beta cell
KW - Characterization
KW - Expression
KW - Isoform
KW - Phosphofructokinase
KW - Rat pancreas
UR - http://www.scopus.com/inward/record.url?scp=0030583177&partnerID=8YFLogxK
U2 - 10.1016/0167-4781(96)00088-7
DO - 10.1016/0167-4781(96)00088-7
M3 - Article
C2 - 8764833
AN - SCOPUS:0030583177
SN - 0167-4781
VL - 1308
SP - 151
EP - 163
JO - Biochimica et Biophysica Acta - Gene Structure and Expression
JF - Biochimica et Biophysica Acta - Gene Structure and Expression
IS - 2
ER -