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

Zhongmin Ma, Sasanka Ramanadham, Kirsten Kempe, Zhiqing Hu, Jack Ladenson, John Turk

Research output: Contribution to journalArticlepeer-review

13 Scopus citations

Abstract

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.

Original languageEnglish
Pages (from-to)151-163
Number of pages13
JournalBiochimica et Biophysica Acta - Gene Structure and Expression
Volume1308
Issue number2
DOIs
StatePublished - Aug 14 1996

Keywords

  • Beta cell
  • Characterization
  • Expression
  • Isoform
  • Phosphofructokinase
  • Rat pancreas

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