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.
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 beta-cells and glycolytic metabolism. Little is known about islet PFK isozymes, but the possibility that PFK-A is expressed in beta-cells is of interest because that isoform is thought to govern glycolytic oscillations and to interact with a metabolically activated beta-cell phospholipase A2 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 beta-cells prepared by fluorescence-activated cell sorting (FACS), 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 alpha-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 FACS-purified islet beta-cells, and suggest that its expression is regulated by a cytokine which influences insulin secretion.
Ma
Z
Z
Division of Endocrinology, Diabetes and Metabolism, Washington University School of Medicine, St. Louis, MO 63110, USA.
Ramanadham
S
S
Kempe
K
K
Hu
Z
Z
Ladenson
J
J
Turk
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002469:Cell Separation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D015723:Gene Library; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000494:pharmacology; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics*; D008297:Male; D008969:Molecular Sequence Data; D010732:Phosphofructokinase-1 / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution
D018076:DNA, Complementary; D007375:Interleukin-1; D007527:Isoenzymes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D010732:Phosphofructokinase-1