Abstract
The glucose-phosphorylating enzyme glucokinase likely plays an important role in regulating glucose-stimulated insulin secretion from the islets of Langerhans and has previously been thought to be expressed only in that tissue and in liver. In this study, we demonstrate high levels of glucokinase mRNA in the anterior pituitary cell line AtT20ins, which has been engineered to secrete correctly processed insulin, as well as in primary anterior pituitary tissue. Unlike islet or liver cells, expression of glucokinase mRNA in anterior pituitary cells was not accompanied by expression of the high Km glucose transporter (GLUT-2) mRNA. The glucokinase transcript in anterior pituitary cells was similar in size to islet glucokinase mRNA, which has a unique, elongated 5'-end relative to the liver glucokinase message. Amplification and sequence analysis of the glucokinase mRNA expressed in islets, RIN1046-38 cells, and anterior pituitary cells confirmed that the glucokinase transcripts in these cell types contain the same 5'-sequence. In addition, a novel alternative transcript was identified that contains a 52-nucleotide deletion and that predicts a 58-amino acid peptide as a result of a frame shift. Both the deleted and undeleted transcripts were found in islets, RIN cells, and AtT20ins cells, whereas only the deleted product was identified in primary anterior pituitary tissue. An antibody prepared against a peptide found at the N terminus of the islet isoform of glucokinase easily detected a protein with a size predicted by the undeleted transcript in extracts prepared from islets, RIN1046-38 cells, and AtT20ins cells. Since both the glucokinase protein and mRNA are naturally expressed in AtT20ins and RIN1046-38 cells, we compared the effect of varying concentrations of glucose on insulin secretion from the two lines. Insulin secretion from RIN1046-38 cells was stimulated by glucose in a dose-dependent manner over the range 0-2.5 mM, where it reached a maximum. AtT20ins cells, in contrast, exhibited no response to glucose at any concentration tested, despite the fact that insulin secretion from both cell lines was stimulated by incubation with dibutyryl cAMP. We conclude that glucokinase expression in AtT20ins cells may be necessary, but is not sufficient to confer glucose-stimulated insulin secretion.
Highlights
AtTeOi,. cells, native islet glucokinase,the variant liver glucokinase of Hay- despite a level of expression of glucokinase protein similar to zer and Iynedjian [37],and the variant mRNA of islets and that found in normal islets, fail to respond to glucose as an anterior pituitary described in bacteria
This study provides direct evidence for expression of glucokinase in a tissue other than liver or islets. Both the islet isoform of the glucokinase proteinandits mRNA are expressed in anterior pituitary cells and cell lines
Glucokinase gene expression in the anterior pituitary may be confined to certain cell types, as indicated by the absence of expression in a cell line derived from prolactin-secreting anterior pituitary cells
Summary
Secretion-Untransfected AtT2O cells as well as AtT2O cells stably system in Ref.; provided by Dr Graeme Bell (University of transfected with a chimeric cDNA/genomic copy of the human insulin Chicago)) This is ahuman probe, there is 97% identity gene [15], constitutively driven by the Rous sarcoma virus long between the human and rat GLUT-1sequences [24,25]. Glucose- sequence to facilitate subcloning of amplified fragments This reacstimulated insulin secretion was measured in six independent wells tion mixture was heated to 65 “Cfor 3 min, transferred to 53 ‘C while by incubating the cells at 37 ‘C in HBSS containing 0, 0.05, 0.1, 0.5, adding 1 pl of avian myeloblastosis virus reverse transcriptase
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