Molecular and functional characterization of insulin receptors present on hamster glucagonoma cells.
Studies using pancreas perfusion techniques point to a physiological inhibition of glucagon release by insulin which should be mediated by A cell-residing insulin receptors. In this study, we have characterized the insulin receptors expressed in a hamster glucagonoma A cell line (INR1G9 cells) which is an accepted tool for A cell studies. In receptor binding assays 125I-insulin was displaced with a Kd of 3 nmol/l. Binding was also dependent upon time, temperature and cell number. Insulin concentration-dependently inhibited glucagon secretion (1 mumol: 59%, 100 nmol/l: 71%, 10 nmol/l: 86% of controls). In transient transfection experiments insulin inhibited proglucagon gene transcription (controls: 100%, 100 nmol/l: 54%, 10 nmol/l: 57%, 1 nmol/l: 72%, 100 pmol/l: 96%). Treatment of INR1G9 cells with insulin for 20 h induced a strong downregulation of insulin receptors (controls: 100%, 100 nmol/l: 30%, 10 nmol/l: 70%, 1 nmol/l: 73%, 100 pmol/l: 75%) and of insulin receptor mRNA levels (controls: 100%, 100 nmol/l: 42%, 10 nmol/l: 82%, 1 nmol/l: 84%, 100 pmol/l: 90%). When INR1G9 cells were transiently transfected with a hybrid gene containing the promotor/enhancer region of the human insulin receptor promotor (1,462 bp) linked to the transcriptional reporter gene chloramphenicol acetyltransferase and were treated with insulin it was demonstrated that insulin did not affect the insulin receptor gene transcription. In conclusion, INR1G9 cells express specific receptors for insulin. Insulin inhibits glucagon secretion and proglucagon gene expression via an inhibition of proglucagon gene transcription. Ligand-induced downregulation of the insulin receptor is not mediated by changes of insulin receptor gene transcription and is most likely regulated by posttranscriptional mechanisms, e.g. destabilization of insulin receptor mRNA.
Fehmann
H C
HC
Department of Internal Medicine, Philipps University of Marburg, FRG.
Strowski
M
M
Lankat-Buttgereit
B
B
Göke
B
B
D000818:Animals; D015152:Blotting, Northern; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D006224:Cricetinae; D003627:Data Interpretation, Statistical; D005786:Gene Expression Regulation / Q000187:drug effects; D005934:Glucagon / Q000235:genetics / Q000378:metabolism; D005935:Glucagonoma; D007328:Insulin / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism / Q000502:physiology*; D008024:Ligands; D052336:Proglucagon; D011498:Protein Precursors / Q000235:genetics; D012313:RNA / Q000302:isolation & purification; D011972:Receptor, Insulin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D007328:Insulin; D008024:Ligands; D011498:Protein Precursors; D052336:Proglucagon; D012313:RNA; D005934:Glucagon; D015500:Chloramphenicol O-Acetyltransferase; D011972:Receptor, Insulin