Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion
| Autor: | Eiichi Araki, S. Isami, Hideaki Jinnouchi, Nobuhiro Miyamura, Tetsuya Shirotani, S. Ura, Hideki Kishikawa, K. Kisanuki, M. Uehara, Motoaki Shichiri |
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| Rok vydání: | 1995 |
| Předmět: |
medicine.medical_specialty
Endocrinology Diabetes and Metabolism medicine.medical_treatment Molecular Sequence Data Gene Expression Sulfur Radioisotopes Polymerase Chain Reaction Estrogen-related receptor alpha Methionine Cricetinae Insulin receptor substrate Internal medicine Tumor Cells Cultured Internal Medicine medicine Animals Humans Insulin RNA Messenger Glucagon-like peptide 1 receptor DNA Primers Insulin-like growth factor 1 receptor Base Sequence biology Blotting Northern Glucagon Immunohistochemistry Receptor Insulin IRS2 Clone Cells Pancreatic Neoplasms Kinetics Insulin receptor Endocrinology Insulin receptor binding biology.protein Insulinoma |
| Zdroj: | Diabetologia. 38:422-429 |
| ISSN: | 1432-0428 0012-186X |
| DOI: | 10.1007/bf00410279 |
| Popis: | In pancreatic alpha cells, the existence and function of the insulin receptor has not yet been fully established. In this study, to confirm the expression of functional insulin receptors in pancreatic alpha cells, we performed: 1) insulin receptor binding assay, 2) Northern blot analysis and RT-PCR (reverse transcription-polymerase chain reaction) amplification of insulin receptor mRNA, 3) immunocytochemical staining, 4) biosynthetic labelling of insulin receptor protein using [35S]methionine, 5) analysis of insulin-stimulated autophosphorylation of the insulin receptor in glucagon secreting cell lines, In-R1-G9 and αTC clone 6 cells. Glucagon secretion decreased with the addition of insulin in both cells. The receptor binding studies using [125I-Tyr-A14] insulin revealed that both cells possessed a significant number of insulin receptors (In-R1-G9: K1=2.1×109mol/l−1, K2=6.2×107 mol/l−1, R1=0.2×104, R2=1.86×104 sites/cell; αTC clone 6: K1=2.1×109 mol/l−1, K2=7.3×107 mol/l−1, R1=0.27×104, R2=1.95×104 sites/cell). Northern blot analysis as well as RT-PCR amplification showed the mRNA specific for insulin receptor in both cells. By immunocytochemical staining using anti-insulin receptor α-subunit antibody, positive immunostaining for insulin receptor was observed in both cells. [35S]Methionine labelling of both cells followed by immunoprecipitation using anti-insulin receptor antibody showed the correct size of the insulin receptor protein. The insulin receptor expressed in these cells underwent autophosphorylation by insulin stimulation. It is concluded that functional insulin receptors are properly expressed in In-R1-G9 and αTC clone 6 cells. |
| Databáze: | OpenAIRE |
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