Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion

Summary

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.

Abbreviations

RT-PCR:

Reverse transcriptase-polymerase chain reaction

FBS:

fetal bovine serum

DMEM:

Dulbecco's modified Eagle's medium

BSA:

bovine serum albumin

PBS:

phosphate buffered saline

SDS:

sodium dodecyl sulphate

SSC:

saline sodium citrate

References

  1. 1.

    Shichiri M, Kawamori R, Abe H (1979) Normalization of the paradoxic secretion of glucagon in diabetics who were controlled by the artificial beta cell. Diabetes 28: 272–275

  2. 2.

    Kawamori R, Shichiri M, Kikuchi M, Yamasaki Y, Abe H (1980) Perfect normalization of excessive glucagon responses to intravenous arginine in human diabetes mellitus with the artificial beta cell. Diabetes 29: 762–765

  3. 3.

    Paolisso G, Sgambato S, Torella R, Varricchio M, Scheen A, D'Onoerio F, Lefebvre PJ (1988) Pulsatile insulin delivery is more efficient than continuous infusion in modulating islet cell function in normal subjects and patients with type 1 diabetes. J Clin Endocrinol Metab 66: 1220–1226

  4. 4.

    Hamaguchi T, Fukushima H, Uehara M et al. (1991) Abnormal glucagon response to arginine and normalization in obese hyperinsulinaemic patients with glucose intolerance: importance of insulin action on pancreatic alpha cells. Diabetologia 34: 801–806

  5. 5.

    Kahn CR, White MF (1988) The insulin receptor and the molecular mechanism of insulin action. J Clin Invest 82: 1151–1156

  6. 6.

    Ebina Y, Araki E, Taira M et al. (1987) Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity. Proc Natl Acad Sci USA 84: 704–708

  7. 7.

    Patel YC (1982) Quantitative electron microscopic autoradiography of insulin, glucagon, and somatostatin binding sites on islets. Science 217: 1155–1156

  8. 8.

    Van Schravendijk CFH, Foriers A, Hooghe-Peters El et al. (1985) Pancreatic hormone receptors on islet cells. Endocrinology 117: 841–848

  9. 9.

    Takaki R, Ono J, Nakamura M et al. (1986) Isolation of glucagon-secreting cell lines by cloning insulinoma cells. In Vitro Cell Dev Biol 22: 120–126

  10. 10.

    Hamaguchi K, Leiter EH (1989) Comparison of cytokine effects on mouse pancreatic α-cell and Β-cell lines. Viability, secretory function, and MHC antigen expression. Diabetes 39: 415–425

  11. 11.

    Powers AC, Efrat S, Mojsov S, Spector D, Habener JF, Hanahan D (1989) Proglucagon processing similar to normal islets in pancreatic α-like cell line derived from transgenie mouse tumor. Diabetes 39: 406–414

  12. 12.

    Scatchard G (1949) The attractions of proteins for small molecules and ions. Ann N Y Acad Sci 51: 660–672

  13. 13.

    Thakur AK, Jaffe ML, Rodbard D (1980) Graphical analysis of ligand-binding systems: evaluation by Monte Carlo studies. Anal Biochem 107: 279–295

  14. 14.

    Rosenthal HE (1967) A graphic method for the determination and presentation of binding parameters in a complex system. Anal Biochem 20: 525–532

  15. 15.

    Chirgwin JM, Przybyla AE, MacDonald RJ, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18: 5294–5299

  16. 16.

    Ebina Y, Ellis L, Jarnagin K et al. (1985) The human insulin receptor cDNA: the structural basis for hormone-activated transmembrane signalling. Cell 40: 747–758

  17. 17.

    Saiki RK, Scharf SJ, Faloona F et al. (1985) Enzymatic amplification of Β-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230: 1350–1354

  18. 18.

    Saiki RK, Gelfand DH, Stoffel S et al. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487–489

  19. 19.

    Flores-Riveros JR, Sibley E, Kastelic T, Lane MD (1989) Substrate phosphorylation catalyzed by the insulin receptor tyrosine kinase: kinetic correlation to autophosphorylation of specific sites in the Β subunit. J Biol Chem 264: 21557–21572

  20. 20.

    Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 74: 5463–5467

  21. 21.

    Kasuga M, Hedo JA, Yamada KM, Kahn CR (1982) The structure of insulin receptor and its subunits. Evidence for multiple nonreduced forms and a 210,000 possible proreceptor. J Biol Chem 257: 10392–10399

  22. 22.

    Nishino T, Kodaira T, Shin et al. (1981) Glucagon immunoassay with use of antiserum to glucagon C-terminal fragment. Clin Chem 27: 1690–1697

  23. 23.

    Shennan KIJ, Holst JJ, Docherty K (1989) Proglucagon expression, posttranslational processing and secretion in SV40-transformed islet cells. Mol Cell Endocrinol 67: 93–99

  24. 24.

    Pipeleers DG, Schuit FC, Van Schravendijk CFH, Van De Winkel M (1985) Interplay of nutrients and hormones in the regulation of glucagon release. Endocrinology 117: 817–823

  25. 25.

    Drucker DJ, Philippe J, Mojsov S (1988) Proglucagon gene expression and post-translational processing in a hamster islet cell line. Endocrinology 123: 1861–1867

  26. 26.

    Samols E, Bonner-Weir S, Weir GC (1986) Intra-islet insulin-glucagon-somatostatin relationships. Clin Endocrinol Metab 15: 33–58

  27. 27.

    Philippe J (1989) Glucagon gene transcription is negatively regulated by insulin in a hamster islet cell line. J Clin Invest 84: 672–677

  28. 28.

    Gammeltoft S, Gliemann J (1973) Binding and degradation of 125I-labelled insulin by isolated rat fat cells. Biochim Biophys Acta 320: 16–32

  29. 29.

    Hidaka H, Howard BV, Ishibashi F et al. (1981) Effect of pH and 3-hydroxybutyrate on insulin binding and action in cultured human fibroblasts. Diabetes 30: 402–406

  30. 30.

    Olefsky JM, Jen P, Reaven GM, Alto P (1974) Insulin binding to isolated human adipocytes. Diabetes 23: 565–571

  31. 31.

    Gavin III JR, Roth J, Jen P, Freychet P (1972) Insulin receptors in human circulating cells and fibroblasts. Proc Natl Acad Sci USA 69: 747–751

  32. 32.

    Goldstein BJ, Muller-Wieland D, Kahn CR (1987) Variation in insulin receptor messenger ribonucleic acid expression in human and rodent tissues. Mol Endocrinol 11: 759–766

  33. 33.

    Ullrich A, Gray A, Tarn AW et al. (1986) Insulin-like growth factor I receptor primary structure: comparison with insulin receptor suggests structural determinants that define functional specificity. EMBO Journal 5: 2503–2512

  34. 34.

    Hedo JA, Kahn CR, Hayashi M, Yamada KM, Kasuga M (1983) Biosynthesis and glycosylation of the insulin receptor. Evidence for a single polypeptide precursor of the two major subunits. J Biol Chem 258: 10020–10026

  35. 35.

    Barr PJ (1991) Mammalian subtilisins: the long-sought dibasic processing endoproteases. Cell 66: 1–3

  36. 36.

    Robertson BJ, Moehring JM, Moehring TJ (1993) Defective processing of the insulin receptor in an endoprotease-deficient Chinese hamster cell strain is corrected by expression of mouse furin. J Biol Chem 268: 24274–24277

  37. 37.

    Yanagita M, Hoshino H, Nakayama K, Takeuchi T (1993) Processing of mutated proinsulin with tetrabasic cleavage site to mature insulin reflects the expression of furin in non-endocrine cell lines. Endocrinology 133: 639–644

  38. 38.

    Boulware SD, Tamborlane WV, Rennert NJ, Gesundheit N, Sherwin RS (1994) Comparison of the metabolic effects of recombinant human insulin-like growth factor-1 and insulin: dose-response relationships in healthy young and middle-aged adults. J Clin Invest 93: 1131–1139

  39. 39.

    Philippe J (1991) Insulin regulation of the glucagon gene is mediated by an insulin-responsive DNA element. Proc Natl Acad Sci USA 88: 7224–7227

Download references

Author information

Correspondence to Dr. K. Kisanuki.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kisanuki, K., Kishikawa, H., Araki, E. et al. Expression of insulin receptor on clonal pancreatic alpha cells and its possible role for insulin-stimulated negative regulation of glucagon secretion. Diabetologia 38, 422–429 (1995). https://doi.org/10.1007/BF00410279

Download citation

Key words

  • Pancreatic alpha cell
  • In-R1-G9
  • αTC clone 6
  • insulin receptor
  • glucagon secretion