Summary
Starved and fed mice were injected intravenously with either fluorescent or125I-insulin and the localization of the hormone was investigated in various tissues. The fluorescent hormone was found in the kidney of starved animals only, whereas the radioactive hormone was found in the proximal convoluted tubule cells of the kidney of both fed and fasted animals, although the latter group appeared to contain more radioactivity than the first. With high resolution techniques the radioactivity in the kidney was found in the brush border, the apical vacuoles, the apical mitochondria and the nucleus, and never in the Golgi apparatus or the lysosomes. All other tissues contained radioactivity due to the hormone, but no difference in either the amount or the localization was found between fed an starved animals. It is concluded that the process of absorption of insulin by the cells of the proximal convoluted tubules is specific and that the hormone is not degraded in a way similar to other proteins, but is likely to be stored and to control cellular metabolic processes from its sites of localization.
Résumé
La localisation d'insuline marquée avec I125 ou avec de l'isothiocyanate de fluorescéine a été étudiée au moyen d'autohistoradiographie au microscope optique, au microscope électronique et au moyen de microscopie à fluorescence dans les tissus de souris à jeun et de souris alimentées normalement. L'hormone fluorescente a été trouvée dans les cellules des tubules proximaux du rein de souris à jeun seulement, tandis que l'hormone radioactive a été repérée aussi dans le rein de souris alimentées, bien qu'en quantité inférieure à celle trouvée chez les souris à jeun. Au microscope électronique la radioactivité est présente dans le rein dans la bordure en brosse, les vacuoles et les mitochondries apicales et le noyau, mais pas dans l'appareil de Golgi ou dans les lysosomes. —L'hormone radioactive est aussi présente dans les autres tissus mais sans différence quantitative entre les souris à jeun et les souris alimentées. Les auteurs concluent que le procédé de ré-absorption de l'insuline par les cellules des tubules proximaux du rein est spécifique: l'hormone n'est pas dégradée de la même façon que les autres protéines mais probablement stockée afin de contrôler les processus métaboliques relevant des organelles subcellulaires dans lesquelles elle se trouve.
Zusammenfassung
Gefütterte und fastende Mäuse erhielten fluoreszierendes oder mit125J markiertes Insulin i.v. Die Lokalisation des Insulins in den Geweben wurde untersucht. Nur bei Tieren im Hungerzustand fand sich das fluoreszierende Hormon in den Nieren, während sich das radioaktive Insulin bei gefütterten und fastenden Tieren in den proximalen Tubulusschlingen der Nieren nachweisen ließ. Die fastenden Tiere schienen dabei mehr Radioaktivität aufzuweisen. Wurden die Nieren unter Verwendung von Techniken mit hohem Auflösungsvermögen untersucht, so fand sich die Radioaktivität im Bürstensaum, in den apikalen Vakuolen und Mitochondrien und dem Kern, jedoch nicht im Golgi-Apparat oder den Lysosomen. Alle anderen Gewebe enthielten Hormon-Radioaktivität, aber weder ihre Menge noch ihre Lokalisation unterschieden sich bei gefütterten und fastenden Tieren. Es wird gefolgert, daß der Prozeß der Insulinabsorption durch die Zellen der proximalen Tubulusschlingen spezifisch ist und daß das Hormon nicht in ähnlicher Weise wie andere Eiweißkörper abgebaut wird. Wahrscheinlich kommt es über eine Speicherung zu einer Steuerung der entsprechenden Stoffwechselprozesse in den subzellulären Organellen, in denen das Hormon sich befindet.
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References
Arquilla, E.R., H. Ooms, andJ. Finn: Genetic differences of combining sites of insulin antibodies and importance of C-terminal portion of the A-chain to biological and immunological activity of insulin. Diabetologia2, 1–13 (1966).
Beck, F.: The distribution of acid phosphatase in the chick blastoderm. Exp. Cell Res.37, 504–508 (1965).
Bromer, W.W., S. Knapp-Sheehan, A.W. Berns, andE.R. Arquilla: Preparation and properties of fluorescein thiocarbamyl insulins. Biochemistry6, 2378–2388 (1967).
Budd, G.C., andS.R. Pelc: The membrane method of electron microscope autoradiography. Stain Technol.39, 295–302 (1964).
Caro, L.G.: High resolution autoradiography. II. The problem of resolution. J. Cell Biol.15, 189–199 (1962).
Elgee, N.J., andR.H. Williams: Degradation of insulin-I131 by liver and kidneyin vivo. Proc. Soc. Exp. Biol.87, 352–355 (1954).
Gambassi, G., V. Maggi, andC. Matarazzo: Sur le problème de l'inactivation hépatique de l'insulinein vivo. Acta med. scand.153, 201–209 (1956).
Glauert, A.M.: In Techniques for electron microscopy, 2nd edition. Editor:Kay, D.H., p. 166. Oxford: Blackwell Scient. Publ. 1965.
Graham, R.C., andM.J. Karnovsky: The early stages of absorption of injected horse-radish peroxidase in the proximal consoluted tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem.14, 291–302 (1966).
Greenwood, F.C., W.M. Hunter, andJ.S. Glover: The preparation of131I-labelled human growth hormone of high specific activity. Biochem. J.89, 114–123 (1963).
Hales, C.N., andP.J. Randle: Immunoassay of insulin with insulin-antibody precipitate. Biochem. J.88, 137–146 (1963).
Izzo, J.L., W.F. Bale, M.J. Izzo, andA. Roncone: High specific activity labelling of insulin with131I. J. biol. Chem.239, 3743–3748 (1964).
—,A. Roncone, M.J. Izzo, andW.F. Bale: Relationship between degree of iodination of insulin and its biological electrophoretic and immunochemical properties. J. biol. Chem.239, 3749–3754 (1964).
Kayes, J., A.B. Maunsbach, andS. Ullberg: Electron microscopic autoradiography of radioiodine in the thyroid using the extranuclear electrons of I125. J. ultrastruct. Res.7, 339–345 (1962).
Lee, N.D.: The specificity of the interaction of insulin-I131 with tissue. Endocrinology65, 347–356 (1959).
—, andR. Wiseman, Jr.: The significance of the binding of insulin-I131 to cytostructural elements of rat liver. Endocrinology65, 442–450 (1959).
Lowry, O.H., N.J. Rosebrough, A.L. Farr, andR.J. Randall: Protein measurement with the Folin phenol reagent. J. biol. Chem.193, 265–275 (1951).
Maggi, V.: The localization of fluorescent insulin in mouse tissues. Exp. Cell Res.44, 672–676 (1966).
—, andM.F. Oddy: Effect of short-term starvation on the cells of the proximal convoluted tubule of the mouse: a cytological and cytochemical study. The Histochemical Journal1, 78–92 (1968).
Maunsbach, A.B.: Absorption of I125-labelled homologous albumin by rat kidney proximal convoluted tubule cells. A study of microperfused single proximal tubules by electron microscopic autoradiography and histochemistry. J. ultrastruct. Res.15, 197–241 (1966a).
—: Absorption of ferritin by rat kidney proximal tubule cells. J. ultrastruct. Res.16, 1–12 (1966b).
Messier, B., andC.P. Leblond: Preparation of coated radioautographs by dipping sections in fluid emulsion. Proc. Soc. exp. Biol.96, 7–10 (1957).
Narahara, H.T., N.B. Everett, B.S. Simmons, andR.H. Williams: Metabolism of insulin-I131 and glucagon-I131 in the kidney of the rat. Amer. J. Physiol.192, 227–231 (1958).
Novikoff, A.B.: Lysosomes in the physiology and pathology of cells; contribution of staining methods. In Ciba Found. Symp. on Lysosomes. London: J. & A. Churchill Ltd. 1963.
—E. Essner, andN. Quintana: Golgi apparatus and lysosomes. Fed. Proc.23, 1010–1022 (1964).
Pelc, S.R.: Autoradiograph technique. Nature160, 749–750 (1947).
Randle, P.J., P.B. Garland, C.N. Hales, E.A. Newsholme, R.M. Denton, andC.I. Pogson: Interactions of metabolism and the physiological role of insulin. Recent Progr. Hormone Res.22, 1–44 (1966).
Rogers, A.W., Techniques of autoradiography. London and New York: Elsevier publ. Comp. Amsterdam 1967.
Schalch, D.S.: Use of I125-labelling in radioimmunoassay. Proc. Soc. exp. Biol.121, 1279–1280 (1966).
Stein, O., andJ. Gross: Localization and metabolism of I131-insulin in the muscle and some other tissues of the rat. Endocrinology65, 707–716 (1959).
Straus, W., Comparative observations on lysosomes and phagosomes in kidney and liver of rats after administration of horse-radish peroxidase. CIBA Found. Symp. on Lysosomes 151–175, 1963.
Tietze, F., G.E. Mortimore, andN.R. Lomax: Preparation and properties of fluorescent insulin derivatives. Biochem. biophys. Acta.59, 336–346 (1962).
Worthington, L.A. Jr.,D. Jones, andM.G. Buse: Autoradiographic study of insulin distribution in the rat with special reference to elastic tissue. Endocrinology74, 914–924 (1964).
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Maggi, V., Franks, L.M., Wilson, P.D. et al. Localization of insulin in mouse tissues using fluorescence microscopy and light microscope and high resolution autoradiography.. Diabetologia 5, 67–78 (1969). https://doi.org/10.1007/BF01212000
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DOI: https://doi.org/10.1007/BF01212000