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Electron Microscopic Demonstration of Insulin Receptors on Adipocyte Plasma Membranes Utilizing a Ferritin-Insulin Conjugate

  • Leonard Jarett
    Affiliations
    From the Division of Laboratory Medicine, Departments of Pathology and Medicine, Washington University School of Medicine and Barnes Hospital, St. Louis, Missouri 63110
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  • Robert M. Smith
    Affiliations
    From the Division of Laboratory Medicine, Departments of Pathology and Medicine, Washington University School of Medicine and Barnes Hospital, St. Louis, Missouri 63110
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Open AccessPublished:November 10, 1974DOI:https://doi.org/10.1016/S0021-9258(19)42161-3
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      Ferritin was conjugated to porcine insulin with glutaraldehyde without altering the immunological or biological properties of the hormone. The former was determined by radioimmunoassays and the latter by measuring stimulation of the glucose oxidation and the inhibition of epinephrine-in duced lipolysis of adipocytes or the stimulation of the Mg2+ ATPase. The conjugate was purified by differential centrifugation, gel filtration on Sephadex G-75, and affinity chromatography to remove essentially all unconjugated insulin and the vast majority of free ferritin. The hormone did not dissociate from the ferritin during storage or incubation. Electron microscopic observations revealed the ferritin-insulin to bind specifically and irregularly to only one side of purified fat cell plasma membranes. This binding was prevented by excess unconjugated insulin while ferritin alone did not bind to the plasma membranes. The ferritin-insulin did not bind to adipocyte mitochondria. The complex was not observed free inside the plasma membrane vesicles, suggesting that the complex was not capable of crossing the membrane. These biochemical and electron microscopic studies support the concept that penetration of the insulin into the cytosol of the cell is not necessary for the hormone to exert its biological effects on adipocytes. The hormone may enter the cell in a specialized compartment since occasionally the ferritin-insulin complex was observed specifically bound to the inside of pinocytotic-like microvesicles arising from the plasma membrane. The physiological significance of this latter observation remains unexplained.

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