Entry of Insulin Into Human Cultured Lymphocytes: Electron Microscope Autoradiographic Analysis

Goldfine, I D; Jones, A. L.; Hradek, Gary T.; Wong, Kam-Fai; Mooney, JS

doi:10.1126/science.715440

Cited by 133 publications

(35 citation statements)

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“…Morphologic evidence suggests that IM-9 lymphocytes may internalize receptor-bound insulin to some degree (17,18). However, the fact that these cells neither accumulate nor degrade appreciable amounts of 125I-insulin suggests that intracellular insulin is released intact at a rate equal to its rate of uptake or the amount of internalized insulin processed through the intracellular degradative pathway is too small to be detected.…”

Section: Resultsmentioning

confidence: 99%

“…This article must therefore be hereby marked "advertisement" in accordance with 18 reagents, and buffer were added to a total volume of 2 ml and the dishes were incubated in a shaking water bath (50 oscillations per minute). After incubation, an aliquot of the buffer was withdrawn for estimation of total radioactivity, and the remaining buffer was aspirated and discarded.…”

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confidence: 99%

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Regulation of insulin receptors: evidence for involvement of an endocytotic internalization pathway.

Baldwin¹,

Prince²,

Marshall³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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Recent reports suggest that, after initial cell surface binding, insulin receptor complexes are internalized by absorptive endocytosis (1, 2). Following this, insulin is degraded at one or more intracellular sites, possibly involving lysosomes (3-7). The fate of the internalized receptor is unknown at present, although it is most likely degraded or recycled back to the plasma membrane or both. Gavin et al. (8) were the first to report that insulin could mediate the regulation of its own receptors; they found that, when IM-9 cultured lymphocytes were incubated with insulin, a marked reduction in insulin binding occurred. Since this initial report, it has been confirmed that insulin can lead to the loss of cell surface receptors in other cell types such as hepatocytes, adipocytes, and fibroblasts (9-11). Several workers have speculated that the internalization pathway for the insulin receptor may mediate the process of insulin-induced receptor loss. In order to examine the relationship between insulin internalization and receptor regulation, we have studied these processes in two cultured cell lines: normal human fibroblasts and the transformed cell line IM-9 lymphocytes.MATERIALS AND METHODS Materials. Minimal essential medium and trypsin were purchased from GIBCO. Fetal calf serum and bovine serum albumin were purchased from Reheis (Kankakee, IL). Hepes, N-tris[(hydroxymethyl)methylglycine] (Tricine), and chloroquine were purchased from Sigma. Na125I (carrier free) was Experimental Procedure. Binding experiments were performed with cultured human fibroblast monolayers in 60 X 15 mm plastic dishes as follows. After aspiration of growth media, monolayers were washed twice with 3 ml of cool (22°C) binding buffer (minimal essential medium, pH 7.4/25 mM Hepes/25 mM Tricine/1% bovine serum albumin). Then, 1251-labeled insulin (1251-insulin) at 0.3-0.4 mg/ml, insulin standards, other reagents, and buffer were added to a total volume of 2 ml and the dishes were incubated in a shaking water bath (50 oscillations per minute). After incubation, an aliquot of the buffer was withdrawn for estimation of total radioactivity, and the remaining buffer was aspirated and discarded. The monolayers were washed four times with 3 ml of ice-cold Hanks' buffer; then, 1.5 ml of 1 M NaOH was added to each dish. The dishes were rotated (100 oscillations per minute) for 15 min and the NaOH was replaced by another 1.5 ml for another 15 min. These two 1.5-ml portions of NaOH, containing all cell-associated radioactivity, were pooled and assayed in an automatic gamma counter with a 7.62-cm (3-inch) crystal. Thus, the amount bound contained all of the '25I-insulin bound from the original entire 2 ml incubation volume. All data are normalized to a cell concentration of 106 cells per dish.'25I-Insulin was prepared by a modification (12) of the method of Freychet et al. (13) as described (6).Cultured Lymphocytes. These cells were grown in suspension culture in modified minimal essential media as described (8). Cells were subcultured ever...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Regulation of insulin receptors: evidence for involvement of an endocytotic internalization pathway.

Baldwin¹,

Prince²,

Marshall³

et al. 1980

Proc. Natl. Acad. Sci. U.S.A.

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“…On the basis of their studies of the binding of insulin to isolated nuclei (44) and the subcellular localization of insulin to nuclei (45), Goldfine et al (46) have suggested that insulin is internalized by the cell and recently they report that internalized insulin localizes to endoplasmic reticulum and nuclear membrane. Our studies differ in several ways for unexplained reasons: we have not found labeled insulin preferentially localized to either nuclei or endoplasmic reticulum in either cultured lymphocytes or isolated hepatocytes.…”

Section: Discussionmentioning

confidence: 99%

Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography.

Carpentier¹,

Görden²,

Freychet³

et al. 1979

J. Clin. Invest.

173

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A B S T R A C T Quantitative electron microscopic autoradiographic studies in cultured human lymphocytes and isolated rat hepatocytes have demonstrated that labeled insulin initially localizes to the plasma membrane and is subsequently internalized to a limited region of the peripheral cytoplasm.When 0.5 nm '251-insulin is incubated with isolated rat hepatocytes, binding to the plasma membrane occurs at both 20°C and 37°C. Under steady-state binding conditions _30-40% of the labeled hormone is internalized to a distance of _ 15% of the radius of the cell. When the localization of the internalized labeled material is analyzed, by 2-5 min of incubation at 37°C there is a fivefold preferential association of autoradiographic grains with lysosomal structures, and by 30-60 min of incubation at 37°C there is a 10-fold preferential association. When the cell-associated radioactivity is extracted and filtered on Sephadex G-50 at each time point of incubation, radioactivity elutes predominantly in the position of 1251-insulin and is predominantly trichloracetic acid precipitable, bindable to talc, and rebindable to liver membranes.With increasing time of association at 37°C the initial rate and absolute amount of labeled material dissociable from the cell is reduced. With increasing time of dissociation both the cell-associated radioactivity and

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“…The fraction of bound radioactivity not dissociated by the subsequent addition of excess nonlabeled insulin may represent 1251-insulin bound irreversibly to the plasma membranes or located intracellularly, either as an intact hormone or as degradation products. Several recent studies demonstrated that insulin may enter the cells as an intact molecule, and such a process may be operative in glomerular cells (24,25).…”

Section: Discussionmentioning

confidence: 99%

Binding of 125I-insulin to the isolated glomeruli of rat kidney.

Kurokawa¹,

Silverblatt²,

Klein³

et al. 1979

J. Clin. Invest.

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A B S T R A C T To investigate a possible action of insulin on the glomerulus, the binding 1251-insulin to the isolated glomeruli prepared from rat kidney was examined. When incubated at 22°C, '251-insulin binding proceeded with time and reached a steady state at 45 min at which time nonspecific binding was <25% of total binding. A small fraction of '25I-insulin was degraded during incubation. This binding was specific to insulin in that it was inhibited by unlabeled porcine and beef insulins and to a lesser extent by porcine proinsulin and desalanine-desasparagine insulin, but not by glucagon, parathyroid hormone, vasopressin, calcitonin, and angiotensin II. Increasing concentrations of nonlabeled insulin displaced 1251-insulin binding in a dose-dependent fashion. Scatchard plot of the data was curvilinear consistent with either two classes of receptors with different affinities or a single class of receptors that demonstrate negative cooperativity. The addition of excess nonlabeled insulin to the glomeruli preincubated with 1251-insulin resulted in a rapid dissociation of -70% of bound I251-insulin. Insulin decreased the increments in glomerular cyclic AMP levels by epinephrine and by prostaglandin E2, but not those by histamine. These data showed the presence of specific insulin receptors in the glomeruli, and that insulin action may be, at least in part, through modulation of glomerular cyclic AMP concentrations. Such action of insulin may underlie the alteration in glo-

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Entry of Insulin Into Human Cultured Lymphocytes: Electron Microscope Autoradiographic Analysis

Cited by 133 publications

References 14 publications

Regulation of insulin receptors: evidence for involvement of an endocytotic internalization pathway.

Regulation of insulin receptors: evidence for involvement of an endocytotic internalization pathway.

Lysosomal association of internalized 125I-insulin in isolated rat hepatocytes. Direct demonstration by quantitative electron microscopic autoradiography.

Binding of 125I-insulin to the isolated glomeruli of rat kidney.

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