Insulin Degradation by Liver Cell Membranes*

Duckworth, William C.

doi:10.1210/endo-104-6-1758

Cited by 54 publications

(29 citation statements)

References 27 publications

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“…Sephadex G-50 chromatography revealed that the insulin associated with the nucleus was equally divided between a peak that was eluted as intact insulin and a peak containing a high molecular weight complex. A similar high molecular weight peak has been reported (27) by many investigators after insulin incubation of cells or plasma membranes and has usually been attributed to insulin-receptor complexes. However, since we failed to detect internalized plasma membrane receptors in the nuclei by two techniques, immunoelectron microscopy and immunoblotting, the high molecular weight peak may represent '25I-labeled insulin complexed with components of the nuclear heterochromatin.…”

Section: Discussionsupporting

confidence: 79%

Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells.

Soler

Thompson

Smith

et al. 1989

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

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Although insulin is known to regulate nuclear-related processes, such as cell growth and gene transcription, the mechanisms involved are poorly understood. Previous studies suggested that translocation of insulin or its receptor to cell nuclei might be involved in some of these processes. The present investigation demonstrated that intact insulin, but not the insulin receptor, accumulated in nuclei of insulin-treated cells. Cell fractionation studies demonstrated that the nuclear accumulation of 12'I-labeled insulin was time-, temperature-, and insulin-concentration-dependent. Electron microscopic immunocytochemistry demonstrated that the insulin that accumulated in the nucleus was immunologically intact and associated with the heterochromatin. Only 1% of the '25I-labeled insulin extracted from isolated nuclei was eluted from a Sephadex G-50 column as 125I-labeled tyrosine. Plasma membrane insulin receptors were not detected in the nucleus by immuno electron microscopy or when wheat germ agglutininpurified extracts of the nuclei were subjected to PAGE, electrotransfer, and immunoblotting with anti-insulin receptor antibodies. These results suggested that internalized insulin dissociated from its receptor and accumulated in the nucleus without its membrane receptor. We propose that some of insulin's effects on nuclear function may be caused by the translocation of the intact and biologically active hormone to the nucleus and its binding to nuclear components in the heterochromatin.Several laboratories have demonstrated that extracellular insulin affects nuclear processes including mRNA efflux (1), protooncogene expression (2), transcription of tyrosine aminotransferase (3) and phosphoenolpyruvate carboxykinase (4) genes, cell growth (5), and phosphate incorporation into the nuclear envelope (6) and nuclear lamins (7). The first step leading to these effects is the binding of insulin to receptors in the plasma membrane. The remaining events are not understood. In particular, it is not known whether insulin binding to receptors generates signals leading to these nuclear-related events or whether insulin is internalized and interacts directly with components of the nucleus. Miller showed (8) that microinjection of insulin into the cytoplasm of Xenopus laevis oocytes increased RNA and protein synthesis. Also, insulin added to isolated nuclei of oocytes stimulated RNA synthesis, suggesting a direct biological effect of insulin on nuclear function.Although some earlier studies reported that insulin could bind to isolated nuclei (9-11) or accumulate in nuclei of intact IM-9 lymphocytes (12) and rat hepatocytes (13), other studies (14, 15) questioned those findings. This laboratory has provided (16) evidence that receptor-mediated internalization of insulin resulted in a time-and temperature-dependent nuclear accumulation of 1251-labeled insulin and ferritin-labeled insulin in 3T3-L1 adipocytes. The ferritin-labeled insulin was associated with the heterochromatin near nuclear pores. The presence of both 125I and ...

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Section: Discussionsupporting

confidence: 79%

Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells.

Soler

Thompson

Smith

et al. 1989

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

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“…The nature of the labeled material after exposure to the hepatocytes was also assessed by chromatography on Sephadex G-50 (1.5 X 90 cm) in 1 M acetic acid (9). In some experiments the cells themselves were extracted with 3 M acetic acid containing 6 M urea, 1 ml/liter Triton X-100 and 0.15 M NaCl, and the extract was chromatographed on Sephadex G-50 in 1 M acetic acid.…”

Section: Methodsmentioning

confidence: 99%

Insulin Degradation by Hepatocytes in Primary Culture*

et al. 1981

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The mechanism by which the liver degrades insulin has not yet been completely clarified. In intact, non-"leaky" cells the primary process seems to be mediated by initial receptor binding. We now demonstrate that isolated rat hepatocytes in primary culture are suitable for examining insulin degradation. Hepatocytes did not leak degrading activity into the medium, and thus, the degradation seen was essentially exclusively cell mediated. [125I]Iodoinsulin degradation by these cells was dependent on time and cell concentration. There was a short lag time before degradation products could be detected in the medium. After incubation with the hepatocytes, three peaks of 125I-labeled material could be separated by chromatography on Sephadex G-50. The same three peaks were seen with 125I-labeled material extracted from the cells. When [3H]insulin, labeled exclusively at the B-1 phenylalanine residue, was incubated with the cells, additional peaks of labeled material were recovered from the column. These additional peaks were intermediate in size between insulin and iodotyrosine, suggesting the production of products smaller than insulin but larger than individual amino acids. In order to begin to characterize the subcellular mechanisms for insulin metabolism, the effect of various potential inhibitors on insulin degradation were examined. The most effective inhibitors were N-ethylmaleimide, bacitracin, and Kunitz pancreatic trypsin inhibitor. Chloroquine decreased degradation only 10%, and NH4Cl had no detectable effect. The effect of the inhibitors on the purified insulin-degrading enzyme, insulin protease, was also examined. The purified enzyme responded essentially identically as the intact cells to the various inhibitors. From all these data it would seem that lysosomal degradation of insulin in the hepatocyte may be a relatively minor pathway and the neutral protease may play a major role.

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“…IDE was initially purified from human red blood cells (37) but is also present in a wide range of nucleated cells (www.genecards.org/cgi-bin/carddisp.pl?gene=IDE). Depending on the type of cell, IDE may be located in the cytosol (38,39), peroxisomes (13,40), endosomes (12,41), mitochondria (15), or on the cell membrane (7)(8)(9)(10)(11)42).…”

Section: Discussionmentioning

confidence: 99%

“…IDE is present in human beings, animals, fungi, and plants (2-4; http://merops.sanger.ac.uk/, see distribution within family M16.002: insulysin) and is reported to be expressed in the liver, adipocytes, muscle cells, erythrocytes, and kidney (3)(4)(5) but also in cell types not responsive to insulin (6) (www.genecards.org/cgi-bin/carddisp.pl?gene=IDE). Despite its predominant presence in the cytosol, IDE is also found in small but significant amounts in subcellular compartments such as the plasma membrane, endosomes, peroxisomes, and mitochondria (7)(8)(9)(10)(11)(12)(13)(14)(15).…”

Section: Introductionmentioning

confidence: 99%

Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues

Yfanti

Mengele²,

Gkazepis³

et al. 1998

Int J Mol Med

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Abstract. Insulin-degrading enzyme (IDE, insulysin, insulinase; EC 3.4.22.11), a thiol metalloendopeptidase, is involved in intracellular degradation of insulin, thereby inhibiting its translocation and accumulation to the nucleus. Recently, protein expression of IDE has been demonstrated in the epithelial ducts of normal breast and breast cancer tissue. Utilizing four different antibodies generated against different epitopes of the IDE molecule, we performed Western blot analysis and immunohistochemical staining on several normal human tissues, on a plethora of tumor cell lines of different tissue origin, and on malignant breast and ovarian tissue. Applying the four IDE-directed antibodies, we demonstrated IDE expression at the protein level, by means of immunoblotting and immunocytochemistry, in each of the tumor cell lines analyzed. Insulin-degrading enzyme protein expression was found in normal tissues of the kidney, liver, lung, brain, breast and skeletal muscle, as well as in breast and ovarian cancer tissues. Immunohistochemical visualization of IDE indicated cytoplasmic localization of IDE in each of the cell lines and tissues assessed. In conclusion, we performed for the first time a wide-ranging survey on IDE protein expression in normal and malignant tissues and cells thus extending our knowledge on the cellular and tissue distribution of IDE, an enzyme which to date has mainly been studied in connection with Alzheimer's disease and diabetes but not in cancer.

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Insulin Degradation by Liver Cell Membranes*

Cited by 54 publications

References 27 publications

Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells.

Immunological demonstration of the accumulation of insulin, but not insulin receptors, in nuclei of insulin-treated cells.

Insulin Degradation by Hepatocytes in Primary Culture*

Expression of metalloprotease insulin-degrading enzyme insulysin in normal and malignant human tissues

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