Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Izzo, Joseph L.; Roncone, A.; Helton, Deirdre L.; Izzo, Mary Jane

doi:10.1016/0003-9861(79)90399-0

Cited by 28 publications

(12 citation statements)

References 39 publications

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“…2). It is presently well established that insulin enters the cell (44)(45)(46). However, the relationship of insulin internalization to the biological effect of insulin remains uncertain.…”

Section: Discussionmentioning

confidence: 99%

Insulin resistance in uremia. Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats.

Kauffman¹,

José²

1983

J. Clin. Invest.

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A B S T R A C T We have developed a model in the rat that leads to a predictable degree of severe uremia to study the role of the liver in the insulin-resistant state of uremia. The uremic animals were euglycemic and had increased serum immunoreactive insulin when compared with their pair-fed controls. Insulin action, binding, internalization, and degradation were characterized in freshly isolated hepatocytes from uremic animals, sham-operated pair-fed, and ad lib.-fed controls.The basal rate of aminoisobutyric acid (AIB) uptake was increased in hepatocytes from both uremic and pair-fed control rats. However, while hepatocytes from uremic animals were refractory to insulin with regard to AIB uptake, there was no significant difference in the absolute increment above basal AIB uptake by hepatocytes from pair-fed and fed ad lib. animals at any insulin concentration studied.125I-Insulin binding at 240C was higher in hepatocytes from uremic rats at every insulin concentration studied when compared with fed ad lib. controls. The time course of '251-insulin binding to the cell and to the fractions that were membrane bound or internalized were studied at 370C. An increase in membranebound '251-insulin at 370C was present also in hepatocytes from uremic animals. The same fraction of membrane-bound 1251-insulin was internalized in hepatocytes from all groups of animals. nalization was studied at 370C by gel chromatography. There was a delayed and decreased rate of 125I-insulin degradation in hepatocytes from uremic rats in the three compartments.We conclude: (a) In chronic uremia the liver is refractory to insulin with regard to AIB uptake. (b) Insulin resistance in uremic rat liver is not due to defects in insulin binding or internalization. (c) Despite the high level of circulating immunoreactive insulin, hepatocytes from uremic rats did not show the expected "down regulation" of their insulin receptors or an increased rate of insulin degradation. These studies further emphasize the primary role of postbinding events in the regulation of insulin binding and degradation. The mechanism as to how the coordinated steps of insulin metabolism in the liver are disrupted in a pathological state is presently unknown. INTRODUCTIONInsulin resistance is widely recognized in patients with chronic renal failure (1). Peripheral insulin insensitivity in uremia is well accepted, but the role of the liver in insulin action, binding, and processing (internalization and degradation) remains controversial or unexplored (2, 3). This is an important problem to define since the liver is a major target organ for insulin action and the main organ for insulin metabolism and degradation (4).In order to study hepatic insulin metabolism in uremia we have developed a model of chronic renal failure in the rat that leads to a predictable degree of severe uremia. In the experimental design, specific consideration was given to the nutritional status of the animals. Two sham-operated control groups, ad lib.-fed and pair-fed animals were included. This al...

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“…2). It is presently well established that insulin enters the cell (44)(45)(46). However, the relationship of insulin internalization to the biological effect of insulin remains uncertain.…”

Section: Discussionmentioning

confidence: 99%

Insulin resistance in uremia. Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats.

Kauffman¹,

José²

1983

J. Clin. Invest.

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“…Fm-Ins was prepared and characterized as described (29 (8,(11)(12)(13)(14) and accumulated in cytoplasmic vesicles or lysosomes (10,11,14) or Golgi (10,(15)(16)(17)(18). Many studies (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) Some cells also were incubated with Fm-Ins at 15'C.…”

Section: Methodsmentioning

confidence: 99%

“…Many studies (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) Some cells also were incubated with Fm-Ins at 15'C. The cells were washed and prepared for electron microscopy as described (25).…”

Section: Methodsmentioning

confidence: 99%

“…Insulin has been shown by both biochemical and ultrastructural techniques to be internalized by numerous cell types (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) and to have effects on isolated nuclei (19,20), leading to the speculation that internalization, intracellular translocation, and/or processing may be physiologically significant, especially for the long-term effects of insulin.…”

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confidence: 99%

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Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process.

Smith¹,

Jarett²

1987

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

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Monomeric ferritin-labeled insulin (Fm-Ins), a biologically active, electron-dense marker of occupied insulin receptors, was used to characterize the internalization of insulin in 3T3-L1 adipocytes. Fm-Ins bound specifically to insulin receptors and was internalized in a time-and temperature-dependent manner. Fm-Ins was found in cytoplasmic vesicles within 5-10 min at 3rC and subsequently was observed in multivesicular bodies and Iysosomes. In addition, small amounts of Fm-Ins were associated with nuclei after 30 min. The number of Fm-Ins particles observed in nuclei continued to increase in a time-dependent manner until at least 90 min. In the nucleus, several Fm-Ins particles usually were found in the same general location-near nuclear pores, associated with the periphery of the condensed chromatin. Addition of a 250-fold excess of unlabeled insulin or incubation at 15°C reduced the number of Fm-Ins particles found in nuclei after 90 min by 99% or 92%, respectively. Nuclear accumulation of unlabeled ferritin was only 2% of that found with Fm-Ins after 90 min at 37°C. Biochemical experiments utilizing 12'I-labeled insulin and subceliular fractionation indicated that intact 3T3-L1 adipocytes internalized insulin rapidly and that =3% of the internalized ligand accumulated in nuclei after 1 hr. These data provide biochemical and high-resolution ultrastructural evidence that 3T3-L1 adipocytes accumulate potentially significant amounts of insulin in nuclei by an insulin receptor-mediated process. The transport of insulin or the insulin-receptor complex to nuclei in this cell or in others may be directly involved in the long-term biological effects of insulin-in particular, in the control of DNA and RNA synthesis.Most of the effects of insulin are observed within a few seconds after the addition of the hormone to cells. As a result, it is accepted that most of insulin's biological effects are generated as a direct result of binding to specific plasma membrane receptors. However, some insulin-responsive processes are not affected for several minutes or hours (for a review, see ref. 1). The mechanisms by which insulin effects these processes are unknown, although several have been postulated (for a review, see ref.2), including intracellular insulin processing (3)(4)(5). Insulin has been shown by both biochemical and ultrastructural techniques to be internalized by numerous cell types (6-18) and to have effects on isolated nuclei (19,20), leading to the speculation that internalization, intracellular translocation, and/or processing may be physiologically significant, especially for the long-term effects of insulin.This laboratory has used high-resolution electron microscopic and biochemical analyses to investigate the binding and subsequent processing ofthe insulin-receptor complex in various cells (11,(21)(22)(23)(24)(25) and has observed cell-specific heterogeneity in receptor organization, distribution, mechanism of internalization, and routes of intracellular processing (for a review, see ref. 26). As part...

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“…Insulin and glucagon were also used, as these peptides are well known to have specific receptors on the liver-cell surface (11,12). We selected somatostatin as a reference peptide, because its specific binding to the liver cell surface has not been detected in in vitro binding experiments.…”

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confidence: 99%

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Sato

Sugiyama

Sawada

et al. 1988

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

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Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of '2MI-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [In (inulin/EGF) versus time] ofthe dilution curve for EGF exhibited an upward straight line initially for a short period of time (-10 sec), whereas the ratio plot [In (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer 30 jag) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kon), the dissociation rate constant of EGF from the EGF-receptor complex (ko)9 and the sequestration rate constant ofthe complex. The kinetic parameters [the dissociation constant (Kd = kl/k,) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones-cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.Many peptide hormones in the circulating blood are eliminated by the liver. Above all, for hormones taken up via receptor-mediated endocytosis, the liver is a major homeostatic regulator in controlling the concentration of peptide hormones in circulating blood (1-3).The interaction between peptide hormones and their specific receptors has been analyzed principally by in vitro studies with isolated or cultured hepatocytes (4) and liver plasma membranes (5). These systems, however, neither maintain cell polarity nor include ligand delivery by the blood flow, thus creating difficulty when the physiological significance of the peptide-receptor interaction is being experimentally evaluated. Use of the liver-perfusion method may effectively resolve this problem, because cell polarity and spatial architecture between hepatocytes and the capillary bed are maintained in this system. However, determination of the microscopic kinetic constan...

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Subcellular distribution of intraportally injected 125I-labeled insulin in rat liver

Cited by 28 publications

References 39 publications

Insulin resistance in uremia. Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats.

Insulin resistance in uremia. Characterization of insulin action, binding, and processing in isolated hepatocytes from chronic uremic rats.

Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process.

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

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