125I-labeled Insulin Research Articles

REgulation of insulin binding to isolated hepatocytes: correction for bound hormone fragments linearizes Scatchard plots.

Fragments of 125I-labeled insulin (125I-insulin) are rapidly produced after the initial cell binding process. After association of 125I-insulin with hepatocytes, hormone fragments remain bound to cells. At 23 degrees C, approximately 20% of the label bound at steady state was soluble in trichloroacetic acid. Correction of saturation experiments for the presence of bound trichloroacetic acid-soluble insulin fragments decreased the number and increased the affinity of 125I-insulin-binding sites. Label extracted from cell pellets recovered from saturation experiments was characterized by gel filtration; 59%, 55%, 40%, and 36% of the bound label was from intact hormone after recovery from incubation mixtures containing 0.18, 0.60, 4.6, and 7.5 nM applied 125I-insulin, respectively. At high applied 125I-insulin concentrations, the hormone predominantly interacted with lower affinity degradation systems. When binding data were corrected to assay for undegraded 125I-insulin only, curvilinear Scatchard plots were linearized. The insulin receptor is therefore not composed of heterogeneous or negatively cooperative sites. It is necessary to correct for retained fragments of 125I-insulin in order to define mechanisms through which hormone binding and cellular response may be regulated.

Degradation of 125I-labelled insulin by isolated rat hepatocytes [proceedings

Degradation of 125I-labelled insulin by isolated rat hepatocytes [proceedings

Characteristics of insulin receptors in the heart muscle Binding of insulin to isolated muscle cells from adult rat heart

Adult rat heart muscle cells obtained by perfusion of the heart with collagenase have been used to characterize the insulin receptors by equilibrium binding and kinetic measurements. Binding of 125I-labelled insulin to heart cells exhibited a high degree of specificity; it was dependent on pH and temperature, binding at steady increased with decreasing temperatures. About 70% of the radioactivity bound at equilibrium at 25°C could be dissociated by addition of an excess of unlabelled insulin. 54 and 40% of 125I-labelled insulin was degraded by isolated heart cells after 2 h at 37°C and 4 h at 25°C, respectively. This degrading activity was effectively inhibited by high concentration of albumin. Equilibrium binding studies were conducted at 25°C using insulin concentrations ranging from 2.5 · 10 −11 mol/l to 10 −6 mol/l. Scatchard analysis of the binding data resulted in a curvilinear plot (concave upward), which was further analyzed using the average affinity profile. The empty site affinity constant was calculated to be 9.5 · 10 7 l/mol with a total receptor concentration of 3.4 · 10 6 sites per cell. The presence of site-site interactions of the negative cooperative type among the insulin receptors has been confirmed by kinetic experiments. The rate of dilution induced dissociation was enhanced in the presence of native insulin (5 · 10 −9 mol/l), both, under conditions of low and high fractional saturation of receptors.

Prolonged effect of insulin on glucose uptake by rat skeletal muscle.

The fate of insulin, as it relates to its action on skeletal-muscle glucose uptake, was studied in non-cyclically perfused rat hindlimbs. Insulin (1m-i.u./ml) with and without (125)I-labelled insulin was infused intra-arterially for 5 or 6min. Net glucose uptake and the release of (125)I-labelled insulin into the venous effluent were evaluated by arteriovenous-difference measurements for an additional 24-32min. The infusion of insulin for 5min promoted glucose uptake, an effect that persisted throughout a subsequent 25min of perfusion in the absence of insulin. The addition of insulin antibody to the perfusate in the presence of insulin blocked the action of insulin on glucose uptake, but it failed to alter insulin action if the muscle tissue had been exposed to insulin before addition of antibody. When (125)I-labelled albumin was infused for 6min, venous effluent radioactivity decayed rapidly and remained HClO(4)-insoluble and there was no significant tissue retension of radioactivity. Comparable experiments in which (125)I-labelled insulin was infused for 6min revealed that the venous effluent radioactivity decayed more slowly, a significant amount of the (125)I-labelled insulin appeared as fragments (HClO(4)-soluble) and there was a significant retention of radioactivity in the tissue. Radioactivity in muscle tissue biopsies obtained 28min after infusion of (125)I-labelled insulin was associated largely with intact insulin and a peptide of mol.wt. 2400. The total radioactivity retained in the muscle at this time was 7% of the amount infused. An insulin bolus (1i.u.) failed to increase the discharge of this tissue-associated radioactivity. These results suggest that insulin and a product of insulin metabolism persists in muscle tissue long after the arterial presence of insulin ends. This tissue residence and processing of insulin may be important components of insulin's prolonged action on glucose uptake by skeletal muscle.

Effect of vinblastine on the insulin-receptor interaction in mammalian heart muscle

Isolated muscle cells from adult rat heart were used to investigate the effect of microtubule modifiers on the insulin-receptor interaction in mammalian heart muscle. Preincubation of freshly isolated cells with increasing concentrations of vinblastine resulted in a dose-related inhibition of specific binding of 125I-labelled insulin, whereas colchicine did not affect the binding reaction. Analysis of equilibrium binding data demonstrated a reduction of receptor number after treatment of cells with vinblastine. The dissociable fraction of 125I-labelled insulin specifically bound at equilibrium was significantly reduced by preincubation with vinblastine. The results suggest involvement of microtubules in the insulin-receptor interaction in cardiac muscle.

Insulin degradation by adipose tissue. Studies at several levels of cellular organization.

A systematic study of the degradation of physiological concentrations of 125I-labelled insulin was performed in intact fat-pads, isolated adipocytes and subcellular fractions of isolated adipocytes. The findings indicate that insulin is rapidly degraded to low-molecular-weight peptides and/or amino acids by the intact tissue and isolated cells. Of the total insulin-degradation products present after incubation with an intact fat-pad, 94% is recovered in the medium, indicating that these products are not retained by the cells or tissue. The plasma membranes do not degrade insulin significantly in the absence of reduced glutathione, and over 99% of the cellular degradative capacity is found in the postmicrosomal supernatant (cytosol). The cytosol degrades insulin to several labelled fragments that are intermediate in size between insulin and insulin A chain, as well as to the low-molecular-weight tissue degradation products. Inclusion of plasma membranes with cytosol accelerates the cleavage of the intermediate fragments to the size of the small products seen with the intact tissue. However, plasma membranes do not increase the initial step in the degradation of insulin when incubated with cytosol, suggesting that the insulin receptor is not involved with the direct cleavage of insulin. This study supports the hypothesis that the bulk of insulin degradation occurs in the adipocyte cytosol, where intermediate-sized fragments are generated and rapidly cleaved to smaller products by the plasma membrane and quickly released into the surrounding medium.

Changes in the number of insulin receptors of isolated rat hepatocytes during pregnancy and lactation

The weight of the rat liver increased during pregnancy and lactation due solely to an increase in hepatocyte size. Insulin receptors were identified using 125I-labelled insulin and isolated hepatocytes in vitro. Scatchard analysis was interpreted in terms of high affinity ( K D 4 nM) and low affinity ( K D 80 nM ) receptors for insulin. No change in the affinity of either receptor site was detected during pregnancy or lactation. There was, however, a significant increase in the number of both types of receptor on the hepatocyte by day 12 of pregnancy which was maintained until at least day 20 of pregnancy. During lactation, the number of receptors declined to values similar to those of virgin rats. Serum insulin concentrations, determined by radioimmunoassay, were elevated during pregnancy, returned to valued similar to virgin rats during early lactation and were significantly reduced compared with virgin rats by day 15 of lactation. These results illustrate that physiological conditions exist whereby the number of insulin receptors may increase, despite elevated serum insulin concentrations, in apparent conflict with the ‘down-receptor’ hypothesis.

The binding and degradation of insulin by rat liver membranes: demonstration of three distinct insulin-binding components in detergent-solubilized material from liver plasma membrane.

The components responsible for insulin-binding and insulin degradation were solubilized from purified liver plasma membranes by treatment with the nonionic detergent Triton X-100. The characteristics of specific insulin binding and insulin degradation were similar to those found with the intact membranes. Polyacrylamide gel electrophoresis of the detergent extract separated the insulin-binding activity into three peaks, designated peaks I, II, and D. The majority of the insulindegrading activity was located in peak D; peak I failed to degrade insulin, while only a small amount of degradation was associated with peak II. Scatchard analysis of the insulin-binding data for peak I showed a curvilinear plot, with the initial high affinity portion of the curve having a Kd of 2 × 10−10 m. The Scatchard plot for peak II was linear, with a Kd of 8 × 10−9 m. Both peaks bound 125I-labeled insulin with high specificity, as determined by competition studies using desoctapeptide insulin, proinsulin, and glucagon. Insul...

Isolation of material displaying insulin-like immunological biological activity from the brain of the blowfly Calliphora vomitoria.

An insulin-like material from the brain of the blowfly Calliphora vomitoria was partially purified by acid alcohol extraction, gel filtration and ion-exchange cellulose chromatography. In addition, the RF value on polyacrylamide-gel electrophoresis was determined. The material was characterized by its ability to cross-react with bovine insulin antibody and by displaying diminished immunoreactivity on dilution. It displaced specifically bound 125I-labelled insulin from rat liver plasma membrane insulin receptors and displayed insulin-like biological activity on the isolated rat fat-cell. Within 30 min of injection into Calliphora, made hypertrehalocaemic and hyperglucaemic as a result of median neurosecretory cell removal, it caused the concentrations of both sugars to return to normal. The hypothesis is put forward that the median neurosecretory cells are the source of the material.

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

Time course studies revealed that at 30 s after intraportal injection of 200 μU of 125I-labeled insulin per 100 g rat 47.9 ± 2.8% of the injected radioactivity was recovered from the liver homogenate by precipitation with trichloroacetic acid. Trichloroacetic acid precipitable radioactivity declined to very low levels during the next 30 min whereas trichloroacetic acid soluble radioactivity reached a peak value of 9.56 ± 1.9% at 5 min and declined gradually thereafter. At 30 s mean peak accumulations ±SE of 6.83 ± 0.42, 5.06 ± 0.27, 14.90 ± 1.85, and 3.58 ± 0.58% of injected radioactivity were recovered in trichloroacetic acid precipitates from the 700 g (nuclei + debris), 10,000 g (mitochondria + lysosome), 105,000 g (microsomes), and supernatant (cytosol) subfractions, respectively. Mean peak values of 0.72 ± 0.08, 0.12 ± 0.02, and 1.11 ± 0.16% of injected radioactivity were recovered in the partially purified mitochondrial fraction, purified nuclei, and plasma membranes, respectively, as trichloroacetic acid precipitable material. Most of the trichloroacetic acid precipitable activities in the subfractions were immunoprecipitable. Trichloroacetic acid soluble radioactivity was found mainly in the cytosol and microsomal fractions. Peak specific activity (percentage of injected dose/mg protein × 10 −3) was highest in the microsomes, intermediate in the plasma membranes, and very low in the purified nuclei and partially purified mitochondrial fraction. The specific activity of the microsomes remained at or near peak levels for 5 min after 125I-labeled insulin injection and then declined, whereas specific activity of the plasma membranes dropped precipitously to 25% of peak values at 5 min. Sephadex gel filtration of the radioactivity in the deoxycholate soluble fraction of microsomes at 5 min after 125I-labeled insulin injection resulted in the elution of a major peak (Peak I) in the region of 125I-labeled insulin and a minor peak (Peak II) in the region of the labeled A and B chains. Incubation of the fraction for 30 min at 37 °C with 3 m m reduced glutathione and 15 m m EDTA resulted in a reciprocal fall in Peak I and rise in Peak II. The data suggest that intraportally injected 125I-labeled insulin is rapidly internalized and concentrated in the rat liver microsomes. The time courses of appearance and disappearance of trichloroacetic acid precipitable radioactivity in plasma membrane and microsomes further suggest, although do not prove, that insulin binds to plasma membranes before it is internalized. They also provide presumptive evidence suggesting that the sequential degradative pathway is operative in vivo.

Intracellular localization of 125 I-labeled insulin in hepatocytes from intact rat liver

Intracellular localization of ¹²⁵ I-labeled insulin in hepatocytes from intact rat liver

Distribution of insulin receptors among mouse liver endomembranes

Specific binding of insulin to highly purified preparations of rough endoplasmic reticulum, Golgi apparatus, and plasma membrane of mouse liver was determined. 125I-labeled insulin bound maximally to the plasma membrane in radio-receptor assays. Golgi apparatus fractions exhibited binding 10–20% that of plasma membrane and rough endoplasmic reticulum exhibited only 1–2% of plasma membrane binding. Binding was proportional to membrane concentration and dose vs. response curves were very similar for the different fractions. Scatchard analysis of the insulin binding data for the plasma membrane and Golgi apparatus fractions showed curvilinear plots yielding similar apparent binding affinities (0.9 and 3.0 · 10 8 M −1, respectively). Purity of the isolated endomembranes was analyzed by morphometry and (Na + + K + + Mg 2+)-ATPase and these preparations displayed less than 1% contamination by plasma membrane. These findings provide important confirmation of the presence of insulin receptors in Golgi apparatus membranes comparable to those located on the plasma membrane. Finally, the present study did not allow us to verify the existence of insulin receptors in the endoplasmic reticulum.

The Handling of Intravenously Injected 125I-Labelled Insulin by Rat Liver

The Handling of Intravenously Injected 125I-Labelled Insulin by Rat Liver

Effect of insulin on glucose oxidation and amino isobutyric acid transport and binding of insulin in chicken thymocytes

In chicken thymocytes isolated from 15–40 day-old chickens, after a 2 h incubation at 37°C, insulin stimulated amino isobutyric acid uptake (maximal response: 40–50% of increase at 1 μg insulin/ml and half maximal response at 60 ng/ml) by specifically stimulating the influx without altering the efflux. Insulin also stimulated glucose oxidation (maximal response: 11% of increase at 1 μg insulin/ml). Binding of 125I-labelled chicken insulin to thymocytes was rapid and higher at 15°C than at 37°C. At steady state, (90 min at 15°C), chicken, porcine and goose insulins were equipotent in inhibiting the binding of 125I-labelled chicken insulin. Maximal binding capacity was estimated at 1250 pg insulin/10 8 cells, i.e., 1250 binding sites/cell with an apparent dissociation constant of 200 ng insulin/ml at 15°C. Degradation of 125I-labelled chicken insulin in the incubation medium was negligible at 15°C but very noticeable at 37°C. Therefore, the low level of insulin binding at 15°C reflects a true scarcity of insulin receptors in chicken thymocytes as compared to rat thymocytes.

Evidence for lysosomal reduction of cystine residues

Previously reported evidence for the existence of a thiol: protein disulphide oxidoreductase in rat liver lysosomes has been re-examined and ambiguous results obtained. However, incubation of purified rat liver lysosomes with 125I-labelled insulin at pH 5.5 shows that cathepsin D and a thiol-dependent enzyme other than cathepsin B or L are important in its digestion. The latter enzyme is most probably a thiol: protein disulphide oxidoreductase.

Intracellular localization of 125I-labeled insulin in hepatocytes from intact rat liver.

We have shown that 125I-labeled insulin initially localizes to the plasma membrane of isolated rat hepatocytes. The ligand is subsequently internalized and preferentially localizes to lysosomal structures. Further, we have observed that labeled insulin localizes to regions of the cell rich in lysosomal and Golgi elements. In the present study in intact rat liver we found that, approximately 10 min after a pulse injection of 125I-labeled insulin, 56% of the label was internalized by the cell. When all grains are considered there is a preferential localization of grains to the biliary pole of the cell and these grains are almost all internalized and preferentially associated with lysosomes. These data, therefore, demonstrate that the lysosome-Golgi-rich area of the isolated hepatocyte corresponds to the biliary pole of the cell and there is a movement of the labeled hormone from its initial binding site on the plasma face of the cell membrane toward the biliary pole of the cell.

Mode of uptake and degradation of 125I-labelled insulin by isolated hepatocytes and H4 hepatoma cells.

The effects of various agents on the binding and degradation of 125I-labelled insulin by isolated rat hepatocytes and cultured H4 hepatoma cells were studied. Various lysosomotropic agents, including chloroquine, ammonium chloride, and the topical anesthetics, lidocaine and procaine inhibited insulin degradation by H4 hepatoma cells but had little effect on the binding of the hormone. Similarly, tosyl-L-lysyl chloromethyl ketone selectively inhibited the degradation of 125I-labelled insulin by isolated hepatocytes, as did the sulfhydryl reagents, p-hydroxy- and p-chloromercuriphenyl sulfonic acid. Inhibitors of energy production, including sodium fluoride, sodium azide, and dinitrophenol, also selectively inhibited the degradation of insulin by hepatocytes, although cyanide had no effect under the conditions used. Lectins and antimicrotubular agents, which are known to affect the mobility of plasma membrane proteins or of intracytoplasmic vesicles, selectively inhibited insulin degradation by hepatocytes to varying degrees, whereas agents which inhibit the function of microfilaments had no effect. At temperatures below 20 degrees C, insulin degradation was negligible but rose rapidly between 20 and 37 degrees C, suggesting that a membrane-related step is rate limiting in the overall degradative process. These results are all consistent with a model of insulin uptake by target tissue involving pinocytosis of receptor-bound hormone followed by intralysosomal degradation.

Modulation of binding and bioactivity of insulin by anti-insulin antibody: relation to possible role of receptor self-aggregation in hormone action.

Incubation of physiological concentrations of 125I-labeled insulin with liver membranes in the presence of anti-insulin IgG results in a 7- to 15-fold increase in the specific binding of the hormone. The low-affinity/high-capacity binding sites are replaced by an apparently homogeneous class of high-affinity sites, and the nonlinear Scatchard plots are converted to linear plots without a change in the maximum number of binding sites. Similarly, the binding of insulin to receptors in 3T3 fibroblasts is increased substantially in the presence of anti-insulin antibody, and the biological activity of subactive concentrations of insulin is enhanced by antibody in these cells. However, the affinity of 125I-labeled epidermal growth factors (EGF) in fibroblasts is not affected by anti-EGF IgG. In adipocytes anti-insulin IgG in the same concentration range only inhibits the binding of insulin and suppresses insulin-mediated glucose oxidation. Monovalent Fab' fragments from anti-insulin IgG inhibit the binding of the hormone, indicating that the enhancement of binding in liver membranes and fibroblasts requires the bivalency of the antibody.

Insulin action in isolated fat cells: II. Effects of divalent cations on stimulation by insulin of protein synthesis, on inhibition of lipolysis by insulin, and on the binding of 125I-labelled insulin to isolated fat cells

The effects of omission of Ca 2+ and Mg 2+ from the incubation medium on three aspects of insulin action in isolated fat cells have been investigated. In the (Ca 2+ + Mg 2+)-free incubation medium incorporation of L-[ 14C]leucine into fat cell protein was reduced in the absence of insulin. Insulin stimulated L-[ 14C]-leucine incorporation only in the presence of added CaCl 2 or MgCl 2. Incubation of the cells in the (Ca 2+ + Mg 2+)-free medium reduced but did not abolish the ability of adrenaline to stimulate lipolysis or the ability of insulin to inhibit the adrenaline-stimulated lipolysis. Specific binding of 125I-labelled insulin to the fat cells was reduced in the absence of Ca 2+ and Mg 2+ but was not abolished, even in the presence of EDTA. Ca 2+ was routinely the most effective divalent cation in supporting these aspects of insulin action, but similar responses were obtained with Mg 2+, Sr 2+ and Ba 2+. Since insulin still binds to the cells under conditions in which some of the cellular effects of the hormone are abolished, it is suggested that divalent cations may have a role, either direct or indirect, in the processes linking the insulin-insulin receptor complex to certain effector systems in the cells. It is tentatively suggested that this action occurs at the level of the fat cell plasma membrane.

Effects of insulin on insulin-binding components extracted from rat fat cell membranes.

WE have previously shown the presence of two distinct insulin-binding species (designated species I and II) in a Triton X-100 extract of fat cell membranes1. They were separable electrophoretically and displayed different insulin-binding characteristics. Scatchard analysis of the insulin-binding data for species I indicated a complex, non-classical association, illustrated by a curvilinear plot. The characteristics of this curve are quite similar to those of the binding curves obtained with unfractionated detergent extract or with intact plasma membranes1. In contrast to these findings, insulin-binding to species II was characterised by a relatively low affinity and a linear Scatchard plot. Although these findings indicate different insulin-binding and electrophoretic properties for the two species, the possibility remains that they are related structurally. For example, the Kd values for the low-affinity portion of the peak I Scatchard plot and for insulin binding to peak II are similar. Furthermore, following the incubation of isolated peak I with 125I-labelled insulin and subsequent electrophoresis, a small amount of radioactivity migrated in the position of peak II (ref. 1). We report here that interconversion occurs between the two binding species and that the conversion is mediated by insulin.