Insulin Binding Kinetics Research Articles

Comparison of metabolic and mitogenic response in vitro of the rapid-acting insulin lispro product SAR342434, and US- and EU-approved Humalog®

SAR342434 is a biosimilar of insulin lispro (Humalog® U-100). Batches of SAR342434 were compared with Humalog® batches of either EU or US origin in a panel of in vitro biological assays that included insulin binding to insulin receptor (IR) isoforms A (IR-A) and B (IR-B) and IR-A/IR-B autophosphorylation. A surface plasmon resonance biosensor-based assay was developed to characterize the kinetics of insulin binding to solubilized full-length IR-A or IR-B. Insulin-dependent metabolic activity assays included inhibition of lipolysis in in vitro differentiated human adipocytes, glucose uptake in L6-myocytes, and repression of glucose-6-phosphatase gene expression in human hepatocytes. Mitogenic activity assays included insulin binding to insulin-like growth factor-1 receptor (IGF1R), IGF1R autophosphorylation, and cell proliferation in MCF-7 cells. Weighted geometric means and their respective 95% confidence intervals (CI) were calculated for all 50% inhibitory or effective concentration values and kinetic binding constants for IR-A and IR-B. Statistical evaluation of the data demonstrated that the 90% CIs of the ratio of geometric means between SAR342434 and Humalog® EU or Humalog® US were within the predefined acceptance limits for each assay. Insulin lispro as SAR342434 solution demonstrated similarity to both US- and EU-approved Humalog® based on a side-by-side biological similarity assessment.

Characterization of insulin-resistance: role of receptor alteration in insulin-dependent diabetes mellitus, essential hypertension and cardiac hypertrophy

Insulin-resistance is associated with a number of disease states such as diabetes, syndrome X, and hypertension. These situations may be coupled to insulin-resistance through the insulin signaling system as a common pathway. The purpose of this study was to investigate the receptor binding alterations in streptozotocin-induced diabetic rats, spontaneously hypertensive rats and aortocaval shunted rats (eccentric cardiac hypertrophy). A physical model describing a 1:1 stoichiometry of ligand binding with its receptor is proposed describing reversible binding of [ 125I]insulin or [ 125I]IGF-1 at the microvascular endothelial as well as with the cardiac myocytes after CHAPS-treatment. Analysis of the collected effluents are curve-fitted with a conservation equation and a first-order Bessel function which allowed the calculation of the forward binding constants ( k n ), the reversible constants ( k − n ), the dissociation constants ( k d) and the residency time constants ( τ). The results showed that streptozotocin-induced diabetic rats showed insulin-resistance through alterations in the kinetics of insulin receptor binding. The normotensive controls of the spontaneously hypertension rats (SHR) carry themselves insulin-resistant receptors whose binding to insulin worsens in the hypertensive SHR. Negative cooperativity between insulin-like growth factor IGF-1 and insulin receptors could be a causative factor predisposing for insulin-resistance in the aortocaval shunted rats to insulin resistance. The defects may be occurring at the receptor level in insulin-dependent diabetes mellitus, Wistar-Kyoto rats and spontaneously hypertensive rats. In conclusion, alterations in the kinetics of insulin binding to its receptor seem to play a central role for the initiation of insulin-resistance during the various pathophysiological states.

Effect of Isoflavone Genistein on Insulin Receptors in Perfused Liver of Ovariectomized Rats

The experiments were carried out on ovariectomized Wistar rats. Their livers were perfused with basic perfusion medium (BPM) or BPM supplemented with isoflavone genistein. insulin or combination of the two factors. The obtained results support the hypothesis that genistein influences the kinetics of insulin binding to cell membranes changing the number of insulin receptors and dissociation constant (Kd) BPM supplementation with genistein decreased number of high affinity insulin receptors (HAIR) both in livers treated and untreated with insulin. The amount of HAIR diminished significantly from 610±77 × 10-15 (no genistein) to 238±72 × 10-13 mol/mg of membrane protein (supplement of genistein). Similarly, genistein reduced slightly the amount of HAIR even when added together with insulin (372±59 × 10-13 mol/mg) in comparison to rats perfused with medium containing insulin but not the isoflavone (421±46 × 10-15 mol/mg). Simultaneously, genistein decreased significantly Kd for HAIR (perfusion with BPM - 1.44±0.18 × 10-9 mol/l; perfusion with BMP + genistein - 0.83±0.20 × 10-9 mol/l). Such effects of genistein during liver perfision did not take place when the liver membranes were in vitro incubated with this xenobiotic.

Insulin-receptor binding characteristics in perfused SHR and WKY rat hearts

This work uses a new heart-perfusion technique to measure 125I-insulin binding on capillary endothelium and myofiber cell membranes in Wistar-Kyoto and spontaneously hypertensive rats. Ringer–Lock buffer was infused at a rate of 1 ml min −1 in the presence of 20 meq l −1 K + and 125I-insulin through an aortic cannula. The effluent was collected through a catheter introduced into the right atrium. The capillary endothelial lining was removed by detergent treatment to expose the cardiac myocyte surfaces. A physical model describing a 1:1 binding stoichiometry of 125I-insulin with its receptors is proposed and the derived mathematical equations allow for the calculation of binding constants ( k n), unbinding constants ( k −n), dissociation constants ( k d), and residency time constants ( τ). The results showed that in the spontaneously hypertensive rats’ hearts significant alterations were not noticed in the kinetics of insulin binding with its receptor at the capillary endothelial site compared to hearts of the normotensive control Wistar-Kyoto rats. However, at the myocyte site and in the spontaneously hypertensive rats, steric, configurational, and/or structural modifications for insulin binding with the receptor were observed as indicated by changes in insulin affinity for its receptor. Hence, alterations in insulin binding rather than reduction in insulin receptor number due to hyperinsulinemia, can be considered among the peculiarities of insulin resistance in the spontaneously hypertensive rats. Hyperinsulinemia, therefore, may be considered an upregulatory process as a consequence of insulin-resistance. The results support the hypothesis that insulin-resistance on the myocytes could be a pathophysiologic defect in insulin-receptor structure, function and affinity, and therefore myocardial function.

Kinetics of insulin binding and kinase activity of the partially purified insulin receptor from human skeletal muscle

The kinetics of insulin binding and kinase activity of soluble, partially purified insulin receptors from human skeletal muscle are considered. An equilibrium for insulin binding was obtained within 2 h at 37°C. At lower temperatures the equilibrium for insulin binding was less clearly defined. Dissociation of 125I-labelled insulin was incomplete unless an excess amount of unlabelled insulin was added. Insulin-stimulatable autophosphorylation of the 95 kDa subunit was verified by gel electrophoresis. The kinase activity was measured with the synthetic polypeptide poly(Glu-Tyr(4:1)) as a phosphoacceptor. The insulin receptor kinase activity correlated significantly ( r = 0.92, P < 0.0001) to the concentration of high-affinity insulin binding sites in the eluate. Autophosphorylation of the insulin receptor was necessary for the activation of the receptor kinase. When activated the receptor kinase activity was stable for at least 60 min at 21°C with a pH optimum of approx. 7.8, similar to the pH optimum for insulin binding. The non-ionic detergent Triton X-100 inhibited the sensitivity of the receptor kinase to insulin. Insulin stimulated the V max of the kinase reaction about 3-fold, decreased the K m for ATP from 35 ± 5 μM (mean ± S.E.) to 8 ± 1 μM ( P < 0.02) and induced a positive cooperativity to ATP with an increase in the Hill coefficient from 1.00 ± 0.02 to 1.37 ± 0.07 ( P < 0.05). According to the Hill plots, insulin itself showed no cooperativity with respect to receptor binding or kinase activation.

Binding kinetics of mutated insulin receptors in transfected cells grown in suspension culture: Application to the Tyr → Phe 960 insulin receptor mutant

Site-directed mutagenesis of the insulin receptor cDNA is now widely used to elucidate the role of various domains and residues of the receptor, particularly in order to examine the functional importance of the β chain - associated tyrosine kinase. However, little has been done to correlate the functional repercussions of such mutations with alterations in the complex insulin binding kinetics. This is due in part to the difficulty of conducting large scale experiments using transfected cells on culture dishes. In an effort to overcome this problem, we have developed a method for culturing Chinese hamster ovary (CHO) cells in suspension culture, which provides a large number of cells and obviates the need for enzymatic or mechanical detachment of cells. The feasibility of this approach is demonstrated in a detailed study of the kinetics of insulin binding to the Tyr → Phe 960 insulin receptor mutant.

Hepatic 123I-insulin binding kinetics in non-insulin-dependent (type 2) diabetic patients after i.v. bolus administration.

Insulin binding kinetics in the liver were studied in non insulin dependent (Type 2) diabetic patients, by i.v. bolus administration of 123I-insulin. Eight Type 2 diabetic patients were compared with six male volunteers. Uptake of 123I-insulin by liver and kidneys was measured by dynamic scintigraphy with a gamma camera during 30 min. Images of liver and kidneys appeared within 2-3 min after administration of 123I-insulin at a dose of 1 mCi (37 MBq). Peak radioactivity for the liver was found 7.5 +/- 0.2 and 6.9 +/- 0.3 min after injection for the healthy and the diabetic subjects, respectively (N.S.). The percentage 123I-insulin hepatic uptake was not significantly different for the diabetic and the healthy subjects. Although a large variation exists for maximal uptake of radioactivity within both groups, the data suggest that binding differences in the liver in Type 2 diabetic patients, as compared to healthy subjects, may not account for hepatic insulin resistance.

Characterization of insulin binding in the UMR-106 rat osteoblastic osteosarcoma cell.

The correlation of insulin receptor occupancy with classic insulin effects, such as stimulation of glucose uptake, have not been examined in osteoblastlike cells. Accordingly, we characterized insulin binding and examined its relationship to stimulation of glucose analog transport in the UMR-106 rat osteoblastic osteosarcoma cell line. Insulin binding in UMR-106 cells was found to be pH sensitive, temperature dependent, saturable, and specific. Proinsulin was 100-fold less effective than insulin in displacing specific [125I]insulin binding in these cells, whereas IGF-I at concentrations between 0.1 and 10 nM produced no displacement of [125I]insulin but did produce significant displacement of insulin binding at 100 and 1000 nM. Insulin receptor downregulation was observed after exposure to 100 nM insulin for 6 h at 37 degrees C and was temperature dependent. Insulin binding was reversible after 24 h at 4 degrees C. Insulin binding correlated directly with stimulation of 2-deoxyglucose uptake at insulin concentrations between 0.1 and 100 nM, with a half-maximal concentration (ED50) of 0.9 nM for both [125I]insulin binding displacement and stimulation of 2-deoxyglucose uptake. Hence, there was no evidence for spare insulin receptors with regard to stimulation of glucose analog transport. Scatchard analysis of insulin binding kinetics yielded a curvilinear plot, suggesting negative cooperativity. Analysis of insulin binding kinetics using a two-site model yielded a KD of 0.9 nM for the apparent high-affinity binding site and an estimated 80,000 high-affinity binding sites per cell. These findings demonstrate that osteoblastlike cells exhibit a relationship between insulin binding and glucose transport stimulation that is similar to that in liver cells and other insulin-sensitive tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

A short incubation time in insulin binding experiments leads to disappearance of negative cooperativity in H35 hepatoma cells

The effects of different periods of incubation (8 min vs 20 min) on insulin binding kinetics were examined in a H35 hepatoma cell line. Scatchard plots from cells incubated for 8 min were linear (r = 0.987 +/- 0.006), in contrast to curvilinear Scatchard plots from cells incubated for 20 min. Hill plots showed a slope of 1.006 +/- 0.024 for the 8 min incubation, whereas the slope was 0.827 +/- 0.0026 (p less than 0.0005) for the 20 min incubation. TCA precipitation of the medium showed minimal insulin degradation products at 8 min with a significant increase at 20 min (1.38 +/- 0.11% vs. 3.06 +/- 0.37%, p less than 0.0005). Internalized insulin was also significantly increased at 20 min as compared to 8 min incubation (48.9 +/- 5.6% vs. 32.4 +/- 3.0%, p less than 0.0005) These data indicate that after 8 min of incubation no appreciable cooperativity of insulin binding was present, while negative cooperativity was present after 20 min of incubation. As significantly more insulin degradation has taken place after prolonged incubation these data support the hypothesis that insulin degradation leads to negative cooperativity of insulin receptors.

Kinetics of insulin binding to rat white fat cells at 15 degrees C.

The kinetics of insulin binding to isolated rat epididymal fat cells was investigated at 15 degrees C, at which temperature the system was simplified by the absence of lysosomal insulin degradation. The data were fit by maximum likelihood criteria with differential equations describing a number of models for the interaction of insulin and cells. Among those models that yielded a fit, the selection criteria were minimization of the Akaike information criterion and compatibility of the overall equilibrium constant for the system calculated from rate constants with the previously obtained experimental value. The results of the analysis indicated that insulin, I, first reversibly bound to cell surface receptors, R, whereupon this initial insulin-receptor complex, RI, reversibly altered its state or cellular location to R'I, according to the following equation. (Formula: see text) No evidence was found that insulin could either associate or dissociate from R'I directly. The association rate constant was kappa 12 = 1.6 x/divided by 1.4 X 10(5) liter mol-1 s-1, a value shown to be incompatible with diffusion control. The other rate constants were: kappa 21 = 3.4 x/divided by 1.6 X 10(-3) s-1, kappa 23 = 3.2 x/divided by 1.5 X 10(-4) s-1, and kappa 32 = 2.0 x/divided by 1.5 X 10(-4) s-1. From these rate constants, an equilibrium constant of 8.4 x/divided by 1.5 nM was calculated, in excellent agreement with the previously measured value of 8.8 x/divided by 1.3 nM (Lipkin, E. W., Teller, D. C., and de Haën, C. (1986) J. Biol. Chem. 260, 1694-1701). The kinetic analysis also yielded receptor numbers similar to those obtained by equilibrium binding studies. The nature of the R'I state is discussed in terms of an internalized state, in terms of insulin receptor complex in caveolae, in terms of receptor aggregates, and in terms of being a Michaelis complex between insulin bound to the receptor and cell surface-bound insulin protease.

Insulin receptors: binding kinetics and structure-function relationship of insulin.

During the last decade, earlier suggestions that insulin acts at the plasma membrane level via combination with receptors have been amply confirmed in studies of 125I-labeled insulin binding kinetics. Efforts have been devoted to the development of homogeneous, stable, and bioactive tracers, and a preparation of monoiodo[TyrA14]insulin showed 100-125% biological activity. The initially simple model of reversible, bimolecular, and noncooperative interaction between receptor and insulin has been revised to include the existence of at least three affinity states that may be linked to modulation of the biological response induced by the insulin-receptor complex. Thus negative cooperativity seems important in reducing oscillations of insulin action with variations in plasma insulin concentration, and formation of a high-affinity state or positive cooperativity may lead to desensitization of receptors. The kinetic phenomena suggest that receptor-binding affinity and function are actively regulated by insulin itself. At present the receptor model is purely functional and does not imply molecular mechanisms. However, recent advances in the analysis of receptor structure and biochemistry promise that the molecular equivalents of the kinetic phenomena may be elucidated in the near future. Furthermore the reaction between receptor and insulin is irreversible because of degradation of receptor-bound insulin, which may result in termination of the metabolic activation. Morphological and biochemical work suggests that internalization of the receptor-insulin complex from the plasma membrane transfers insulin to intracellular organelles like the lysosomes, the Golgi apparatus, or nucleus, where degradation by insulin protease takes place, whereas the receptor is recycled back to the membrane. Recent advances in the studies of biosynthesis and cellular dynamics of receptors indicate that intracellular processing and redistribution of binding sites may play a role in the mechanism of insulin action. Insulin receptors are widely distributed in all cell types, but evidence has accumulated that receptors show tissue and species variations in their functional properties regarding binding affinity, insulin specificity, cooperativity, and insulin degradation and in structural properties such as antigenic determinants and glycosidic composition. Perhaps these differences reflect cellular adaptations and variations in the physiological role of insulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of RNA synthesis in isolated nuclei by an insulin-induced factor in liver.

Insulin stimulates the transport of small molecules as well as the synthesis of triglyceride, glycogen, DNA, RNA and protein. Although it is widely accepted that its differing actions are consequent to the binding to surface receptors, there is suggestive evidence that it may also act intracellularly. In 1975, we reported two well defined intracellular insulin binding sites in rat liver: the nucleus and the endoplasmic reticulum1. Specific binding sites for insulin on intact nuclei were shown by an immunofluorescent and autoradiographic technique, and the kinetics of insulin binding to isolated nuclear membranes were described2,3. Similar results have been obtained by other investigators4–6, and the more recent reports on the internalization of insulin7–9 give further support for an intracellular site of action of intact insulin or of its metabolic product. Investigation of the stimulatory effect of insulin on the synthesis of DNA and RNA in diabetic rats and isolated hepatocytes10–13 suggests that the stimulation of RNA synthesis precedes that of DNA, and is likely to be an early event in the stimulation of macromolecular synthesis. I report here evidence of a low molecular weight substance in rat liver perfused with insulin which can stimulate the synthesis of RNA in isolated nuclei. These results suggest that insulin may function by binding to the cell surface, initiating or enhancing endocytosis14 and entering the cell via a micropinocytotic route9. Once inside the cell, the active principle could be insulin itself, an intracellular degradation product of insulin, or a metabolic product of insulin action.

Insulin receptors in transformed fibroblasts and in adipocytes: a comparative study

This study compares the receptor for insulin in rat adipocytes with an insulin receptor from transformed AL/N mouse embryo fibroblasts (SVS AL/N). In many respects the receptors from the two tissue sources are very similar. Crude solubilized receptor preparations from both fat cells and SVS AL/N cells exhibit complicated insulin-binding kinetics (two binding plateaus) as well as an apparent chromatographic heterogeneity (Sepharose 6B); each receptor preparation yields two insulin-binding components having apparent Stokes radii of about 7.2 and 3.8 nm. In contrast, the receptors isolated from both cell types by affinity chromatography behave as single chromatographic components (Sepharose 6B) having apparent stokes radii of 3.8 nm and exhibit simple insulin-binding kinetics without evidence for receptor–ligand co-operativity. The isolated receptor from SVS AL/N cells (KD about 1.2 nM) possesses a slightly lower insulin affinity than does the receptor from fat cells (KD about 0.8 nM); nonetheless, the SVS AL/N receptor exhibits an appropriate ligand specificity for insulin and insulin analogues. Receptors from both cell types are similarly sensitive to trypsin and exhibit an identical dependence on pH for insulin binding. Differences in the two receptor preparations are observed in the effect of NaCl and phospholipase C, reagents which augment insulin binding in adipocyte membranes but do not affect the binding by SVS AL/N cell membranes. Additionally, iodoacetamide, N-ethylmaleimide, and ethanol markedly reduce the binding by SVS AL/N membranes, whereas the binding by adipocyte membranes is unaffected. It is concluded that certain differences between the receptors from the two sources may be attributed to differences in the membrane environment in which the receptors reside. However, in view of the many similarities between the two receptor preparations, it is suggested that the same receptor macromolecule may participate in the response of a variety of cell types not only to insulin but also to other insulin-like polypeptides.

Initial lag-phase in the action of insulin on glucose transport and cAMP levels in fat cells

Whereas insulin binding to plasma membrane receptors is a well-studied phenomenon, comparatively little is known about the transmission of the hormone signal from the receptor to enzyme systems associated with the plasma membrane. The isolated fat cell lends itself well as a model to the assessment of insulin action upon two major membrane functions, glucose transport and control of CAMP levels. It is not clear whether the transmission of the hormone signal occurs through a common initial mechanism or by direct coupling of hormone-receptor complexes to enzyme systems. Comparison of doseresponse curves of glucose oxidation and antilipolysis [ 1] suggests different mechanisms of action. Here, the initial kinetics of insulin binding are compared with membrane functions between 5 s and 4 min after the first contact of the hormone.

Characteristics of the solubilized insulin receptor of human placenta

A soluble human insulin receptor can be obtained in high yield from placenta membranes, using the detergent Ammonyx-LO. In crude soluble preparations, the placenta receptor exhibits complex insulin binding kinetics (two binding plateaus, half-saturated at about 40 pM and 700 pM insulin) and an apparent chromatographic heterogeneity (Sepharose 6B) with two insulin binding components having apparent Stokes radii of 72Å and 38Å. However, subsequent to purification by affinity chromatography on insulin-Sepharose, the placenta receptor exhibits a simple insulin binding isotherm, without evidence for binding cooperativity (K D about 830 pM), and upon chromatography behaves as a single component with a Stokes radius of 38Å A. When combined with a previously described non-receptor glyco-protein preparation isolated from liver membranes, the affinity-purified plancenta receptor undergoes an increase in its Stokes radius from 38Å to 72Å. Because of the physicochemical similarities between the placenta receptor and the insulin receptor previously isolated from liver cell membranes, it is concluded that the placenta receptor is representative of the insulin receptor present in other traditional target tissues for insulin. The study underscores a possible role for insulin in placental physiology and provides for the large scale isolation of the human receptor from a readily available source.