Structural Investigations of Full-Length Insulin Receptor Dynamics and Signalling

Insulin regulates glucose homeostasis via binding and activation of the insulin receptor dimer at two distinct pairs of binding sites 1 and 2. Here, we present cryo-EM studies of full-length human insulin receptor (hIR) in an active state obtained at non-saturating, physiologically relevant insulin conditions. Insulin binds asymmetrically to the receptor under these conditions, occupying up to three of the four possible binding sites. Deletion analysis of the receptor together with site specific peptides and insulin analogs used in binding studies show that both sites 1 and 2 are required for high insulin affinity. We identify a homotypic interaction of the fibronectin type III domain (FnIII-3) of IR resulting in tight interaction of membrane proximal domains of the active, asymmetric receptor dimer. Our results show how insulin binding at two distinct types of sites disrupts the autoinhibited apo-IR dimer and stabilizes the active dimer. We propose an insulin binding and activation mechanism, which is sequential, exhibits negative cooperativity, and is based on asymmetry at physiological insulin concentrations with one to three insulin molecules activating IR.     

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Blood glucose lowering assay proved that [B16Ala]insulin and [B26Ala]insulin exhibit potency of acute blood glucose lowering in normal pigs, which demonstrates that they are fast- acting insulin. Single-chain precursor of [B16Ala]insulin and [B26Ala]insulin is [B16Ala]PIP and [B26Ala]PIP, respectively, which are suitable for gene expression. Secretory expression level of the precursors in methylotrophic yeast Pichia pastoris was quite high, 650 mg/L and 130 mg/L, respectively. In vivo biological assay showed that the two fast-acting insulins have full or nearly full biological activity. So both [B16Ala]insulin and [B26Ala]insulin can be well developed as fast-acting insulin for clinic use.     

胰岛素蛋白质工程研究进展

近年来,胰岛素蛋白质工程研究进展很快,主要介绍具有临床意义的速效长效和高效胰岛素研究概况,包括分子设计基础,生物活性和应用前景等.此外,还讨论了胰岛素的受体结合部位及胰岛素与其受体相互作用的研究近况．     

Protein engineering of insulin: Two novel fast-acting insulins [B16Ala]insulin and [B26Ala]insulin

Insulin has been successfully used in clinic treatment of diabetes for more than 80 years. However, the clinic practice has shown that regular insulin preparation used in clinic exhibits several intrinsic problems that have existed for a long time. One of the major problems is that insulin has a potency of self-association when its concentration is higher than physiological concentration (10-8—10-10 mol/L)[1,2]. The concentration of the regular insulin is higher than 10-4 mol/L. At such a hi…     

Overexpression of membrane glycoprotein PC-1 can influence insulin action at a post-receptor site

An elevated content of membrane glycoprotein PC-1 has been observed in cells and tissues of insulin resistant patients. In addition, in vitro overexpression of PC-1 in cultured cells induces insulin resistance associated with diminished insulin receptor tyrosine kinase activity. We now find that PC-1 overexpression also influences insulin receptor signaling at a step downstream of insulin receptor tyrosine kinase, independent of insulin receptor tyrosine kinase. In the present studies, we employed Chinese hamster ovary cells that overexpress the human insulin receptor (CHO IR cells; ∼10⁶ receptors per cell), and transfected them with human PC-1 c-DNA (CHO IR PC-1). In CHO IR PC-1 cells, insulin receptor tyrosine kinase activity was unchanged, following insulin treatment of cells. However, several biological effects of insulin, including glucose and amino acid uptake, were decreased. In CHO IR PC-1 cells, insulin stimulation of mitogen-activated protein (MAP) kinase activity was normal, suggesting that PC-1 overexpression did not affect insulin receptor activation of Ras, which is upstream of MAP kinase. Also, insulin-stimulated phosphatidylinositol (PI)-3-kinase activity was normal, suggesting that PC-1 overexpression did not interfere with the activation of this enzyme by insulin receptor substrate-1. In these cells, however, insulin stimulation of p70 ribosomal S6 kinase activity was diminished. These studies suggest, therefore, that, in addition to blocking insulin receptor tyrosine kinase activation, PC-1 can also block insulin receptor signaling at a post-receptor site. J. Cell. Biochem. 68:366–377, 1998. © 1998 Wiley-Liss, Inc.     

Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors

Madin-Darby canine kidney (MDCK) cells were previously shown to have few or no plasma membrane insulin binding sites (Hofmann et al: J Biol Chem 258:11774, 1983]. Accordingly, neither insulin-stimulated incorporation of [14C]glucose into glycogen, nor insulin-induced uptake of radiolabeled alpha-aminoisobutyrate ([3H]AIB) could be demonstrated. To probe for receptors, MDCK cultures were surface-labeled with Na125I or were labeled with [35S]methionine. When solubilized cells were immunoprecipitated with sera containing antibodies to the insulin receptor, and immunoprecipitates were analyzed on SDS-gel electrophoresis, no evidence for insulin receptor components was found. Also, when intact MDCK cells wee incubated first with serum containing antibodies to the insulin receptor and then with 125I-protein A, no radiolabeling of insulin receptors occurred. Various agents reported to have insulin-like activity were tested on MDCK cells. The insulinomimetic lectins concanavalin A and wheat germ agglutinin as well as hydrogen peroxide enhanced incorporation of [14C]glucose into glycogen and induced stimulated [3H]AIB uptake, whereas trypsin, vanadate, and serum containing antibodies to the insulin receptor were without effects. Altogether, these results showed that MDCK cells had few or no insulin receptors and were correspondingly insulin-insensitive. However, since insulin-associated responses could be elicited by some insulin mimickers, the post-receptor limb of response in MDCK cells was apparently intact.     

Lysosomal proteolysis: effects of aging and insulin

Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.     

Insulin receptor internalization and signalling

The insulin receptor kinase (IRK) is a tyrosine kinase whose activation, subsequent to insulin binding, is essential for insulin-signalling in target tissues. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-IRK complexes into the endosomal apparatus (EN) of the cell. Internalization of insulin into target organs, especially liver, is implicated in effecting insulin clearance from the circulation. Internalization mediates IRK downregulation and hence attenuation of insulin sensitivity although most internalized IRKs readily recycle to the plasma membrane at physiological levels of insulin. A role for internalization in insulin signalling is indicated by the accumulation of activated IRKs in ENs. Furthermore, the maximal level of IRK activation has been shown to exceed that attained at the cell surface. Using an in vivo rat liver model in which endosomal IRKs are exclusively activated has revealed that IRKs at this intracellular locus are able by themselves to promote IRS-1 tyrosine phosphorylation and induce hypoglycemia. Furthermore, studies with isolated rat adipocytes reveal the EN to be the principle site of insulin-stimulated IRS-1 tyrosine phosphorylation and associated PI3K activation. Key steps in the termination of the insulin signal are also operative in ENs. Thus, an endosomal acidic insulinase has been identified which limits the extent of IRK activation. Furthermore, IRK dephosphorylation is effected in ENs by an intimately associated phosphotyrosine phosphatase(s) which, in rat liver, appears to regulate IRK activity in both a positive and negative fashion. Thus, insulin-mediated internalization of IRKs into ENs plays a crucial role in effecting and regulating signal transduction in addition to modulating the levels of circulating insulin and the cellular concentration of IRK in target tissues.     

Inputs and outputs of insulin receptor

The insulin receptor (IR) is an important hub in insulin signaling and its activation is tightly regulated. Upon insulin stimulation, IR is activated through autophosphorylation, and consequently phosphorylates several insulin receptor substrate (IRS) proteins, including IRS1-6, Shc and Gab1. Certain adipokines have also been found to activate IR. On the contrary, PTP, Grb and SOCS proteins, which are responsible for the negative regulation of IR, are characterized as IR inhibitors. Additionally, many other proteins have been identified as IR substrates and participate in the insulin signaling pathway. To provide a more comprehensive understanding of the signals mediated through IR, we reviewed the upstream and downstream signal molecules of IR, summarized the positive and negative modulators of IR, and discussed the IR substrates and interacting adaptor proteins. We propose that the molecular events associated with IR should be integrated to obtain a better understanding of the insulin signaling pathway and diabetes.     

Approaches to the molecular cloning of protein-tyrosine phosphatases in insulin-sensitive tissues

The intrinsic tyrosyl kinase activity of the insulin receptor is regulated by a balance between insulin-induced receptor autophosphorylation, which stimulates the receptor kinase, and enzymatic dephosphorylation of the receptor, which deactivates its kinase activity. The cellular protein-tyrosine phosphatase (PTPase) enzymes responsible for reversing the activated state of the insulin receptor have not been characterized. Our laboratory is interested in identifying and cloning the specific PTPase(s) that regulate the phosphorylation state of the insulin receptor. This chapter will summarize the design and results of our initial molecular cloning studies to identify specific PTPases in insulin-sensitive tissues that may have a potential physiological role in insulin action and clinical insulin resistance.     

BIG3 inhibits insulin granule biogenesis and insulin secretion

While molecular regulation of insulin granule exocytosis is relatively well understood, insulin granule biogenesis and maturation and its influence on glucose homeostasis are relatively unclear. Here, we identify a novel protein highly expressed in insulin-secreting cells and name it BIG3 due to its similarity to BIG/GBF of the Arf-GTP exchange factor (GEF) family. BIG3 is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments. BIG3-deficient insulin-secreting cells display increased insulin content and granule number and elevated insulin secretion upon stimulation. Moreover, BIG3 deficiency results in faster processing of proinsulin to insulin and chromogranin A to β-granin in β-cells. BIG3-knockout mice exhibit postprandial hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance. Collectively, these results demonstrate that BIG3 negatively modulates insulin granule biogenesis and insulin secretion and participates in the regulation of systemic glucose homeostasis.     

胰岛素蛋白质工程：高活性［B10Asp］人胰岛素

用基因定位突变方法将胰岛素Ｂ链第１０位的Ｈｉｓ变为Ａｓｐ,获得高活力胰岛素──［Ｂ１０Ａｓｐ］人胰岛素。其受体结合能力和离体生物活力分别为猪胰岛素的２６２％和２３５％;体内生物活力也明显高于猪胰岛素;它的促细胞生长能力为猪胰岛素的１７４％。     

Effects of phorbol esters on insulin receptor function and insulin action in hepatocytes: evidence for heterogeneity

We investigated the effect of phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator on insulin receptors and insulin action in freshly isolated and primary cultures of rat hepatocytes. PMA (1 x 10^–7 M) did not alter insulin receptor numbers or affinity either acutely or chronically but within 60 minute inactivated insulin stimulated tyrosine kinase of the insulin receptor. PKC activation inhibitied insulin (1 x 10^–7M) stimulation of glycogen and lipid synthesis with a decrease or no change in basal glycogenesis and lipogenesis respectively. However, PKC activation did not alter insulin stimulated or basal amino acid transport even though PCK activation inhibited insulin stimulation of the insulin. receptor tyrosine kinase. Thus, within one tissue, PKC activation has differential effect on insulin action depending on which pathway is examined. Furthermore, insulin stimulation of the insulin receptor tyrosine kinase may not be a necessary step for all insulin signaling pathways.     

Prolonged elevation of insulin content in the unicellular tetrahymena after insulin treatment: induction of insulin production or storage?

In the unicellular organism, Tetrahymena, the first encounter with an exogeneously given hormone results in hormonal imprinting. This causes an increase of the binding capacity of receptors and the production of the appropriate hormone in the progeny generations of the treated cell. In the present experiments the quantity (using radioimmunoassay) and localization (using confocal laser scanning microscopy) of the immunologically insulin‐like material (hereafter insulin) were studied for 10 days after 4 h or 24 h 10⁻⁶ m insulin treatment (hormonal imprinting). Forty‐eight hours after both insulin treatments a high quantity of insulin was present in the cells. This value was also significantly increased after 96 h. After 8 days the difference to the control was significant only in the 24 h treated group. Confocal microscopy (using antibody to pig insulin) localized insulin in the cell body. The oral field contained extremely high quantities of the endogeneous hormone. Insulin treatment (after 48 and 96 h) caused an elevation of insulin content in general, and specific accumulation in the posterior sections of the cell, around the nucleus and in the periphery were observed. Ten days after both treatments only the peripheral region of the cell body and the ciliary row contained more insulin than the control. This means that after insulin treatment the quantity of insulin increases for a lengthy time period which is followed by the expression of insulin in the peripheral region. Insulin contained by Tetrahymena 48 h after imprinting stimulated glucose uptake of rat diaphragm. Copyright © 1999 John Wiley & Sons, Ltd.     

No Decrease in Free IGF‐I with Increasing Insulin in Obesity‐Related Insulin Resistance

Objective: Different facts suggest that the insulin growth factor (IGF)/ insulin growth factor‐binding protein (IGFBP) system may be regulated by factors other than growth hormone. It has been proposed that, in healthy subjects, free IGF‐I plays a role in glucose metabolism. The role of free IGF‐I in glucose homeostasis in insulin resistance is poorly understood. This study was undertaken to evaluate the effects of acute changes in plasma glucose and insulin levels on free IGF‐I and IGFBP‐1 in obese and non‐obese subjects. Research Methods and Procedures: Nineteen lean and 24 obese subjects were investigated. A frequently sampled intravenous glucose tolerance test was performed. Free IGF‐I and IGFBP‐1 were determined at 0, 19, 22, 50, 100, and 180 minutes. Results: Basal free IGF‐I levels tended to be higher and IGFBP‐1 lower in obese than in lean subjects. IGFBP‐1 levels inversely correlated with basal insulin concentration. To determine the effects of insulin on the availability of free IGF‐I and IGFBP‐1, changes in their plasma concentrations were measured during a frequently sampled intravenous glucose tolerance test. After insulin administration, a significant suppression of free IGF‐I at 22% was observed in lean subjects. In contrast, plasma‐free IGF‐I levels remained essentially unchanged in the obese group. The differences between both groups were statistically significant at 100 minutes (p < 0.01) and 180 minutes (p < 0.05). Serum IGFBP‐1 was suppressed to a similar extent in both groups. Discussion: These data suggest that the concentrations of free IGF‐I and IGFBP‐1 are differentially regulated by obesity. Obesity‐related insulin resistance leads to unsuppressed free IGF‐I levels.     

Molecular mechanism of insulin resistance

Free fatty acids are known to play a key role in promoting loss of insulin sensitivity, thereby causing insulin resistance and type 2 diabetes. However, the underlying mechanism involved is still unclear. In searching for the cause of the mechanism, it has been found that palmitate inhibits insulin receptor (IR) gene expression, leading to a reduced amount of IR protein in insulin target cells. PDK1-independent phosphorylation of PKC_ε causes this reduction in insulin receptor gene expression. One of the pathways through which fatty acid can induce insulin resistance in insulin target cells is suggested by these studies. We provide an overview of this important area, emphasizing the current status.     

Insulin-stimulated cell growth in insulin receptor substrate-1–deficient ZR-75-1 cells is mediated by a phosphatidylinositol-3-kinase–independent pathway

In many human breast cancers and cultured cell lines, insulin receptor expression is elevated, and insulin, via its own insulin receptor, can stimulate cell growth. It has recently been demonstrated that the enzyme phosphatidylinositol-3-kinase (PI3-K) mediates various aspects of insulin receptor signaling including cell growth. In order to understand the mechanisms for insulin-stimulated cell growth in human breast cancer, we measured insulin-stimulable PI3-K activity in a non-transformed breast epithelial cell line, MCF-10A, and in two malignantly transformed cell lines, ZR-75-1 and MDA-MB157. All three cell lines express comparable amounts of insulin receptors whose tyrosine autophosphorylation is increased by insulin, and in these cell lines insulin stimulates growth. In MDA-MB157 and MCF-10A cells, insulin stimulated PI3-K activity three- to fourfold. In ZR-75-1 cells, however, insulin did not stimulate PI3-K activity. In ZR-75-1 cells PI3-K protein was present, and its activity was stimulated by epidermal growth factor, suggesting that there might be a defect in insulin receptor signaling upstream of PI3-K and downstream of the insulin receptor. Next, we studied insulin receptor substrate-1 (IRS-1), a major endogenous substrate for the insulin receptor which, when tyrosine is phosphorylated by the insulin receptor, interacts with and activates PI3-K. In ZR-75-1 cells, there were reduced levels of protein for IRS-1. In these cells, both Shc tyrosine phosphorylation and mitogen-activated protein kinase (MAP-K) activity were increased by the insulin receptor (indicating that the p21^ras pathway may account for insulin-stimulated cell growth in ZR-75-1 cells). The PI3-K inhibitor LY294002 (50 μM) reduced insulin-stimulated growth in MCF-10A and MDA-MB157 cell lines, whereas it did not modify insulin effect on ZR-75-1 cell growth. The MAP-K/Erk (MEK) inhibitor PD98059 (50 μM) consistently reduced insulin-dependent growth in all three cell lines. Taken together, these data suggest that in breast cancer cells insulin may stimulate cell growth via PI3-K–dependent or–independent pathways. J. Cell. Biochem. 70:268–280, 1998. © 1998 Wiley-Liss, Inc.     

The effect of insulinomimetic agents on protein degradation in H35 hepatoma cells

A wide variety of agents are shown to mimic insulin action and inhibit rates of intracellular protein degradation in H35 hepatoma cells. For oxidizing agents such as NaNO₂, H₂O₂ and oxidized glutathione, inhibition of protein breakdown is reversed by adding catalase. Phenylhydrazine behaves like an oxidant and mimics insulin action in a manner potentiated by superoxide dismutase and reversed by catalase. Similarly the effect of insulin itself is increased by superoxide dismutase and reduced by catalase. Sulfhydryl reagents also mimic insulin action: inhibition of protein breakdown is seen following addition of 2-mercaptoethanol or a brief pre-treatment with N-ethylmaleimide or iodoacetate. Mild pre-treatment with trypsin also inhibits subsequent rates of protein breakdown. A model is proposed suggesting that these insulinomimetic actions involve a common mechanism which links the generation of active oxygen species through the redox potential of the cell to the activation of a proteinase.     

Structural models of viral insulin-like peptides and their analogs

The insulin receptor (IR), the insulin-like growth factor-1 receptor (IGF1R), and the insulin/IGF1 hybrid receptors (hybR) are homologous transmembrane receptors. The peptide ligands, insulin and IGF1, exhibit significant structural homology and can bind to each receptor via site-1 and site-2 residues with distinct affinities. The variants of the Iridoviridae virus family show capability in expressing single-chain insulin/IGF1 like proteins, termed viral insulin-like peptides (VILPs), which can stimulate receptors from the insulin family. The sequences of VILPs lacking the central C-domain (dcVILPs) are known, but their structures in unbound and receptor-bound states have not been resolved to date. We report all-atom structural models of three dcVILPs (dcGIV, dcSGIV, and dcLCDV1) and their complexes with the receptors (μIR, μIGF1R, and μhybR), and probed the peptide/receptor interactions in each system using all-atom molecular dynamics (MD) simulations. Based on the nonbonded interaction energies computed between each residue of peptides (insulin and dcVILPs) and the receptors, we provide details on residues establishing significant interactions. The observed site-1 insulin/μIR interactions are consistent with previous experimental studies, and a residue-level comparison of interactions of peptides (insulin and dcVILPs) with the receptors revealed that, due to sequence differences, dcVILPs also establish some interactions distinct from those between insulin and IR. We also designed insulin analogs and report enhanced interactions between some analogs and the receptors.     

Resistance to trophic neurite outgrowth of sensory neurons exposed to insulin

Insulin offers trophic support through receptors expressed widely on peripheral neurons. In this work, we studied whether peripheral sensory neurons demonstrate resistance to its trophic properties, a property relevant during type 2 diabetes mellitus or following supraphysiological therapy. Insulin receptors were not only localized to neuronal membranes and cytoplasm but also had a unique, previously unrecognized localization to neuronal nuclei. We confirmed that nanomolar doses increased neurite outgrowth of adult sensory neurons, but in response to micromolar doses of insulin, even following a brief 2-h exposure, survival and outgrowth of neurites were blunted. Neurons exposed to picomolar insulin concentrations in their media for 5 days had resistance to the impact of later nanomolar doses of insulin. Using a stripe assay seeded with insulin, neurites were more likely to reject higher doses of insulin. Insulin down-regulated mRNAs of the insulin receptor β subunit and up-regulated levels of GSK-3β, both potential mechanisms of insulin resistance, while down-regulating the protein expression of pAkt and pGSK-3β. Overall, these studies identify neuronal nuclear targeting of insulin and evidence for insulin-induced resistance to its trophic properties. The findings have implications for the understanding of the actions of insulin in the treatment of diabetes and neurological disorders.