共查询到20条相似文献,搜索用时 31 毫秒
1.
Cellular responses elicited by insulin mimickers in cells lacking detectable plasma membrane insulin receptors 总被引:2,自引:0,他引:2
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. 相似文献
2.
《Journal of molecular biology》2022,434(5):167458
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. 相似文献
3.
胰岛素蛋白质工程研究进展 总被引:1,自引:0,他引:1
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. 相似文献
4.
Xu B Hua QX Nakagawa SH Jia W Chu YC Katsoyannis PG Weiss MA 《Journal of molecular biology》2002,316(3):435-441
The hydrophobic core of vertebrate insulins contains an invariant isoleucine residue at position A2. Lack of variation may reflect this side-chain's dual contribution to structure and function: Ile(A2) is proposed both to stabilize the A1-A8 alpha-helix and to contribute to a "hidden" functional surface exposed on receptor binding. Substitution of Ile(A2) by alanine results in segmental unfolding of the A1-A8 alpha-helix, lower thermodynamic stability and impaired receptor binding. Such a spectrum of perturbations, although of biophysical interest, confounds interpretation of structure-activity relationships. To investigate the specific contribution of Ile(A2) to insulin's functional surface, we have employed non-standard mutagenesis: inversion of side-chain chirality in engineered monomer allo-Ile(A2)-DKP-insulin. Although the analogue retains native structure and stability, its affinity for the insulin receptor is impaired by 50-fold. Thus, whereas insulin's core readily accommodates allo-isoleucine at A2, its activity is exquisitely sensitive to chiral inversion. We propose that the Ile(A2) side-chain inserts within a chiral pocket of the receptor as part of insulin's hidden functional surface. 相似文献
5.
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. 相似文献
6.
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. 相似文献
7.
D. Kh. Zubaerova N. I. Larionova 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2008,2(4):346-355
The review considers commercial insulin formulations. Special attention is paid to difficulties and strategies of the development of alternative hormone delivery systems (buccal, transdermal, intranasal, pulmonary and oral). At the moment there is only one approved formulation of the noninvasive insulin in the world. 相似文献
8.
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… 相似文献
9.
10.
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. 相似文献
11.
Li-Yu Huang Yu-Ping Wang Bao-Feng Wei Jian Yang Ji-Qiu Wang Bing-Hao Wu Zhuang-Zhuang Zhang Ying-Yong Hou Wei-Ming Sun Ren-Ming Hu Guang Ning Ze-Guang Han 《Cell research》2013,23(11):1310-1321
IRTKS encodes a member of the IRSp53/MIM homology domain family, which has been shown to play an important role in the formation of plasma membrane protrusions. Although the phosphorylation of IRTKS occurs in response to insulin stimulation, the role of this protein in insulin signaling remains unknown. Here we show that IRTKS-deficient mice exhibit insulin resistance, including hyperglycemia, hyperinsulinemia, glucose intolerance, decreased insulin sensitivity, and increased hepatic glucose production. The administration of ectopic IRTKS can ameliorate the insulin resistance of IRTKS-deficient and diabetic mice. In parallel, the expression level of IRTKS was significantly decreased in diabetic mouse model. Furthermore, DNA hypermethylation of the IRTKS promoter was also observed in these subjects. We also show that IRTKS, as an adaptor of the insulin receptor (IR), modulates IR-IRS1-PI3K-AKT signaling via regulating the phosphorylation of IR. These findings add new insights into our understanding of insulin signaling and resistance. 相似文献
12.
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. 相似文献
13.
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. 相似文献
14.
Singh B Xu Y McLaughlin T Singh V Martinez JA Krishnan A Zochodne DW 《Journal of neurochemistry》2012,121(2):263-276
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. 相似文献
15.
Mohammad Shamsul Ola 《Current issues in molecular biology》2022,44(12):6015
Obesity has become a serious health problem in the world, with increased morbidity, mortality, and financial burden on patients and health-care providers. The skeletal muscle is the most extensive tissue, severely affected by a sedentary lifestyle, which leads to obesity and type 2 diabetes. Obesity disrupts insulin signaling in the skeletal muscle, resulting in decreased glucose disposal, a condition known as insulin resistance. Although there is a large body of evidence on obesity-induced insulin resistance in various skeletal muscles, the molecular mechanism of insulin resistance due to a disruption in insulin receptor signaling, specifically in the gastrocnemius skeletal muscle of obese Zucker rats (OZRs), is not fully understood. This study subjected OZRs to a glucose tolerance test (GTT) to analyze insulin sensitivity. In addition, immunoprecipitation and immunoblotting techniques were used to determine the expression and tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and insulin receptor-β (IRβ), and the activation of serine-632-IRS-1 phosphorylation in the gastrocnemius muscle of Zucker rats. The results show that the GTT in the OZRs was impaired. There was a significant decrease in IRS-1 levels, but no change was observed in IRβ in the gastrocnemius muscle of OZRs, compared to Zucker leans. Obese rats had a higher ratio of tyrosine phosphorylation of IRS-1 and IRβ than lean rats. In obese rats, however, insulin was unable to induce tyrosine phosphorylation. Moreover, insulin increased the phosphorylation of serine 632-IRS-1 in the gastrocnemius muscle of lean rats. However, obese rats had a low basal level of serine-632-IRS-1 and insulin only mildly increased serine phosphorylation in obese rats, compared to those without insulin. Thus, we addressed the altered steps of the insulin receptor signal transduction in the gastrocnemius muscle of OZRs. These findings may contribute to a better understanding of human obesity and type 2 diabetes. 相似文献
16.
Barry J. Goldstein Wei-Ren Zhang Naotake Hashimoto C. Ronald Kahn 《Molecular and cellular biochemistry》1992,109(2):107-113
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. 相似文献
17.
Insulin receptor internalization and signalling 总被引:5,自引:0,他引:5
Di Guglielmo Gianni M. Baass Patricia C. Authier François Posner Barry I. Bergeron John J.M. 《Molecular and cellular biochemistry》1998,182(1-2):59-63
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. 相似文献
18.
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. 相似文献
19.
Summary Insulin binding to human placenta membranes treated at pH 7.6 or 8.5 in the presence or absence of 2.0mm DTT for 5 min, followed by the simultaneous removal of the DTT and pH adjustment to pH 7.6, displayed curvilinear (heterogeneous) insulin binding plots when analyzed by the method of Scatchard. However, Triton X-100 solubilization followed by Bio-Gel A-1.5m gel filtration chromatography of the placenta membranes previously treated with DTT at pH 8.5 generated a nearly straight line (homogeneous) Scatchard plot.125I-insulin affinity crosslinking studies coupled with Bio-Gel A-1.5m gel filtration chromatography demonstrated that the alkaline pH and DTT treatment of placenta membranes followed by detergent solubilization generated an heterodimeric insulin receptor complex from the 22 heterotetrameric disulfide-linked state. The ability of alkaline pH and DTT to produce a functional heterodimeric insulin receptor complex was found to be time dependent with maximal formation and preservation of tracer insulin binding occurring at 5 min. These data demonstrate that (i) a combination of alkaline pH and DTT treatment of placenta membranes can result in the formation of a functional heterodimeric insulin receptor complex. (ii) the heterodimeric complex displays homogeneous insulin binding. (iii) the insulin receptor membrane environment maintains the 22 association state, which displays heterogeneous insulin binding, despite reduction of the critical domains that are responsible for the covalent interaction between the heterodimers.Abbreviations used are ATP
adenosine 5-triphosphate
- DTT
dithiothreitol
- SDS
sodium dodecyl sulfate
- DSS
disuccinimidyl suberate
- NEM
N-ethylmaleimide
- IGF-I
insulin-like growth factor-I
- EDTA
ethylenediaminetetraacetic acid
- HEPES
4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid 相似文献
20.
Hongyu Li Shunhui Wei Kenneth Cheng Natalia V Gounko Russell E Ericksen Aimin Xu Wanjin Hong Weiping Han 《EMBO reports》2014,15(6):714-722
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. 相似文献