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WO1998021592A1 - Dosages relatifs au diabete non insulinodependant - Google Patents

Dosages relatifs au diabete non insulinodependant Download PDF

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Publication number
WO1998021592A1
WO1998021592A1 PCT/US1997/020870 US9720870W WO9821592A1 WO 1998021592 A1 WO1998021592 A1 WO 1998021592A1 US 9720870 W US9720870 W US 9720870W WO 9821592 A1 WO9821592 A1 WO 9821592A1
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WO
WIPO (PCT)
Prior art keywords
irs
activity
protein
candidate
insulin
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Application number
PCT/US1997/020870
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English (en)
Inventor
Ulf Smith
Christina M. Rondinone
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Receptron Corporation
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Application filed by Receptron Corporation filed Critical Receptron Corporation
Priority to AU71811/98A priority Critical patent/AU7181198A/en
Publication of WO1998021592A1 publication Critical patent/WO1998021592A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors

Definitions

  • the present invention relates to screening methods using insulin receptor substrate molecules.
  • insulin In mammals, insulin is the principal hormone controlling blood glucose levels and acts by stimulating glucose intake and metabolism in muscle and adipocytes. Insulin action is mediated through the insulin receptor, a transmembrane glycoprotein with protein tyrosine kinase activity. Insulin binds to the subunit of the insulin receptor, which activates the tyrosine kinase in the ⁇ subunit. The kinase activity appears to mediate the insulin response through phosphorylation of the receptor itself and substrates like insulin receptor substrate- 1
  • IRS-1 insulin receptor tyrosine kinase receptor 1
  • SH2 Src homology 2 domain-containing proteins
  • the insulin receptor does not appear to bind directly to SH2 proteins. Rather, the insulin receptor phosphorylates IRS-1, which in turn recognize and bind to the SH2 domains of various signal transduction proteins, including Syp, Nek, Grb2, Pyn, and the SH2 domain of the subunit of the receptor itself.
  • IRS-1 deficient mice display impaired insulin-stimulated glucose disposal in vivo as well as glucose transport in vitro (Tamemoto, H., et al., Nature 372:182-186 (1994), Araki, E., et al., Nature 372:186-190 (1994)) but their survival and residual insulin sensitivity is dependent on the presence of IRS-2 (Sun, X-J., et al., Nature 377:173-177 (1995), Patti, M. E., et al., J Biol. Chem. 270:24670-24673 (1995)).
  • NIDDM Non-insulin dependent diabetes mellitus
  • the present invention provides methods for screening for a bioactive agent capable of binding to an insulin receptor substrate (IRS) molecule.
  • the method comprises adding a candidate bioactive agent to a sample of the IRS molecule. The presence or absence of binding of the candidate agent to the IRS molecule is then determined.
  • IRS insulin receptor substrate
  • the present invention provides methods for screening for a bioactive agent capable of modulating the activity of an IRS molecule.
  • the method comprises adding a candidate bioactive agent to a cell population.
  • the activity of the IRS molecules in the cell population is then measured.
  • the invention also provides bioactive agents identified using the methods of the invention.
  • the present invention provides methods of diagnosing individuals at risk for type II diabetes mellitus.
  • the method comprises measuring the amount of IRS-1 in adipocytes in a first individual, and comparing the amount to an amount of IRS-1 in adipocytes from a second unaffected individual. When the activity of IRS-1 from the first individual is less than the activity of IRS-1 in the second individual, the first individual is at risk for type II diabetes mellitus.
  • the present invention is based on the discovery that IRS-1 levels in non-insulin dependent diabetes mellitus (NIDDM) individuals are significantly reduced (50-90%) as compared to the levels in comparable healthy individuals. This provides for the first time both a potential diagnostic tool for NIDDM as well as a target for candidate drug screening to elevate the levels of either IRS-1 or IRS-2. Prior to this discovery, no correlation between NIDDM and a biochemical characteristic such as protein expression was known. Thus, the present invention allows diagnostic assays for NIDDM, and, importantly, target molecules for use in screening drug candidates.
  • NIDDM non-insulin dependent diabetes mellitus
  • the present invention provides methods for screening candidate bioactive agents which are capable of modulating the activity of IRS-1 and IRS-2 (collectively, IRS molecules). That is, since the present discovery that individuals with NIDDM have decreased levels of IRS-1 expression, bioactive agents that increase the levels or activity of IRS-1 are desirable. Similarly, since previous work has shown that IRS-2 can, at least in part, compensate for low levels of IRS-1 , bioactive agents that increase the levels or activity of
  • IRS-2 may also be desirable.
  • the present invention is directed to methods of screening candidate bioactive agents.
  • candidate bioactive agents or “candidate drugs” or grammatical equivalents herein is meant any molecule, e.g. polypeptides (including proteins and peptides), small organic or inorganic molecules, polysaccharides, polynucleotides, etc. which may be tested for the ability to directly or indirectly bind to either or both IRS-1 or IRS-2, and/or preferably modulate their activity, as defined below.
  • Candidate agents encompass numerous chemical classes. For example, since increasing transcription of IRS-1 or IRS-2 may increase the total activity present in a cell, preferred candidate agents include known transcription factors or transcription factor candidates, including both proteinaceous and nucleic acid (usually RNA) transcription factors.
  • the candidate agents are organic molecules, particularly small organic molecules, comprising functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more chemical functional groups.
  • Candidate agents are obtained from a wide variety of sources, as will be appreciated by those in the art, including libraries of synthetic or natural compounds. Any number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications to produce structural analogs.
  • candidate bioactive agents may be assayed or screened in a number of ways.
  • candidate bioactive agents may be preliminarily screened for their ability to directly bind to IRS-1 or IRS-2, using techniques well known in the art. These techniques may include, but are not limited to, labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like.
  • labeled herein is meant a compound that has at least one element, isotope or chemical compound attached to enable the detection of the compound.
  • labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
  • the labels may be incorporated into the compound at any position.
  • the binding assays utilize IRS molecules.
  • the IRS molecule is from human, although other IRS proteins may be used.
  • the IRS-1 molecules is highly conserved between species.
  • IRS molecules both IRS-1 and IRS-2) from other species may be used, preferably other mammalian species such as rat and mouse.
  • IRS proteins are made using techniques well known in the art. For example, recombinant IRS nucleic acids are utilized to make recombinant IRS proteins.
  • sequence of both IRS-1 and IRS-2 are known (see for example Sun et al., Nature 377:173 (1995), and references cited therein, all of which are expressly incorporated by reference herein).
  • recombinant protein is a protein made using recombinant techniques, i.e. through the expression of a recombinant nucleic acid.
  • a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
  • the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
  • an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
  • a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
  • the definition includes the production of a IRS protein from one organism in a different organism or host cell.
  • the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
  • the protein may be in a form not normally found in nature, as in the addition of an epitope tag, a purification signal such as His 6 , or amino acid substitutions, insertions and deletions, as discussed below.
  • the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome.
  • these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the IRS protein.
  • "Operably linked" in this context means that the transcriptional and translational regulatory DNA is positioned relative to the coding sequence of the IRS protein in such a manner that transcription is initiated. Generally, this will mean that the promoter and transcriptional initiation or start sequences are positioned 5' to the IRS protein coding region.
  • the transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the IRS protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the IRS protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
  • the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • the regulatory sequences include a promoter and transcriptional start and stop sequences.
  • the expression vector may comprise additional elements, including, for example, two replication systems, thus allowing it to be maintained in two organisms; at least one sequence homologous to the host cell genome to allow recombination, for integrating vectors; and/or selectable marker genes to allow the selection of transformed host cells.
  • the IRS proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a IRS protein, under the appropriate conditions to induce or cause expression of the IRS protein.
  • the conditions appropriate for IRS protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
  • the IRS proteins may be produced in a number of cell types, including, but not limited to, yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, and adipocytes.
  • the IRS protein may also be made as a fusion protein, using techniques well known in the art.
  • the IRS protein may be made as a fusion protein to increase expression, for ease in purification, or for other reasons.
  • amino acid sequence variants are also included within the definition of IRS proteins of the present invention. These variants fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the IRS protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above.
  • variant IRS protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques.
  • Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the IRS protein amino acid sequence.
  • the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be appreciated by those in the art.
  • the IRS protein is purified or isolated after expression for use in the binding assays of the invention.
  • IRS proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse- phase HPLC chromatography, and chromatofocusing.
  • the IRS protein may be purified using a standard anti-IRS antibody column. If purification sequences are included, such as the His 6 tag, suitable methods are used, such as a metal-containing column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY (1982).
  • the IRS proteins are used in screening assays.
  • candidate bioactive agents are added to a sample of IRS-1 or IRS-2, made as described above, and then the mixture is evaluated to determine whether the candidate agent binds to the IRS molecules to form a complex. This is generally done by using either labeled candidate agent or labeled IRS molecules, with the unbound species being separated or purified away, to allow detection of the complex.
  • either the IRS molecules or the candidate agents may be bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate).
  • the IRS molecules are attached to the insoluble support, and labeled candidate agents are added, the excess unbound material is washed away, and detection of agent binding to the IRS molecule is done via detection of the label.
  • the candidate agents may be bound to the support (they may even be synthesized on the support) and then labeled IRS molecules are added and assayed as above.
  • the insoluble supports may be made of any composition to which the agents and/or IRS molecules may be bound, are readily separated from soluble material, and are otherwise compatible with the overall method of screening.
  • the surface of such supports may be solid or porous and of any convenient shape.
  • suitable insoluble supports include surface arrays such as are known in the art, microtiter plates, membranes and beads. These are typically made of glass, plastic (e.g. polystyrene), polysaccharides, nylon or nitrocellulose, which have some sort of suitable binding functionalities on the surface. Microtiter plates and surface arrays are especially convenient because a large number of assays may be run simultaneously, using small amounts of reagents and samples.
  • the particular manner of binding of the agent and/or IRS molecules is not critical as long as it is compatible with the reagents and other components of the system, maintains the activity of the agent or IRS molecule and is nondiffusable.
  • the assays are conducted under approximately physiological conditions. Following binding of the agent and/or IRS molecules, excess unbound material is removed by washing. Any unreacted binding functionalities may be blocked, if necessary, as is known in the art, for example through the addition of neutral or carrier proteins (e.g. bovine serum albumin) so as to prevent direct binding of the other component of the complex to the insoluble support rather than forming a complex.
  • neutral or carrier proteins e.g. bovine serum albumin
  • the other component is added, preferably in a labeled form (i.e. if candidate agents are bound to the support, the labeled IRS molecules are added; if IRS molecules are bound to the support, labeled candidate agents are added).
  • the label afterwards; for example, after the addition of IRS molecules to bound candidate agents, it is possible to add labeled antibodies or other binding moieties, to the IRS molecules.
  • other proteins known to bind to the IRS molecules may be labeled and used to detect the IRS molecules. Incubation of the samples is for a time sufficient to allow binding to occur. Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, labeled material determined. Suitable positive and negative controls are also preferably run.
  • the present invention is directed also to drug screening assays.
  • Drug screening assays utilize the discovery that NIDDM patients have decreased amounts of IRS-1 proteins to identify agents that modulate the activity or amount of IRS-1 or IRS-2.
  • the method comprises the steps of adding or contacting a candidate bioactive agent to or with a cell, and measuring the activity of IRS-1 in the cell.
  • the present invention is directed to methods of screening bioactive agents capable of modulating the activity of IRS molecules, i.e. IRS-1 and IRS-2.
  • modulating the activity herein is meant that the biological activity of the protein is altered, i.e. either increased or decreased.
  • the biological activity is increased. This may be done in several ways, for example, the total amount of the protein may be increased, i.e. the expression of the protein is increased, and thus the measured biological activity increases due to more protein being present. This may be done either by increasing the level or rate of transcription and/or translation of the protein in the cell. Alternatively, the specific activity of the protein may be increased; that is, the protein is made more active by the presence of the bioactive agent, and thus an increase in the total biological activity is seen.
  • the activity of the protein is decreased. This may be important to help elucidate the mechanism of action, for example, or to provide structural information about the sites of protein interaction of the molecule. Similarly, agents which decrease the level of activity of the protein may be useful to define further candidate bioactive agents for testing.
  • Suitable cells for use in the invention include, but are not limited to, adipocytes.
  • Preferred cells include adipocytes from either healthy or NIDDM individuals, with cells from NIDDM individuals being preferred.
  • cells from NIDDM individuals that show a large decrease in the amount of IRS-1 protein are used (e.g., 90%); in other embodiments, testing may be done with cells from several different individuals.
  • Tissue is surgically harvested from patients using well known techniques, and the adipocytes isolated as is known in the art (see Smith et al., J. Lipid Res. 13:822-827 (1972), hereby expressly incorporated by reference). This is generally done using collagenase to separate the cells. The cells may then be used in the assays.
  • the cells are immortalized, i.e. transformed to form cell lines.
  • Methods suitable to make cell lines are well known in the art.
  • reagents in addition to the cells and candidate agents, may be included in the screening assay. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc., which may be used to facilitate optimal protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficacy of the assay, such as inhibitors (protease, nuclease, etc.), anti-microbial agents, etc. may be used.
  • the mixture of components may be added in any order that provides for the interaction of the candidate agent and the cell.
  • the candidate agents may be accompanied by or fused to targeting or signaling molecules that facilitate the uptake or targeting of the agent to different cellular localizations.
  • targeting or signaling molecules that facilitate the uptake or targeting of the agent to different cellular localizations.
  • nuclear localization signals are preferably used.
  • the components are allowed to incubate with the cells under suitable thermal conditions, generally between 4°C and 40°C, with physiological temperatures (about 37°C) being preferred, for a suitable amount of time. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening.
  • the activity of the IRS molecule is measured.
  • the modulation of the activity of the protein is measured in any number of ways, as will be appreciated by those in the art.
  • the activity is measured by measuring the glucose-transport activity in response to different concentrations of insulin, as is outlined in the Examples and described in Rondinone et al., J Biol. Chem. 271 :18148-18153 (1996), hereby expressly incorporated by reference. Briefly, the cells are incubated with different doses of insulin in the presence of radiolabelled glucose for a period of time, generally from about 15 minutes to 2 hours, with about 1 hour being preferred.
  • activity is measured as a function of amount of protein; that is, the activity is measured by measuring the amount of IRS-1 or IRS-2 protein, using techniques well known in the art.
  • the methods utilize immunoprecipitation techniques, such as are outlined in the examples, to determine the amount of protein.
  • the methods utilize enzyme-linked immunosorbent assays (ELISA) techniques for the detection and quantification of the proteins. ELISA techniques are well known in the art; see
  • the activity of the IRS molecule may be measured prior to the addition of the candidate agent. Generally, this is done periodically on the cells or cell line being used, and thus may not need to be done prior to every assay. That is, generally the starting range of IRS activity is initially measured and then known, with the appropriate controls being run as necessary. Alternatively, negative controls may be run with every batch of candidate agents. Alternatively, the activity of the IRS molecule is determined prior to the addition of the candidate agents.
  • the level of IRS activity after the addition of the candidate agent is then compared to the level of activity prior to the addition. If the post-addition activity is different than the initial activity, the candidate agent is capable of modulating the activity of the IRS molecule. As outlined above, this difference may be either an increase or a decrease, and generally will be at least about 5% different, with at least about 10% different being preferred, at least about 20% being particularly preferred and at least about 40-50% being especially preferred.
  • the present invention provides methods for diagnosing individuals at risk for type II diabetes mellitus, i.e. NIDDM, or for confirming a diagnosis of NIDDM.
  • the screen may serve to identify individuals who may not have the full-blown symptoms of NIDDM but may be at risk to develop such symptoms in the future, as outlined in the examples.
  • adipocytes are obtained from a patient, as is known in the art, for example using needle biopsies, and purified if necessary.
  • the amount of IRS protein is then determined, using techniques known in the art and outlined herein, and compared to IRS levels in a matched, healthy individual.
  • Decreases in IRS protein, preferably IRS-1 protein, of about 50%-90% have been shown to correlate with NIDDM or prediabetic conditions as outlined in the Examples.
  • a reduction in IRS levels of at least about 40%-50%, more preferably about 60%-70%, and more preferably about 80%-90%, as compared to healthy matched individuals is indicative of patients either at risk for type II diabetes or with the disease.
  • a decrease of at least about 50% in IRS-1 levels is indicative of these conditions.
  • the methods of the invention allow the diagnostic differentiation between insulin dependent diabetes mellitus (IDDM) and NIDDM.
  • IDDM insulin dependent diabetes mellitus
  • NIDDM insulin dependent diabetes mellitus
  • these tests allow the correct identification of the diabetic type, to verify or correct initial diagnosis.
  • the method comprises measuring the activity of IRS-1 in adipocytes from an individual being tested, and comparing this result to the activity of IRS-1 in adipocytes from a second, unaffected individual, preferably matched as a control individual.
  • the amount of IRS-1 is measured, using the techniques outlined above or others known in the art.
  • the activity from the test subject is less than the activity from the unaffected subject, the test subject is at risk for NIDDM, and in fact may already have it.
  • a 50% or greater reduction in IRS levels is the indicator of status.
  • the cells were separated from the incubation medium by centrifugation through silicone oil and the radioactivity associated with the cells was measured by scintillation counting.
  • Cells were preincubated with or without 6.9 nM insulin for 10 min, immediately separated by centrifugation through silicone oil and lysed in 0.4 ml lysis buffer containing 25 mM Tris-HCl, pH 7.4, 0.5 mM EGTA, 25 mM NaCl, 1% Nonidet P--40, 1 mM Na3VO4, 10 mM NaF, 0.2 mM leupeptin, 1 mM benzamidine and 0.5 mM 4- (2-aminoethyl)-benzenesulfonylfluoride hydrochlorine (AEBSF) and rocked for 40 min at 4°C.
  • AEBSF 2-aminoethyl)-benzenesulfonylfluoride hydrochlorine
  • Proteins were transferred from the gel to nitrocellulose sheets and blocked in 5% milk.
  • the blots were probed with the different primary antibodies: anti-IRS-1 C-terminal, anti-p85 (whole antiserum), anti-SHPTP2/syp, anti-Grb2 and 4G10 anti-PY antibodies (UBI); anti-IR (Transduction Laboratories), anti-IRS-1 (NH-2 terminal), anti pi 10 (Santa Cruz Biotechnology) according to the recommendations of the manufacturer or anti-IRS-2 (1:500 v/v) and the proteins were detected by enhanced chemiluminescence using horseradish peroxidase-labeled second antibodies (Amersham). The intensity of the bands was quantitated with a laser densitometer (Molecular Dynamics).
  • PI 3-kinase assay was performed directly on the immunoprecipitates as described (Rondinone et al., J. Biol. Chem, 271 :18148 (1996); Auger et al., Cell 57:167 (1989), both of which are expressly incorporated by reference).
  • IRS-1 was reduced by 50 to 90% (average, 70+ 6%) in adipocytes from NIDDM patients compared to those from non-diabetic subjects. This was confirmed by using two different NG2- and C-terminal antibodies. However, the insulin receptor, the p85 subunit of PI-3 kinase and the phosphotyrosine phosphatase SHPTP2/Syp were unchanged. Similarly, the IRS-1 protein content was not reduced in adipocytes from IDDM subjects. As expected, in IRS-1 or PI-3 kinase (p85 and pi 10 subunits) in response to insulin could hardly be detected.
  • IRS-2 tyrosine phosphorylated protein at MW-185 kDa suggesting the presence of another protein at that MW.
  • the likely candidate was the recently identified docking protein IRS-2 (Sun, X- J., et al., Nature 377:173-177 (1995)).
  • IRS-2 was present in a similar extent in adipocytes from both NIDDM and healthy subjects; IRS-2 was also tyrosine phosphorylated in an insulin-dependent fashion.
  • IRS-1 and IRS-2 were tyrosine phosphorylated by insulin but quantification of the immunoblots showed that the effect of a maximal insulin concentration was 50% greater for IRS-1 than for IRS-2. Therefore, immunoprecipitates with anti-IRS-1 or anti-IRS-2 antibodies showed that at all insulin concentrations IRS-1 was the main docking protein for PI 3-kinase. However, IRS-2 could also bind PI 3-kinase but this was, for a given insulin concentration, less (-40%) than that bound to IRS-1. This provides evidence for differences between IRS-1 and IRS-2 that may be related to a different interaction between IRS-1 and IRS-2 and the insulin receptor (He, W., et al., J. Biol. Chem.
  • IRS-1 PI 3-kinase lipid kinase activity
  • IRS-2 was the main source of PI 3-kinase activity in adipocytes from NIDDM subjects.
  • IRS-1 protein expression is markedly reduced in adipocytes from NIDDM subjects in comparison with both healthy subjects and individuals with IDDM.
  • the 50% or greater reduction in IRS-1 content was consistently seen in the 12 NIDDM subjects studied. So far, more than 20 NIDDM patients have been examined and only a smaller reduction (-30%) was seen in two subjects. This reduction can not be accounted for by obesity since the control and NIDDM subjects had a similar BMI or by hyperinsulinemia/hyperglycemia since the IDDM were unaffected.
  • IRS-2 levels were unchanged in NIDDM cells. Skeletal muscle from morbidly obese individuals has also been shown to have a moderate reduction in IRS-1 protein content (Goodyear, L., et al., J. Clin. Inves. pp 2195-2204 (1995) but it is not clear whether this reduction was confined to subjects with an impaired glucose tolerance.
  • IRS-1 seems to be the main docking protein for PI 3-kinase and the associated increase in glucose uptake in normal human adipocytes as also demonstrated in other cells (Quon, M. J., et al., Biol. Chem. 269:27920-27924 (1994), Har, K., et al., Proc. Natl. Acad. Sci. USA 91 :74155-7419 (1994), Quon, M. J., et al., Mol. Cell. Biol. 15:5403-5411 (1995)).
  • IRS-2 seems to be able to replace IRS-1 as the main docking protein for binding and activation of PI 3-kinase.
  • IRS-2 requires a higher insulin concentration than IRS-1 for similar binding and activation of PI 3-kinase. This is in parallel to the ability of insulin to increase total tyrosine phosphorylation which also was reduced in IRS-2.

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Abstract

L'invention porte sur des procédés de recherche faisant appel à des molécules de substrats récepteurs de l'insuline.
PCT/US1997/020870 1996-11-13 1997-11-13 Dosages relatifs au diabete non insulinodependant WO1998021592A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001015689A1 (fr) * 1999-08-27 2001-03-08 Centre National De La Recherche Scientifique (Cnrs) Utilisation d'acides amines pour la fabrication de medicaments destines au traitement des insulino-resistances
WO2001094410A1 (fr) * 2000-06-08 2001-12-13 Metcon Medicin Ab Substance regulee par l'insuline (irs-2) induite par la pioglitazone, dosage et utilisation de cette derniere
US6835819B2 (en) 2000-06-08 2004-12-28 Metcon Medicin Ab Sequences and their use

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010629A1 (fr) * 1994-10-03 1996-04-11 Joslin Diabetes Center, Inc. Famille des genes irs

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010629A1 (fr) * 1994-10-03 1996-04-11 Joslin Diabetes Center, Inc. Famille des genes irs

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001015689A1 (fr) * 1999-08-27 2001-03-08 Centre National De La Recherche Scientifique (Cnrs) Utilisation d'acides amines pour la fabrication de medicaments destines au traitement des insulino-resistances
EP1421937A1 (fr) * 1999-08-27 2004-05-26 Innodia Utilisation d'acides amines pour la fabrication de medicaments destinés au traitement des insulino-resistances
US7402611B1 (en) 1999-08-27 2008-07-22 Innodia Inc. Use of amino acids for making medicines for treating to insulin-resistance
WO2001094410A1 (fr) * 2000-06-08 2001-12-13 Metcon Medicin Ab Substance regulee par l'insuline (irs-2) induite par la pioglitazone, dosage et utilisation de cette derniere
US6835819B2 (en) 2000-06-08 2004-12-28 Metcon Medicin Ab Sequences and their use

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