WO2003033676A2 - Gene expression associated with glucose tolerance - Google Patents

Gene expression associated with glucose tolerance Download PDF

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Publication number
WO2003033676A2
WO2003033676A2 PCT/US2002/033524 US0233524W WO03033676A2 WO 2003033676 A2 WO2003033676 A2 WO 2003033676A2 US 0233524 W US0233524 W US 0233524W WO 03033676 A2 WO03033676 A2 WO 03033676A2
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impaired glucose
gene expression
genes
gene
diabetes mellitus
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PCT/US2002/033524
Inventor
Cecilia Lindgren
Joel Hirschhorn
Pablo Tamayo
Mark Daly
Eric Lander
David Altshuler
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Massachusetts Gen Hospital
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Publication of WO2003033676A2 publication Critical patent/WO2003033676A2/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Type 2 diabetes mellitus is a metabolic disorder characterized by insulin resistance and pancreatic ⁇ -cell dysfunction. Insulin is the primary modulator of glucose, and failure of the normal action of insulin in its main target tissue (skeletal muscle) contributes to the pathogenesis of type 2 diabetes.
  • the hyperglycemia of type 2 diabetes is thought to be a consequence of impaired insulin secretion from the ⁇ -cells and /or skeletal muscle insulin resistance. The primary lesions that cause type 2 diabetes are not known.
  • genes that show abnormal expression in skeletal muscle in subjects with varying degrees of glucose tolerance have been identified.
  • genes whose expression is triggered by the acute effect of insulin infusion in skeletal muscle in vivo were also identified.
  • 209 genes were found to be differentially expressed in skeletal muscle from subjects with type 2 diabetes and abnormal glucose tolerance compared to normoglycemic controls (p ⁇ 0.05), and insulin infusion triggered the expression of 89 genes in skeletal muscle (p ⁇ 0.05).
  • the present invention features methods of identifying individuals with varying degrees of glucose tolerance and methods of identifying individuals likely to have or to develop type 2 diabetes mellitus, particularly by assessing the expression profile of one or more of the genes disclosed herein.
  • the present invention also relates to methods for identifying compounds that modulate glucose tolerance and/or homeostasis, insulin response and/or type 2 diabetes mellitus, and oligonucleotide microarrays containing probes for genes involved in glucose tolerance and/or homeostasis, insulin response and/or type 2 diabetes mellitus.
  • the invention features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a nucleic acid sample derived from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control. Increased expression of the informative gene in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the nucleic acid sample is derived from skeletal muscle tissue.
  • the gene expression product is DNA or mRNA.
  • the gene expression profile is determined utilizing specific hybridization probes.
  • the gene expression profile may be determined utilizing oligonucleotide microarrays.
  • the gene expression product is a polypeptide.
  • the gene expression profile is determined utilizing antibodies.
  • the one or more informative genes are selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • the invention also features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a polypeptide sample from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control, where the gene expression product is a polypeptide.
  • Increased expression of the gene expression product in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the polypeptide sample is derived from skeletal muscle tissue.
  • the gene expression profile is determined utilizing antibodies.
  • the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • the invention features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a nucleic acid sample from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control. Decreased expression of the gene in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the nucleic acid sample is derived from skeletal muscle tissue.
  • the gene expression product is DNA or mRNA.
  • the gene expression profile is determined utilizing specific hybridization probes.
  • the gene expression profile may be determined utilizing oligonucleotide microarrays.
  • the gene expression product is a polypeptide.
  • the gene expression profile is determined utilizing antibodies.
  • the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • the invention also features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a polypeptide sample from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control, where the gene expression product is a polypeptide. Decreased expression of the gene expression product in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the polypeptide sample is derived from skeletal muscle tissue.
  • the gene expression profile is determined utilizing antibodies.
  • the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • the invention also features a method of identifying a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising the steps of: a) providing a cell or cell lysate sample; b) contacting the cell or cell lysate sample with a candidate compound; and c) detecting an increase in expression of at least one informative gene having decreased expression in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a candidate compound that increases the expression of the informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the cell or cell lysate sample is derived from skeletal muscle tissue.
  • the cell or cell lysate sample is derived from a cultured cell.
  • gene expression is determined by assessing the DNA or mRNA level of the gene.
  • the DNA or mRNA level is determined utilizing specific hybridization probes.
  • the DNA or mRNA level may be determined utilizing oligonucleotide microarrays.
  • gene expression is determined by assessing the polypeptide level encoded by the informative gene.
  • gene expression is determined utilizing antibodies.
  • the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • the invention features a method of identifying a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising the steps of: a) providing a cell or cell lysate sample; b) contacting the cell or cell lysate sample with a candidate compound; and c) detecting a decrease in expression of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus.
  • a candidate compound that decreases the expression of the informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the cell or cell lysate sample is derived from skeletal muscle tissue. In another embodiment, the cell or cell lysate sample is derived from a cultured cell. In other embodiments, gene expression is determined by assessing the DNA or mRNA level of the gene. Preferably, the DNA or mRNA level is determined utilizing specific hybridization probes. For example, the DNA or mRNA level may be determined utilizing oligonucleotide microarrays.
  • gene expression is determined by assessing the polypeptide level encoded by the informative gene.
  • gene expression is determined utilizing antibodies.
  • the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • the invention also features a method for modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus in a subject (e.g., an individual) by down-regulating in the subject at least one informative gene shown to be expressed in or expressed at increased levels in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, but not in normal individuals.
  • the invention also features a method for modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus in a subject (e.g., an individual) by up-regulating in the subject at least one informative gene shown not to be expressed in or expressed at reduced levels in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to normal individuals.
  • the invention also features oligonucleotide microarrays having immobilized thereon a plurality of oligonucleotide probes specific for one or more informative genes selected from the group consisting of the genes in Table 1 and the genes in Table 2.
  • Figure 1 shows the expression profile of the 54 muscle biopsies from individuals with normal glucose tolerance versus individuals with disturbances in their glucose tolerance (type 2 diabetes and impaired glucose tolerance) (Group A).
  • the plot shows the number of genes within various neighborhoods of the glucose tolerance distinction (+) together with curves indicating the mean (-), 1% ( ⁇ ) and 5%( " " ) significance levels for the number of genes within corresponding neighborhoods of the randomly permuted class distinctions.
  • Genes more highly expressed in tissue from control subjects with normal glucose homeostasis compared to the expression levels in individuals with disturbances in the glucose homeostasis is shown in the left panel.
  • Those genes more highly expressed in tissue from individuals with disturbances in their glucose homeostasis compared to the expression levels in the control subjects with normal glucose homeostasis is shown in the right panel.
  • Figure 2 shows the expression profile of the 28 muscle biopsies from individuals before the euglycemic hyperinsulinemic clamp versus individuals after the euglycemic hyperinsulinemic clamp (Group B.).
  • the plot shows the number of genes within various neighborhoods of the glucose tolerance distinction (+) together with curves indicating the mean (-), 1%( ) and 5%( "" ) significance levels for the number of genes within corresponding neighborhoods of the randomly permuted class distinctions.
  • Genes more highly expressed in tissue from subjects before the euglycemic hyperinsulinemic clamp compared to the expression levels in tissue taken after the euglycemic hyperinsulinemic clamp is shown in the left panel.
  • Type 2 diabetes mellitus is a metabolic disorder characterized by insulin resistance and pancreatic ⁇ -cell dysfunction.
  • Muscle biopsies were obtained from 28 normoglycemic male subjects (age 66 ⁇ 9) and 26 male subjects with impaired glucose homeostasis (age 65 ⁇ 9) from Sweden taken during an euglycemic hyperinsulinemic clamp (Group A).
  • muscle biopsies were also obtained from 7 monozygotic twin pairs (4 female/10 male) before and during a euglycemic hyperinsulinemic clamp (Group B).
  • the present invention is directed to methods for predicting phenotypic classes of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, such as the presence or absence of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, or the identification of compounds that modulate impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, based on gene expression profiles.
  • the invention involves identifying an an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus by obtaining a nucleic acid or polypeptide sample from said individual, and determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus relative to a control. Increased expression of the informative gene is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • identification of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus may be made by obtaining a nucleic acid or polypeptide sample from said individual, and determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control. Decreased expression of the informative gene is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • gene expression profile is meant the level or amount of gene expression of particular genes, for example, informative genes, as assessed by methods described herein.
  • the gene expression profile can comprise data for one or more informative genes and can be measured at a single time point or over a period of time.
  • the gene expression profile can be determined using a single informative gene, or it can be determined using two or more informative genes, three or more informative genes, five or more informative genes, ten or more informative genes, twenty-five or more informative genes, or fifty or more informative genes.
  • a gene expression profile may include expression levels of genes that are not informative, as well as informative genes.
  • Phenotype classification can be made by comparing the gene expression profile of the sample with respect to one or more informative genes with one or more gene expression profiles of a control (e.g., in a database). Using the methods described herein, expression of numerous genes can be measured simultaneously. The assessment of numerous genes provides for a more accurate evaluation of the sample because there are more genes that can assist in classifying the sample.
  • a gene expression profile may involve only those genes that are increased in expression in a sample, only those genes that are decreased in expression in a sample, or a combination of genes that are increased and decreased in expression in a sample.
  • “informative genes” refers to a gene or genes whose expression correlates with a particular phenotype.
  • Expression profiles obtained for informative genes can be used to determine, for example, whether an individual has, or is at risk of developing, impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, or whether a candidate compound increases or decreases gene expression in a sample.
  • Samples can be classified according to their broad expression profile, or according to the expression levels of particular informative genes.
  • the genes that are relevant for classification are referred to herein as "informative genes.” Not all informative genes for a particular class distinction must be assessed in order to classify a sample.
  • the set of informative genes that characterize one phenotypic effect may or may not be the same as the set of informative genes for a different phenotypic effect.
  • a subset of the informative genes that demonstrate a high correlation with a class distinction can be used in identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • This subset can be, for example, one or more genes, two or more genes, three or more genes, five or more genes, ten or more genes, twenty-five or more genes, or fifty or more genes.
  • the informative genes that characterize other classification categories such as, for example, a candidate compound that modulates impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, can be the same or different from the informative genes that identify an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the accuracy of the classification increases with the number of informative genes that are assessed.
  • Informative genes include, but are not limited to, the particular genes shown in Table 1 and Table 2.
  • gene expression products are proteins, polypeptides, or nucleic acid molecules (e.g., mRNA, tRNA, rRNA, cDNA, or cRNA) that result from transcription or translation of genes.
  • the present invention can be used effectively to analyze proteins, polypeptides, or nucleic acid molecules that are the result of transcription or translation, particularly of informative genes identified herein.
  • the nucleic acid molecule levels measured can be derived directly from the gene or, alternatively, from a corresponding regulatory gene or regulatory sequence element. All forms of gene expression products can be measured. For example, the nucleic acid molecule can be transcribed to obtain an RNA gene expression product.
  • the transcript can be translated using, for example, standard in vitro translation methods to obtain a polypeptide gene expression product.
  • Polypeptide gene expression products can be used in protein binding assays, for example, antibody assays, or in nucleic acid binding assays, routinely used in the art. Additionally, variants of genes and gene expression products including, for example, spliced variants and polymorphic alleles, can be measured. Similarly, gene expression can be measured by assessing the level of a polypeptide or protein or derivative thereof translated from mRNA. The sample to be assessed can be any sample that contains a gene expression product.
  • Suitable sources of gene expression products can include intact cells, lysed cells, cellular material for determining gene expression, or material containing gene expression products.
  • samples can include skeletal muscle tissue, cells derived from skeletal muscle, nucleic acids or polypeptides derived from skeletal muscle tissue, blood, plasma, lymph, urine, tissue, mucus, sputum, saliva, or other cell samples. Methods of obtaining such samples are known in the art.
  • the increase is by at least 1.5-fold, more preferably the increase is at least 2-fold, 5-fold, or 10-fold, and most preferably, the increase is at least 20-fold, relative to a control.
  • the decrease is at least 25%, more preferably, the decrease is at least 50%, 60%, 70%, 80%), or 90%) and most preferably, the decrease is at least one-fold, relative to a control sample.
  • the gene expression product is a protein or polypeptide.
  • polypeptide any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
  • polypeptides include, but are not limited to, proteins.
  • the determination of the gene expression profile is made using techniques for protein detection and quantitation known in the art.
  • antibodies that specifically interact with the protein or polypeptide expression product of one or more informative genes can be obtained using methods that are routine in the art.
  • the specific binding of such antibodies to protein or polypeptide gene expression products can be detected and measured by methods known in the art, for example, Western blot analysis or ELISA techniques.
  • the gene expression product is a nucleic acid, for example, DNA or mRNA
  • the gene expression levels are obtained by contacting the sample with a suitable microarray on which one or more probes specific for all or a subset of the informative genes have been immobilized, and dete ⁇ nining the extent of hybridization of the nucleic acid in the sample to the probes on the microarray.
  • a suitable microarray on which one or more probes specific for all or a subset of the informative genes have been immobilized, and dete ⁇ nining the extent of hybridization of the nucleic acid in the sample to the probes on the microarray.
  • Such microairays are also within the scope of the invention. Examples of methods of making oligonucleotide microarrays are described, for example, in WO 95/1 1995. Other methods are readily known to the skilled artisan.
  • the gene expression value measured or assessed is the numeric value obtained from an apparatus that can measure gene expression levels.
  • Gene expression levels refer to the amount of expression of the gene expression product, as described herein.
  • the values are raw values from the apparatus, or values that are optionally re-scaled, filtered and/or normalized. Such data is obtained, for example, from a GeneChip® probe array or Microarray (Affymetrix, Inc.; U.S. Patent Nos.
  • nucleic acids e.g., mRNA or DNA
  • probes on the chip hybridize to the probes on the chip.
  • the nucleic acid to be analyzed (e.g., the target) is isolated, amplified and labeled with a detectable label, (e.g., 32 P or fluorescent label) prior to hybridization to the arrays.
  • a detectable label e.g. 32 P or fluorescent label
  • the arrays are inserted into a scanner that can detect patterns of hybridization. These patterns are detected by detecting the labeled target now attached to the microarray, e.g., if the target is fluorescently labeled, the hybridization data are collected as light emitted from the labeled groups.
  • Affymetrix seamier Affymetrix, Santa Clara, CA .
  • the gene expression levels of the sample are obtained, the levels are compared or evaluated against a model or control sample(s), and then the sample is classified, for example, based one whether a particular informative gene in the sample exhibits increased or decreased expression.
  • the evaluation of the sample determines whether or not the sample is assigned to a particular phenotypic class.
  • the present invention also features methods for identifying compounds that modulate impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • Novel compounds identified as described herein are also the subject of the invention. Such methods involve contacting a sample, for example a cell, cell lysate, tissue, or tissue lysate, with a candidate compound, and detecting an increase in expression of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a candidate compound that increases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a compound that modulates impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can be identified by contacting a sample, for example, a cell, cell lysate, tissue, or tissue lysate with a candidate compound, and detecting a decrease in expression of at least one informative gene having increased expression in an an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a candidate compound that decreases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • An increase or decrease in an informative gene may be identified using any of the methods described herein (or any analogous method known in the art). For example, oligonucleotide array systems described herein may be used to determine whether the addition of a test compound to a sample increases or decreases expression of an informative gene in that sample.
  • modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus is meant increasing or decreasing the likelihood that impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus will occur or develop in a subject.
  • the modulation of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus may be the result of contacting a sample (for example, a cell, tissue, cell or tissue lysate, nucleic acid, or polypeptide) with a candidate compound.
  • a sample for example, a cell, tissue, cell or tissue lysate, nucleic acid, or polypeptide
  • the sample is derived from skeletal muscle tissue. It will be appreciated that the degree of modulation provided by a candidate compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change or a therapeutically effective change in the degree or rate of disease development.
  • candidate compound a molecule, be it naturally-occurring or artificially derived, that is surveyed for its effects on the gene expression profile of an informative gene, employing methods described herein.
  • candidate compounds include, but are not limited to peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, and combinations thereof.
  • increasing gene expression is meant raising the level of expression, and/or the activity, of one or more informative genes in a cell, tissue, cell lysate, or tissue lysate sample relative to a control sample.
  • An increase in gene expression may occur, for example, when the sample is contacted with a candidate compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the control sample may be a cell, tissue, cell lysate, or tissue lysate that was not contacted with the candidate compound or that was contacted with candidate compound vehicle only.
  • the increase is at least 1.5-fold, more preferably the increase is at least 2-fold, 5-fold, or 10-fold, and most preferably, the increase is at least 20-fold, relative to a control sample.
  • decreasing gene expression is meant lowering the level or expression of, and/or the activity of, one or more informative genes in a cell, tissue, cell lysate, or tissue lysate sample relative to a control sample.
  • a decrease in gene expression may occur, for example, when the sample is contacted with a candidate compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the control sample may be a cell, tissue, cell lysate, or tissue lysate that was not contacted with the candidate compound or that was contacted with candidate compound vehicle only.
  • the decrease in gene expression of an informative gene is at least 25%>, more preferably, the decrease is at least 50%>, 60%o, 70%), 80%), or 90%) and most preferably, the decrease is at least one-fold, relative to a control sample.
  • the expression level of an info ⁇ native gene may be modulated by modulating transcription, translation, or mRNA or protein turnover, or the activity of the gene expression product, and such modulation may be detected using known methods for measuring mRNA and protein levels and activities, e.g., oligonucleotide microarray hybridization, RT-PCR, and ELISA and nucleic acid and protein binding assays.
  • a compound that increases the expression level of a gene that is decreased in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can be useful for treating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a compound that decreases the expression level of a gene that is increased in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can also be useful for treating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • candidate compound screening methods are designed primarily to identify candidate compounds that may be used to decrease impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus
  • identification of candidate compounds that increase impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus is also a feature of the present invention.
  • Such candidate compound identification methods involve contacting a sample, for example, a cell, cell lysate, tissue, or tissue lysate with a candidate compound, and detecting an increase in expression of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus.
  • a candidate compound that increases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a compound that modulates impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can be identified by contacting a sample, for example, a cell, cell lysate, tissue, or tissue lysate with a candidate compound, and detecting a decrease in expression of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • a candidate compound that decreases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • candidate compound identification methods may be used for identifying compounds that increase impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, or the risk of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • Such compounds may be identified as compounds to which exposure should be minimized in order to decreased one's likelihood of developing impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • novel drugs for modulation of disease development can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention.
  • any number of chemical extracts or compounds can be screened using the exemplary methods described herein.
  • extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.
  • Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-. peptide-, and nucleic acid-based compounds.
  • Synthetic compound libraries are commercially available, e.g., Chembridge (San Diego, CA).
  • libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA).
  • Biotics Sussex, UK
  • Xenova Slough, UK
  • Harbor Branch Oceangraphics Institute Ft. Pierce, FL
  • PharmaMar, U.S.A. PharmaMar, U.S.A.
  • any library or compound is readily modified using standard chemical, physical, or biochemical methods.
  • the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having an activity that increases or deceases.
  • the same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value maybe subsequently analyzed using animal models for diseases.
  • Informative genes identified as described herein can also be targeted in methods of modulating impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus.
  • expression of at least one informative gene shown to be expressed in or expressed at increased levels in, an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, but not in normal individuals can be down-regulated in a method of inhibiting impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • expression of at least one informative gene shown not to be expressed in, or which are expressed at reduced levels in, an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, relative to normal intestinal samples can be upregulated in a method of inhibiting impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus.
  • Compounds identified by methods described herein, for example, can be utilized in methods of treatment of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
  • the present invention also features arrays, for example, microarrays that have a plurality of oligonucleotide probes involved in impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus immobilized thereon.
  • the oligonucleotide probe may be specific for one or more informative genes, selected from those in Table 1 and Table 2.
  • Methods for making oligonucleotide microarrays are well known in the art, and are described, for example, in WO 95/11995, the entire teachings of which are hereby incorporated by reference.
  • the present invention also provides information regarding the genes that are important in impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, thereby providing additional targets for diagnosis and therapy. It is clear that the present invention can be used to generate databases comprising informative genes that will have many applications in medicine, research and industry; such databases are also within the scope of the invention.
  • High density oligonucleotide arrays The mRNA is converted into biotin labeled cRNA in a reverse transcription followed by an in vitro transcription. Subsequently, the labeled cRNA is hybridized onto a high density oligonucleotide array where 3' end specific probes from more than 6800 genes are immobilized on a solid phase (glass). The biotin labeled cRNA is the labeled by staining with a fluorescent streptavidin conjugate. The level of expression is then analyzed as intensity of fluorescence in a laser confocal scanner. Intensity values were scaled so that the overall fluorescence intensity of each chip was equivalent.

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Description

GENE EXPRESSION ASSOCIATED WITH GLUCOSE TOLERANCE
RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application 60/330,147, filed on October 17, 2001, the entire teachings of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
Type 2 diabetes mellitus is a metabolic disorder characterized by insulin resistance and pancreatic β-cell dysfunction. Insulin is the primary modulator of glucose, and failure of the normal action of insulin in its main target tissue (skeletal muscle) contributes to the pathogenesis of type 2 diabetes. The hyperglycemia of type 2 diabetes is thought to be a consequence of impaired insulin secretion from the β-cells and /or skeletal muscle insulin resistance. The primary lesions that cause type 2 diabetes are not known.
SUMMARY OF THE INVENTION
As described herein, genes that show abnormal expression in skeletal muscle in subjects with varying degrees of glucose tolerance have been identified. As further described, genes whose expression is triggered by the acute effect of insulin infusion in skeletal muscle in vivo were also identified. In summary, 209 genes were found to be differentially expressed in skeletal muscle from subjects with type 2 diabetes and abnormal glucose tolerance compared to normoglycemic controls (p<0.05), and insulin infusion triggered the expression of 89 genes in skeletal muscle (p<0.05).
The present invention features methods of identifying individuals with varying degrees of glucose tolerance and methods of identifying individuals likely to have or to develop type 2 diabetes mellitus, particularly by assessing the expression profile of one or more of the genes disclosed herein. The present invention also relates to methods for identifying compounds that modulate glucose tolerance and/or homeostasis, insulin response and/or type 2 diabetes mellitus, and oligonucleotide microarrays containing probes for genes involved in glucose tolerance and/or homeostasis, insulin response and/or type 2 diabetes mellitus.
In one aspect, the invention features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a nucleic acid sample derived from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control. Increased expression of the informative gene in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In one embodiment, the nucleic acid sample is derived from skeletal muscle tissue. In other embodiments, the gene expression product is DNA or mRNA. Preferably, when the gene expression product is DNA or mRNA, the gene expression profile is determined utilizing specific hybridization probes. For example, the gene expression profile may be determined utilizing oligonucleotide microarrays.
In another embodiment of the first aspect of the invention, the gene expression product is a polypeptide. Preferably, when the gene expression product is a polypeptide, the gene expression profile is determined utilizing antibodies.
In a preferred embodiment, the one or more informative genes are selected from the group consisting of the genes in Table 1 and the genes in Table 2.
The invention also features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a polypeptide sample from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control, where the gene expression product is a polypeptide. Increased expression of the gene expression product in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In one embodiment, the polypeptide sample is derived from skeletal muscle tissue. In another embodiment, the gene expression profile is determined utilizing antibodies. In a preferred embodiment, the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
In addition, the invention features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a nucleic acid sample from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control. Decreased expression of the gene in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In one embodiment, the nucleic acid sample is derived from skeletal muscle tissue. In other embodiments, the gene expression product is DNA or mRNA. Preferably, when the gene expression product is DNA or mRNA, the gene expression profile is determined utilizing specific hybridization probes. For example, the gene expression profile may be determined utilizing oligonucleotide microarrays.
In another embodiment, the gene expression product is a polypeptide. Preferably, when the gene expression product is a polypeptide, the gene expression profile is determined utilizing antibodies.
In a preferred embodiment, the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
The invention also features a method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising obtaining a polypeptide sample from said individual; and determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control, where the gene expression product is a polypeptide. Decreased expression of the gene expression product in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In one embodiment, the polypeptide sample is derived from skeletal muscle tissue. In another embodiment, the gene expression profile is determined utilizing antibodies. In a preferred embodiment, the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
The invention also features a method of identifying a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising the steps of: a) providing a cell or cell lysate sample; b) contacting the cell or cell lysate sample with a candidate compound; and c) detecting an increase in expression of at least one informative gene having decreased expression in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. A candidate compound that increases the expression of the informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In one embodiment, the cell or cell lysate sample is derived from skeletal muscle tissue. In another embodiment, the cell or cell lysate sample is derived from a cultured cell. In other embodiments, gene expression is determined by assessing the DNA or mRNA level of the gene. Preferably, the DNA or mRNA level is determined utilizing specific hybridization probes. For example, the DNA or mRNA level may be determined utilizing oligonucleotide microarrays.
In another embodiment, gene expression is determined by assessing the polypeptide level encoded by the informative gene. Preferably, gene expression is determined utilizing antibodies.
In a preferred embodiment, the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
In addition, the invention features a method of identifying a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising the steps of: a) providing a cell or cell lysate sample; b) contacting the cell or cell lysate sample with a candidate compound; and c) detecting a decrease in expression of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus. A candidate compound that decreases the expression of the informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In one embodiment, the cell or cell lysate sample is derived from skeletal muscle tissue. In another embodiment, the cell or cell lysate sample is derived from a cultured cell. In other embodiments, gene expression is determined by assessing the DNA or mRNA level of the gene. Preferably, the DNA or mRNA level is determined utilizing specific hybridization probes. For example, the DNA or mRNA level may be determined utilizing oligonucleotide microarrays.
In another embodiment, gene expression is determined by assessing the polypeptide level encoded by the informative gene. Preferably, gene expression is determined utilizing antibodies.
In a preferred embodiment, the one or more informative genes is selected from the group consisting of the genes in Table 1 and the genes in Table 2.
The invention also features a method for modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus in a subject (e.g., an individual) by down-regulating in the subject at least one informative gene shown to be expressed in or expressed at increased levels in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, but not in normal individuals.
The invention also features a method for modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus in a subject (e.g., an individual) by up-regulating in the subject at least one informative gene shown not to be expressed in or expressed at reduced levels in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to normal individuals.
The invention also features oligonucleotide microarrays having immobilized thereon a plurality of oligonucleotide probes specific for one or more informative genes selected from the group consisting of the genes in Table 1 and the genes in Table 2.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows the expression profile of the 54 muscle biopsies from individuals with normal glucose tolerance versus individuals with disturbances in their glucose tolerance (type 2 diabetes and impaired glucose tolerance) (Group A). The plot shows the number of genes within various neighborhoods of the glucose tolerance distinction (+) together with curves indicating the mean (-), 1% (■ ) and 5%(" ") significance levels for the number of genes within corresponding neighborhoods of the randomly permuted class distinctions. Genes more highly expressed in tissue from control subjects with normal glucose homeostasis compared to the expression levels in individuals with disturbances in the glucose homeostasis is shown in the left panel. Those genes more highly expressed in tissue from individuals with disturbances in their glucose homeostasis compared to the expression levels in the control subjects with normal glucose homeostasis is shown in the right panel.
Figure 2 shows the expression profile of the 28 muscle biopsies from individuals before the euglycemic hyperinsulinemic clamp versus individuals after the euglycemic hyperinsulinemic clamp (Group B.). The plot shows the number of genes within various neighborhoods of the glucose tolerance distinction (+) together with curves indicating the mean (-), 1%( ) and 5%("") significance levels for the number of genes within corresponding neighborhoods of the randomly permuted class distinctions. Genes more highly expressed in tissue from subjects before the euglycemic hyperinsulinemic clamp compared to the expression levels in tissue taken after the euglycemic hyperinsulinemic clamp is shown in the left panel. Those genes more highly expressed in tissue taken after the euglycemic hyperinsulinemic clamp compared to the expression levels in tissue from subjects before the euglycemic hyperinsulinemic clamp is shown in the right panel. DETAILED DESCRIPTION OF THE INVENTION
Type 2 diabetes mellitus is a metabolic disorder characterized by insulin resistance and pancreatic β-cell dysfunction. As described herein, the transcriptional signature associated in vivo with impaired glucose homeostasis was investigated. Muscle biopsies were obtained from 28 normoglycemic male subjects (age 66±9) and 26 male subjects with impaired glucose homeostasis (age 65±9) from Sweden taken during an euglycemic hyperinsulinemic clamp (Group A). To evaluate the acute effect of insulin, muscle biopsies were also obtained from 7 monozygotic twin pairs (4 female/10 male) before and during a euglycemic hyperinsulinemic clamp (Group B). Labeled cRNA from these individuals was analyzed on high density oligonucleotide arrays including over 6800 genes. Results from Group A were analyzed using k-NN statistics and Group B by a pair- wise comparison of each sample before and after insulin infusion. All p-values were assessed using permutations, although it should be noted that the number of permutations in Group B is limited due to thet smaller numbers of samples. The results show that the expression profile in skeletal muscle differs significantly between individuals with normal and impaired glucose homeostasis. These differentially expressed candidate genes may reflect transcriptional events important in the development and maintenance of impaired glucose homeostasis.
The present invention is directed to methods for predicting phenotypic classes of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, such as the presence or absence of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, or the identification of compounds that modulate impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, based on gene expression profiles. In one aspect, the invention involves identifying an an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus by obtaining a nucleic acid or polypeptide sample from said individual, and determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus relative to a control. Increased expression of the informative gene is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. Alternatively, identification of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus may be made by obtaining a nucleic acid or polypeptide sample from said individual, and determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a control. Decreased expression of the informative gene is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
By "gene expression profile" is meant the level or amount of gene expression of particular genes, for example, informative genes, as assessed by methods described herein. The gene expression profile can comprise data for one or more informative genes and can be measured at a single time point or over a period of time. For example, the gene expression profile can be determined using a single informative gene, or it can be determined using two or more informative genes, three or more informative genes, five or more informative genes, ten or more informative genes, twenty-five or more informative genes, or fifty or more informative genes. A gene expression profile may include expression levels of genes that are not informative, as well as informative genes. Phenotype classification can be made by comparing the gene expression profile of the sample with respect to one or more informative genes with one or more gene expression profiles of a control (e.g., in a database). Using the methods described herein, expression of numerous genes can be measured simultaneously. The assessment of numerous genes provides for a more accurate evaluation of the sample because there are more genes that can assist in classifying the sample. A gene expression profile may involve only those genes that are increased in expression in a sample, only those genes that are decreased in expression in a sample, or a combination of genes that are increased and decreased in expression in a sample. As used herein, "informative genes," refers to a gene or genes whose expression correlates with a particular phenotype. Expression profiles obtained for informative genes can be used to determine, for example, whether an individual has, or is at risk of developing, impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, or whether a candidate compound increases or decreases gene expression in a sample. Samples can be classified according to their broad expression profile, or according to the expression levels of particular informative genes. The genes that are relevant for classification are referred to herein as "informative genes." Not all informative genes for a particular class distinction must be assessed in order to classify a sample. Similarly, the set of informative genes that characterize one phenotypic effect may or may not be the same as the set of informative genes for a different phenotypic effect. For example, a subset of the informative genes that demonstrate a high correlation with a class distinction (e.g., impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus) can be used in identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. This subset can be, for example, one or more genes, two or more genes, three or more genes, five or more genes, ten or more genes, twenty-five or more genes, or fifty or more genes. The informative genes that characterize other classification categories such as, for example, a candidate compound that modulates impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, can be the same or different from the informative genes that identify an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. Typically the accuracy of the classification increases with the number of informative genes that are assessed.
Informative genes include, but are not limited to, the particular genes shown in Table 1 and Table 2.
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As used herein, "gene expression products" are proteins, polypeptides, or nucleic acid molecules (e.g., mRNA, tRNA, rRNA, cDNA, or cRNA) that result from transcription or translation of genes. The present invention can be used effectively to analyze proteins, polypeptides, or nucleic acid molecules that are the result of transcription or translation, particularly of informative genes identified herein. The nucleic acid molecule levels measured can be derived directly from the gene or, alternatively, from a corresponding regulatory gene or regulatory sequence element. All forms of gene expression products can be measured. For example, the nucleic acid molecule can be transcribed to obtain an RNA gene expression product. If desired, the transcript can be translated using, for example, standard in vitro translation methods to obtain a polypeptide gene expression product. Polypeptide gene expression products can be used in protein binding assays, for example, antibody assays, or in nucleic acid binding assays, routinely used in the art. Additionally, variants of genes and gene expression products including, for example, spliced variants and polymorphic alleles, can be measured. Similarly, gene expression can be measured by assessing the level of a polypeptide or protein or derivative thereof translated from mRNA. The sample to be assessed can be any sample that contains a gene expression product. Suitable sources of gene expression products, e.g., samples, can include intact cells, lysed cells, cellular material for determining gene expression, or material containing gene expression products. Examples of such samples are skeletal muscle tissue, cells derived from skeletal muscle, nucleic acids or polypeptides derived from skeletal muscle tissue, blood, plasma, lymph, urine, tissue, mucus, sputum, saliva, or other cell samples. Methods of obtaining such samples are known in the art.
By "increased expression" is meant the level of a gene expression product is made higher and/or the activity of the gene expression product is enhanced. Preferably, the increase is by at least 1.5-fold, more preferably the increase is at least 2-fold, 5-fold, or 10-fold, and most preferably, the increase is at least 20-fold, relative to a control.
By "decreased expression" is meant the level of a gene expression product is made lower and/or the activity of the gene expression product is lowered. Preferably, the decrease is at least 25%, more preferably, the decrease is at least 50%, 60%, 70%, 80%), or 90%) and most preferably, the decrease is at least one-fold, relative to a control sample.
Genes that are particularly relevant for classification, i.e., demonstrate a different expression profile in different classification categories, have been identified as a result of work described herein and are shown in Table 1 and Table 2.
In one embodiment, the gene expression product is a protein or polypeptide. As used herein, by "polypeptide" is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation. Examples of polypeptides include, but are not limited to, proteins. In this embodiment the determination of the gene expression profile is made using techniques for protein detection and quantitation known in the art. For example, antibodies that specifically interact with the protein or polypeptide expression product of one or more informative genes can be obtained using methods that are routine in the art. The specific binding of such antibodies to protein or polypeptide gene expression products can be detected and measured by methods known in the art, for example, Western blot analysis or ELISA techniques. hi a preferred embodiment, the gene expression product is a nucleic acid, for example, DNA or mRNA, and the gene expression levels are obtained by contacting the sample with a suitable microarray on which one or more probes specific for all or a subset of the informative genes have been immobilized, and deteπnining the extent of hybridization of the nucleic acid in the sample to the probes on the microarray. Such microairays are also within the scope of the invention. Examples of methods of making oligonucleotide microarrays are described, for example, in WO 95/1 1995. Other methods are readily known to the skilled artisan.
The gene expression value measured or assessed is the numeric value obtained from an apparatus that can measure gene expression levels. Gene expression levels refer to the amount of expression of the gene expression product, as described herein. The values are raw values from the apparatus, or values that are optionally re-scaled, filtered and/or normalized. Such data is obtained, for example, from a GeneChip® probe array or Microarray (Affymetrix, Inc.; U.S. Patent Nos. 5,631,734, 5,874,219, 5,861,242, 5,858,659, 5,856,174, 5,843,655, 5,837,832, 5,834,758, 5,770,722, 5,770,456, 5,733,729, 5,556,752, all of which are incorporated herein by reference in their entirety), and the expression levels are calculated with software (e.g., Affymetrix GENECHIP software). For example, nucleic acids (e.g., mRNA or DNA) from a sample that has been subjected to particular stringency conditions hybridize to the probes on the chip. The nucleic acid to be analyzed (e.g., the target) is isolated, amplified and labeled with a detectable label, (e.g., 32P or fluorescent label) prior to hybridization to the arrays. After hybridization, the arrays are inserted into a scanner that can detect patterns of hybridization. These patterns are detected by detecting the labeled target now attached to the microarray, e.g., if the target is fluorescently labeled, the hybridization data are collected as light emitted from the labeled groups. Since labeled targets hybridize, under appropriate stringency conditions known to one of skill in the art, specifically to complementary oligonucleotides contained in the microarray, and since the sequence and position of each oligonucleotide in the array are known, the identity of the target nucleic acid applied to the probe is determined.
Quantitation of gene profiles from the hybridization of a labeled nucleic acid microarray can be performed by scanning the microarray to measure the amount of hybridization at each position on the microarray with an Affymetrix seamier (Affymetrix, Santa Clara, CA ). For each stimulus a time series of nucleic acid levels (C={Cl,C2,C3,...Cn}) and a corresponding time series of nucleic acid levels (M={Ml,M2,M3,...Mn|) in control medium in the same experiment as the stimulus is obtained. Quantitative data is then analyzed. Hybridization analysis using microarray is only one method for obtaining gene expression values. Other methods for obtaining gene expression values known in the art or developed in the future can be used with the present invention. Once the gene expression values are determined, the sample can be classified.
Once the gene expression levels of the sample are obtained, the levels are compared or evaluated against a model or control sample(s), and then the sample is classified, for example, based one whether a particular informative gene in the sample exhibits increased or decreased expression. The evaluation of the sample determines whether or not the sample is assigned to a particular phenotypic class.
The correlation between gene expression and class distinction can be determined using a variety of methods. Methods for defining classes and classifying samples are described, for example, in U.S. Patent Application Serial No. 09/544,627, filed April 6, 2000 by Golub et al., the teachings of which are incorporated herein by reference in their entirety. The information provided by the present invention, alone or in conjunction with other test results, aids in sample classification.
The present invention also features methods for identifying compounds that modulate impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. Novel compounds identified as described herein are also the subject of the invention. Such methods involve contacting a sample, for example a cell, cell lysate, tissue, or tissue lysate, with a candidate compound, and detecting an increase in expression of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. A candidate compound that increases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. Alternatively, a compound that modulates impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can be identified by contacting a sample, for example, a cell, cell lysate, tissue, or tissue lysate with a candidate compound, and detecting a decrease in expression of at least one informative gene having increased expression in an an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. A candidate compound that decreases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. An increase or decrease in an informative gene may be identified using any of the methods described herein (or any analogous method known in the art). For example, oligonucleotide array systems described herein may be used to determine whether the addition of a test compound to a sample increases or decreases expression of an informative gene in that sample.
By "modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus" is meant increasing or decreasing the likelihood that impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus will occur or develop in a subject. The modulation of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus may be the result of contacting a sample (for example, a cell, tissue, cell or tissue lysate, nucleic acid, or polypeptide) with a candidate compound. Preferably, the sample is derived from skeletal muscle tissue. It will be appreciated that the degree of modulation provided by a candidate compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change or a therapeutically effective change in the degree or rate of disease development.
By a "candidate compound" is meant a molecule, be it naturally-occurring or artificially derived, that is surveyed for its effects on the gene expression profile of an informative gene, employing methods described herein. Examples of candidate compounds include, but are not limited to peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, and combinations thereof.
By "increasing gene expression" is meant raising the level of expression, and/or the activity, of one or more informative genes in a cell, tissue, cell lysate, or tissue lysate sample relative to a control sample. An increase in gene expression may occur, for example, when the sample is contacted with a candidate compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. The control sample may be a cell, tissue, cell lysate, or tissue lysate that was not contacted with the candidate compound or that was contacted with candidate compound vehicle only. Preferably, the increase is at least 1.5-fold, more preferably the increase is at least 2-fold, 5-fold, or 10-fold, and most preferably, the increase is at least 20-fold, relative to a control sample.
By "decreasing gene expression" is meant lowering the level or expression of, and/or the activity of, one or more informative genes in a cell, tissue, cell lysate, or tissue lysate sample relative to a control sample. A decrease in gene expression may occur, for example, when the sample is contacted with a candidate compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. The control sample may be a cell, tissue, cell lysate, or tissue lysate that was not contacted with the candidate compound or that was contacted with candidate compound vehicle only. Preferably, the decrease in gene expression of an informative gene is at least 25%>, more preferably, the decrease is at least 50%>, 60%o, 70%), 80%), or 90%) and most preferably, the decrease is at least one-fold, relative to a control sample.
The expression level of an infoπnative gene may be modulated by modulating transcription, translation, or mRNA or protein turnover, or the activity of the gene expression product, and such modulation may be detected using known methods for measuring mRNA and protein levels and activities, e.g., oligonucleotide microarray hybridization, RT-PCR, and ELISA and nucleic acid and protein binding assays.
A compound that increases the expression level of a gene that is decreased in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can be useful for treating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. In addition, a compound that decreases the expression level of a gene that is increased in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can also be useful for treating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
While the above described candidate compound screening methods are designed primarily to identify candidate compounds that may be used to decrease impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, identification of candidate compounds that increase impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus is also a feature of the present invention. Such candidate compound identification methods involve contacting a sample, for example, a cell, cell lysate, tissue, or tissue lysate with a candidate compound, and detecting an increase in expression of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus. A candidate compound that increases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
Alternatively, a compound that modulates impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus can be identified by contacting a sample, for example, a cell, cell lysate, tissue, or tissue lysate with a candidate compound, and detecting a decrease in expression of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. A candidate compound that decreases expression of such an informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. These candidate compound identification methods may be used for identifying compounds that increase impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, or the risk of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. Such compounds may be identified as compounds to which exposure should be minimized in order to decreased one's likelihood of developing impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. hi general, novel drugs for modulation of disease development can be identified from large libraries of natural products or synthetic (or semi-synthetic) extracts or chemical libraries according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the exemplary methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-. peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available, e.g., Chembridge (San Diego, CA). Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FL), and PharmaMar, U.S.A. (Cambridge, MA). In addition, natural and synthetically produced libraries are generated, if desired, according to methods known in the art, e.g., by standard extraction and fractionation methods. Furthermore, if desired, any library or compound is readily modified using standard chemical, physical, or biochemical methods.
In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their modulatory activities should be employed whenever possible.
When a crude extract is found to modulate (i.e., stimulate (increase) or inhibit (decrease)) impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, further fractionation of the positive lead extract is desirable to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having an activity that increases or deceases. The same assays described herein for the detection of activities in mixtures of compounds can be used to purify the active component and to test derivatives thereof. Methods of fractionation and purification of such heterogenous extracts are known in the art. If desired, compounds shown to be useful agents for treatment are chemically modified according to methods known in the art. Compounds identified as being of therapeutic value maybe subsequently analyzed using animal models for diseases.
Informative genes identified as described herein can also be targeted in methods of modulating impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus. For example, expression of at least one informative gene shown to be expressed in or expressed at increased levels in, an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, but not in normal individuals can be down-regulated in a method of inhibiting impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus. Alternatively, expression of at least one informative gene shown not to be expressed in, or which are expressed at reduced levels in, an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, relative to normal intestinal samples can be upregulated in a method of inhibiting impaired glucose tolerance, impaired glucose homeostasis and or type 2 diabetes mellitus. Compounds identified by methods described herein, for example, can be utilized in methods of treatment of impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
The present invention also features arrays, for example, microarrays that have a plurality of oligonucleotide probes involved in impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus immobilized thereon. The oligonucleotide probe may be specific for one or more informative genes, selected from those in Table 1 and Table 2. Methods for making oligonucleotide microarrays are well known in the art, and are described, for example, in WO 95/11995, the entire teachings of which are hereby incorporated by reference.
The present invention also provides information regarding the genes that are important in impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, thereby providing additional targets for diagnosis and therapy. It is clear that the present invention can be used to generate databases comprising informative genes that will have many applications in medicine, research and industry; such databases are also within the scope of the invention.
The invention will be further described with reference to the following non- limiting examples. The teachings of all the patents, patent applications and all other publications and websites cited herein are incorporated by reference in their entirety. EXEMPLIFICATION Methods
RNA Extraction. mRNA from all 82 biopsies (Group A, n=54 and Group B, n=28) was extracted from the tissue using the guadinium thiocyanate method.
High density oligonucleotide arrays. The mRNA is converted into biotin labeled cRNA in a reverse transcription followed by an in vitro transcription. Subsequently, the labeled cRNA is hybridized onto a high density oligonucleotide array where 3' end specific probes from more than 6800 genes are immobilized on a solid phase (glass). The biotin labeled cRNA is the labeled by staining with a fluorescent streptavidin conjugate. The level of expression is then analyzed as intensity of fluorescence in a laser confocal scanner. Intensity values were scaled so that the overall fluorescence intensity of each chip was equivalent.
To evaluate the variation in the transcriptional process which may be associate diwth differences in glucose tolerance, variation in gene transcripts in a sample of 54 skeletal muscle biopsies taken from age-, gender- and BMI-matched subjects were studied. The biopsies were taken during a 2-hour euglycemic hyperinsulinemic clamp to control for the effects of intra-individual differences in glucose and insulin levels. Results of the case/control designed sample of men with different glucose tolerance were analyzed using Nearest Neighbor statistics. 209 genes were found to be differentially expressed in skeletal muscle from subjects with impaired glucose homeostasis compared to normoglycemic controls (p<0.05). All p- values were assessed using permutations.
To evaluate the effect of insulin on the transcriptome in in vivo skeletal muscle, expression profiles from skeletal muscle biopsies taken before and during a 2 or 3 hour euglycemic hyperinsulinemic clamp from 7 monozygotic twin pairs (4 women/ 10 men) discordant for their glucose status were compared. In the sample of pair-wise matched samples taken before and after the euglycemic hyperinsulinemic clamp, the results were analyzed by a pair- wise comparison of each sample before and after insulin infusion. Insulin infusion was found to trigger express of 89 genes (p<0.05). All p-values were assessed using permutations.

Claims

ClaimsWhat is claimed:
1. A method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising: a) obtaining a nucleic acid sample derived from said individual; and b) determining a gene expression profile from a gene expression product of at least one informative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a normal individual; wherein increased expression of the informative gene in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
2. The method of Claim 1, wherein the nucleic acid sample is derived from skeletal muscle tissue.
3. The method of Claim 1, wherein the gene expression product is DNA or mRNA.
4. The method of Claim 1, wherein the gene expression profile is determined utilizing specific hybridization probes.
5. The method of Claim 1, wherein the gene expression profile may be determined utilizing oligonucleotide microarrays.
6. The method of Claim 1, wherein the gene expression product is a polypeptide.
7. The method of Claim 1, wherein the gene expression profile is determined utilizing antibodies.
8. The method of Claim 1, wherein the one or more informative genes are selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
9. A method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising: a) obtaining a polypeptide sample from said individual; and b) determining a gene expression profile from a gene expression product of at least one infonτiative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a normal individual, wherein increased expression of the gene expression product in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
10. The method of Claim 9, wherein the gene expression product is a polypeptide.
11. The method of Claim 9, wherein the polypeptide sample is derived from skeletal muscle tissue.
12. The method of Claim 9, wherein the gene expression profile is determined utilizing antibodies.
13. The method of Claim 9, wherein one or more informative genes is selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
14. A method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising: a) obtaining a nucleic acid sample from said individual; and b) determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a normal individual; wherein decreased expression of the gene in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
15. The method of Claim 14, wherein the nucleic acid sample is derived from skeletal muscle tissue.
16. The method of Claim 14, wherein the gene expression product is DNA or mRNA.
17. The method of Claim 14, wherein the gene expression profile is determined utilizing specific hybridization probes.
18. The method of Claim 14, wherein the gene expression profile may be determined utilizing oligonucleotide microarrays.
19. The method of Claim 14, wherein the gene expression product is a polypeptide.
20. The method of Claim 14, wherein the gene expression profile is determined utilizing antibodies.
21. The method of Claim 14, wherein one or more informative genes is selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
22. A method of identifying an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising: a) obtaining a polypeptide sample from said individual; and b) determining a gene expression profile from a gene expression product of at least one informative gene having decreased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to a normal individual; wherein decreased expression of the gene expression product in the sample is indicative of an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
23. The method of Claim 22, wherein where the gene expression product is a polypeptide.
24. The method of Claim 22, wherein the polypeptide sample is derived from skeletal muscle tissue.
25. The method of Claim 22, wherein the gene expression profile is determined utilizing antibodies.
26. The method of Claim 22, wherein one or more informative genes is selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
27. A method of identifying a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising: a) providing a cell or cell lysate sample; b) contacting the cell or cell lysate sample with a candidate compound; and c) detecting an increase in expression of at least one informative gene having decreased expression in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus; wherein a candidate compound that increases the expression of the informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
28. The method of Claim 27, wherein the cell or cell lysate sample is derived from skeletal muscle tissue.
29. The method of Claim 27, wherein the cell or cell lysate sample is derived from a cultured cell.
30. The method of Claim 27, wherein gene expression is determined by assessing the DNA or mRNA level of the gene.
31. The method of Claim 27, wherein the DNA or mRNA level is determined utilizing specific hybridization probes.
32. The method of Claim 27, wherein the DNA or mRNA level is determined utilizing oligonucleotide microarrays.
33. The method of Claim 27, wherein gene expression is determined by assessing the polypeptide level encoded by the informative gene.
34. The method of Claim 27, wherein gene expression is determined utilizing antibodies
35. The method of Claim 27, wherein one or more informative genes is selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
36. A method of identifying a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus, comprising: a) providing a cell or cell lysate sample; b) contacting the cell or cell lysate sample with a candidate compound; and c) detecting a decrease in expression of at least one infoπnative gene having increased expression in an individual having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus; wherein a candidate compound that decreases the expression of the informative gene is a compound for use in modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus.
37. The method of Claim 36, wherein the cell or cell lysate sample is derived from skeletal muscle tissue.
38. The method of Claim 36, wherein the cell or cell lysate sample is derived from a cultured cell.
39. The method of Claim 36, wherein gene expression is determined by assessing the DNA or mRNA level of the gene.
40. The method of Claim 36, wherein the DNA or mRNA level is determined utilizing specific hybridization probes.
41. The method of Claim 36, wherein the DNA or mRNA level is determined utilizing oligonucleotide microarrays.
42. The method of Claim 36, wherein gene expression is determined by assessing the polypeptide level encoded by the informative gene.
43. The method of Claim 36, wherein gene expression is determined utilizing antibodies
44. The method of Claim 36, wherein one or more informative genes is selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
45. A method for modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus in an individual by down-regulating in the individual at least one informative gene shown to be expressed in or expressed at increased levels in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to normal individuals.
46. A method for modulating impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus in an individual by up-regulating in the individual at least one informative gene shown not to be expressed in or expressed at reduced levels in individuals having impaired glucose tolerance, impaired glucose homeostasis and/or type 2 diabetes mellitus relative to normal individuals.
7. An oligonucleotide microarrays having immobilized thereon a plurality of oligonucleotide probes specific for one or more informative genes selected from the group consisting of the genes in Figures 4A-4Q and the genes in Figures 5A-5R.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096875A1 (en) * 2009-02-27 2010-09-02 Verva Pharmaceuticals Ltd A drug identification protocol for type 2 diabetes based on gene expression signatures

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010096875A1 (en) * 2009-02-27 2010-09-02 Verva Pharmaceuticals Ltd A drug identification protocol for type 2 diabetes based on gene expression signatures

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