EP2575860A1 - Applications de diagnostic, de dépistage et thérapeutiques d'outils à base de la protéine ocab - Google Patents

Applications de diagnostic, de dépistage et thérapeutiques d'outils à base de la protéine ocab

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Publication number
EP2575860A1
EP2575860A1 EP11789025.1A EP11789025A EP2575860A1 EP 2575860 A1 EP2575860 A1 EP 2575860A1 EP 11789025 A EP11789025 A EP 11789025A EP 2575860 A1 EP2575860 A1 EP 2575860A1
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EP
European Patent Office
Prior art keywords
ocab
insulin resistance
individual
adipogenesis
glucose intolerance
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11789025.1A
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German (de)
English (en)
Other versions
EP2575860A4 (fr
Inventor
Frédéric PICARD
Sophie Carter
Louise Boivin
Evelyne Rondeau
Catherine Roy-Bellavance
Stéphanie MIARD
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Universite Laval
Original Assignee
Universite Laval
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Publication date
Application filed by Universite Laval filed Critical Universite Laval
Publication of EP2575860A1 publication Critical patent/EP2575860A1/fr
Publication of EP2575860A4 publication Critical patent/EP2575860A4/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/50—Mutagenesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4703—Regulators; Modulating activity
    • G01N2333/4706—Regulators; Modulating activity stimulating, promoting or activating activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/042—Disorders of carbohydrate metabolism, e.g. diabetes, glucose metabolism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/04—Endocrine or metabolic disorders
    • G01N2800/044—Hyperlipemia or hypolipemia, e.g. dyslipidaemia, obesity
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations

Definitions

  • Adipose tissue is a major contributor of inflammation in obesity. Since the demonstration of the presence of macrophages in white adipose tissue (WAT) of obese individuals, studies have shown that blocking macrophage infiltration in WAT prevents and/or attenuates the development of insulin resistance and adipocyte dysfunction brought by a high calorie feeding regimen. T lymphocytes precede and drive Th1 macrophage influx in WAT. How WAT responds to overnutrition by producing pro- inflammatory cytokines and chemokines has been an important step towards linking obesity and insulin resistance. This process activates the recruitment, infiltration and adhesion of many cell types involved in host defense, including Th1 macrophages and T lymphocytes, which occurs most aggressively in visceral WAT (vWAT).
  • WAT white adipose tissue
  • the gold standard for measuring insulin resistance is the hyperinsulinemic/euglycemic clamp.
  • This assay requires the infusion of insulin and glucose simultaneously, at a constant glycemic level. If high levels of glucose have to be infused to keep the glycemia level constant, then it is considered that the individual responds normally to insulin. However, if little glucose can be infused to keep the glycemia constant, it is concluded that the individual is resistant to insulin. This assay is cumbersome, labor-intensive and cannot be amenable to high-throughput methods.
  • Another contributing factor to diabetes is the accumulation of fat in an individual (also referred to as adipogenesis).
  • adipogenesis Another contributing factor to diabetes is the accumulation of fat in an individual (also referred to as adipogenesis).
  • a novel therapeutic target associated with insulin resistance and/or adipogenesis could be used to assess insulin resistance and/or adipogenesis in an individual. Such assessment can be useful to engage early treatment to prevent the onset of diabetes.
  • the novel therapeutic target could also be used to screen for novel compounds for the treatment of insulin resistance and/or adipogenesis.
  • the present application identifies a marker, OcaB, which is downregulated before or during adipogenesis and/or insulin resistance.
  • This marker can be used in various diagnostic methods as well as in screening assays. The modulation of expression of this marker can also provide therapeutic effects.
  • the present application provides a method of characterizing an individual's susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the method comprises : measuring a parameter of an OcaB- based reagent in a biological sample from the individual to provide a test value; comparing the test value to a control value to determine if the test value is higher than, equal to or lower than the control value; and characterizing the individual.
  • the individual is characterized as being susceptible to develop adipogenesis, insulin resistance and/or glucose intolerance if the test value is lower than the control value; and as lacking susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance if the test value is equal to or higher than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in a biological sample of a control individual lacking susceptibility to develop adipogenesis and/or insulin resistance, and a pre-determined value associated with a lack of susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the individual is a human.
  • the OcaB-based reagent is an OcaB polypeptide or a biologically active variant thereof.
  • the parameter of the OcaB-based reagent is the level of expression of the OcaB polypeptide or the biologically active variant thereof.
  • the parameter of the OcaB-based reagent is the level of activity of the OcaB polypeptide or the biologically active variant thereof.
  • the biological activity is the formation of a complex between the OcaB polypeptide or the biologically active variant thereof and at least one of the following partner: Oct-1 , Oct-2, SRC-1 , RXR and PPARy.
  • the activity is a decrease in the expression of at least one of the following genes: aP2, LPL, PPARy, Glut4, ATGL, Adiponectin, Leptin, C/ ⁇ , Perilipin and HSL.
  • the OcaB-based reagent is a nucleotide encoding an OcaB polypeptide or a biologically active variant thereof.
  • the parameter of the OcaB-based reagent is the level of expression of the nucleotide encoding the OcaB polypeptide or the biologically active variant thereof.
  • the parameter of the OcaB-based reagent is the level or stability of the nucleotide encoding the OcaB polypeptide or the biologically active variant thereof.
  • the biological sample is a cell.
  • the cell is an adipocyte, such as an adipocyte from white adipose tissue.
  • the present application provides a method of diagnosing insulin resistance and/or glucose intolerance in an individual.
  • the method comprises measuring a parameter of an OcaB-based reagent in a biological sample from the individual to provide a test value; comparing the test value to a control value to determine if the test value is higher than, equal to or lower than the control value; and characterizing the presence or absence of insulin resistance and/or glucose intolerance in the individual.
  • the individual is considered having insulin resistance and/or glucose intolerant when the test value is lower than the control value; or as being insulin responsive and/or glucose tolerant if the test value is equal to or higher than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in a biological sample of a control individual lacking insulin resistance and/or glucose intolerance, and a pre-determined value associated with a lack of insulin resistance and/or glucose intolerance.
  • the present application provides a method of characterizing the effectiveness of an agent for the prevention, treatment and/or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance in an individual.
  • the method comprises measuring a parameter of an OcaB-based reagent in a biological sample from the individual to provide a test value; comparing the test value to a control value to determine if the test value is higher than, equal to or lower than the control value; and characterizing the agent.
  • the agent is characterized as being effective for the prevention, treatment and/or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance if the test value is higher than the control value; or as lacking effectiveness for the prevention, treatment and/or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance if the test value is equal to or lower than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in a further biological sample obtained from the individual prior the biological sample, the parameter of the OcaB-based reagent in the absence of the agent, the parameter of the OcaB-based reagent in the presence of a control agent lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance and a pre-determined value associated with a lack of ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • Embodiments concerning the individual, the OcaB-based reagent, the parameter of the OcaB-based reagent and the biological sample described above can be applied in the method described herein.
  • the present application provides method of characterizing an agent's ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in an individual.
  • the method comprises combining an agent with an OcaB-based reagent; measuring a parameter of the OcaB-based reagent in the presence of the agent to provide a test value; comparing the test value with a control value to determine if the test value is higher than, equal to or lower than the control value; and characterizing the agent.
  • the agent is characterized as having the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance if the test value is higher than the control value; and as lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance if the test value is lower than or equal to the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in the absence of the agent, the parameter of the OcaB-based reagent in the presence of a control agent lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual and a pre-determined value associated with a lack of prevention, treatment and/or alleviation of the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • Embodiments concerning the individual, the OcaB-based reagent, the parameter of the OcaB-based reagent and the biological sample described above can be applied in the method described herein.
  • the present application provides a method of treating or alleviating the symptoms associated with adipogenesis, insulin resistance and/or glucose intolerance in an individual in need thereof, said method comprising administering to the individual an effective amount of an agent capable of increasing the expression of an ocab nucleic acid and/or the activity of an OcaB polypeptide thereby treating or alleviating the symptoms associated with adipogenesis and/or insulin resistance in the individual.
  • the individual is a human.
  • the agent is at least one of a nucleotide encoding an OcaB polypeptide, and OcaB polypeptide, pioglitazone and combinations thereof.
  • the present application provides an agent capable of increasing the expression of an ocab nucleic acid and/or the activity of an OcaB polypeptide for the treatment or the alleviation of the symptoms associated with adipogenesis, insulin resistance and/or glucose intolerance in an individual.
  • the individual is a human.
  • the agent is at least one of a polynucleotide encoding an OcaB polypeptide, an OcaB polypeptide, pioglitazone and combinations thereof.
  • the present application provides a software product embodied on a computer readable medium and comprising instructions for characterizing an individual's susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the instructions comprise: a receiving module for receiving a test value of a parameter of an OcaB-based reagent in a biological sample of the individual; a comparison module for determining if the test value is lower than, equal to or higher than a control value and generating a corresponding output; and a characterization module receiving the corresponding output from the comparison module and adapted to determine the individual's susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the individual is characterized as susceptible to develop adipogenesis, insulin resistance and/or glucose intolerance if the test value is lower than the control value; and as lacking susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance if the test value is equal to or higher than the control value.
  • the control value is at least one of: the parameter of the OcaB-based reagent in a biological sample of an individual lacking susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance, and a pre-determined value associated with a lack of susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the present application provides a software product embodied on a computer readable medium and comprising instructions for diagnosing insulin resistance and/or glucose intolerance in an individual.
  • the product comprises a receiving module for receiving a test value of a parameter of an OcaB- based reagent in a biological sample of the individual; a comparison module for determining if the test value is lower than, equal to or higher than a control value and generating a corresponding output; and a characterization module receiving the corresponding output from the comparison module and adapted to determine the presence or absence of insulin resistance and/or glucose intolerance in the individual.
  • the insulin resistance and/or glucose intolerance is considered present in the individual if the test value is lower than the control value; and the insulin resistance and/or glucose intolerance is considered absent in the individual if the test value is equal to or higher than the control value.
  • the control value is at least one of: the parameter of the OcaB-based reagent in a biological sample of an individual lacking insulin resistance and/or glucose intolerance, and a predetermined value associated with a lack of insulin resistance and/or glucose intolerance.
  • the present application provides a software product embodied on a computer readable medium and comprising instructions for characterizing the effectiveness of an agent for the prevention, treatment and/or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance in an individual.
  • the product comprises: a receiving module for receiving a test value of a parameter of an OcaB-based reagent in a biological sample of the individual; a comparison module for determining if the test value is lower than, equal to or higher than a control value and generating a corresponding output; and a characterization module receiving the corresponding output from the comparison module and adapted to determine the effectiveness of an agent for the prevention, treatment and/or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • the agent is characterized as effective to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual if the test value is higher than the control value; and the agent is characterized as lacking effectiveness to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual if the test value is equal to or lower than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in a further biological sample obtained from the individual prior the biological sample, the parameter of the OcaB-based reagent in the absence of the agent, the parameter of the OcaB-based reagent in the presence of a control agent lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance and a pre-determined value associated with a lack of ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • Embodiments concerning the individual, the OcaB-based reagent, the parameter of the OcaB-based reagent and the biological sample described above can be applied in the method described herein.
  • the present application provides a software product embodied on a computer readable medium and comprising instructions for characterizing an agent's ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • the product comprises: a receiving module for receiving a test value of a parameter of an OcaB- based reagent in a biological sample of the individual; a comparison module for determining if the test value is lower than, equal to or higher than a control value and generating a corresponding output; and a characterization module receiving the corresponding output from the comparison module and adapted to determine the ability of the agent to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • the agent is characterized as able to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual if the test value is higher than the control value; and the agent is characterized as lacking ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual if the test value is equal to or lower than the control value.
  • control value is at least one of: the parameter of the OcaB- based reagent in the absence of the agent, the parameter of the OcaB-based reagent in the presence of a control agent lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual and a pre-determined value associated with a lack of prevention, treatment and/or alleviation of the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • Embodiments concerning the individual, the OcaB-based reagent, the parameter of the OcaB-based reagent and the biological sample described above can be applied in the method described herein.
  • the present application provides a diagnostic system for characterizing an individual's susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the diagnostic system comprises: a reaction vessel adapted to receive an OcaB-based reagent and a biological sample from the individual; the OcaB-based reagent; a processor in a computer system; a memory accessible by the processor; and at least one application coupled to the processor.
  • the at least on application is configured for: receiving a test value of a parameter of the OcaB-based reagent in the presence of the biological sample; comparing the test value with a control value to determine if the test value is lower than, equal to or higher than the control value; and characterizing the individual as susceptible to develop adipogenesis, insulin resistance and/or glucose intolerance if the test value is lower than the control value; and as lacking susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance if the test value is equal to or higher than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in a biological sample of an individual lacking susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance, and a pre-determined value associated with a lack of susceptibility to develop adipogenesis, insulin resistance and/or glucose intolerance.
  • the individual, the OcaB-based reagent, the parameter of the OcaB-based reagent and the biological sample described above can be applied in the method described herein.
  • the present application provides a diagnostic system for diagnosing insulin resistance and/or glucose intolerance in an individual.
  • the diagnostic system comprises: a reaction vessel adapted to receive an OcaB-based reagent and a biological sample from the individual; the OcaB-based reagent; a processor in a computer system; a memory accessible by the processor; and at least one application coupled to the processor.
  • the at least one application is configured for: receiving a test value of a parameter of the OcaB-based reagent in the presence of the biological sample; comparing the test value with a control value to determine if the test value is lower than, equal to or higher than the control value; and characterizing the insulin resistance and/or glucose intolerance as present in the individual if the test value is lower than the control value; and as absent from the individual if the test value is equal to or higher than the control value.
  • the control value is at least one of: the parameter of the OcaB-based reagent in a biological sample of an individual lacking insulin resistance and/or glucose intolerance, and a pre-determined value associated with a lack of insulin resistance and/or glucose intolerance.
  • the present application provides a diagnostic system for characterizing the effectiveness of an agent for the prevention, treatment and/or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance in an individual.
  • the diagnostic system comprises a reaction vessel adapted to receive an OcaB-based reagent and a biological sample from the individual; the OcaB- based reagent; a processor in a computer system; a memory accessible by the processor; and at least one application coupled to the processor.
  • the at least one application is configured for: receiving a test value of a parameter of the OcaB-based reagent in the presence of the biological sample; comparing the test value with a control value to determine if the test value is lower than, equal to or higher than the control value; and characterizing the agent as effective in the prevention, treatment and/or alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance if the test value is higher than the control value; and as lacking effectiveness in the prevention, treatment and/or alleviation of symptoms in the individual if the test value is equal to or lower than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in a further biological sample obtained from the individual prior the biological sample, the parameter of the OcaB-based reagent in the absence of the agent, the parameter of the OcaB-based reagent in the presence of a control agent lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance and a pre-determined value associated with a lack of ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • the present application provides a screening system for characterizing an agent's ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in an individual.
  • the screening system comprises: a reaction vessel adapted to receive an OcaB-based reagent and the agent; the OcaB-based reagent; a processor in a computer system; a memory accessible by the processor; and at least one application coupled to the processor.
  • the at least one application is configured for: receiving a test value of a parameter of the OcaB-based reagent in the presence of the agent; comparing the test value with a control value to determine if the test value is lower than, equal to or higher than the control value; and characterizing the agent as able to prevent, treat and/or alleviate adipogenesis, insulin resistance and/or glucose intolerance in the individual if the test value is higher than the control value; and as lacking ability to prevent, treat and/or alleviate adipogenesis, insulin resistance and/or glucose intolerance in the individual if the test value is equal to or lower than the control value.
  • control value is at least one of: the parameter of the OcaB-based reagent in the absence of the agent, the parameter of the OcaB-based reagent in the presence of a control agent lacking the ability to prevent, treat and/or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual and a predetermined value associated with a lack of prevention, treatment and/or alleviation of the symptoms of adipogenesis, insulin resistance and/or glucose intolerance.
  • Embodiments concerning the individual, the OcaB-based reagent, the parameter of the OcaB-based reagent and the biological sample described above can be applied in the method described herein.
  • Fig. 1 illustrates the reduction of OcaB expression in obesity in WAT. Reduction of OcaB expression in obesity in WAT.
  • n 8 d.
  • HFHS high-fat, high-sucrose diet
  • Fig 2. illustrates the modulation of OcaB upon adipogenesis.
  • 3T3-L1 cells were stimulated to differentiate into adipocytes.
  • Cells were harvested at different time-points during the differentiation process to quantify mRNA (a) and protein (b) levels of OcaB.
  • results are shown as relative OcaB expression levels in function of cells that reached confluency (C) or of the number of days after the inductions of differentiation. * indicates a significant difference compared to the confluence stage (p ⁇ 0.05).
  • results are shown for the various proteins (OcaB, PPARy, Oct-1 , tubulin) in proliferating cells (P), cells that reached confluency (C) or in cells submitted for a number of days after inductions of differentiation. Note that OcaB expression is negatively associated with that of PPARy.
  • Fig. 3 illustrates that OcaB represses adipogenesis
  • a Oil Red O staining of differentiated mouse embryonic fibroblasts (MEFs) isolated from +/+, +/- and -/- OcaB embryos
  • b Oil Red O staining of differentiated pre-adipocytes isolated from visceral WAT of +/+ and -/- OcaB mice.
  • c Oil Red O staining of differentiated 3T3-L1 cells infected with control (pB) or OcaB retrovirus (pB-OcaB). Staining was performed on day 6 of the differentiation process. Magnification is 10X.
  • d-e Oil Red O staining of differentiated mouse embryonic fibroblasts isolated from +/+, +/- and -/- OcaB embryos
  • c Oil Red O staining of differentiated pre-adipocytes isolated from visceral WAT of +/+ and -/- OcaB mice.
  • pB pro
  • mRNA levels of adipocyte-related genes measured in cells described in a and b, respectively. Results are shown as relative mRNA levels in function of the various genes in +/+ (white bar) and -/- (grey bar) OcaB mouse. In d, * indicates a significant difference compared to wild-type (+/+) (p ⁇ 0.05).
  • Fig. 4 illustrates the binding of OcaB on the octamer sequence of promoters of adipocyte genes
  • EMSA in 3T3-L1 cells showing DNA binding of OcaB on the promoter of the adipocyte genes aP2 and leptin.
  • EMSA were performed using nuclear extracts from 3T3-L1 adipocytes and radiolabeled aP2-octamer and leptin-octamer probes as detailed in Table 2.
  • Non-radioactive (cold) probes were used for competition, ratio with respect to hot probe is shown.
  • OcaB interacts with PPARy.
  • Total protein extracts from 3T3-L1 cultured adipocytes (b) or subcutaneous white adipose tissue from human obese patients (c) were incubated in the presence of OcaB or PPARv antibodies to immunoprecipitate specific complexes. The presence of OcaB in these complexes was then revealed by western blotting.
  • IP with IgG is shown as negative control, d.
  • OcaB interacts with SRC-1 .
  • Total protein extracts from 3T3-L1 cultured adipocytes were used and co-IP described as above, e.
  • OcaB represses the transcriptional activity of PPARv.
  • Fig. 5 illustrates that OcaB promotes insulin sensitivity in vitro and in vivo.
  • a Lipolysis assay using adipocytes freshly isolated from +/+ (WT) and -/- (OcaB -/-) mouse WAT. Results are shown as mmol FFA release/10 6 cells for unstimulated control cells (white bar), norepinephrin-stimulated cells (grey bar) and for norepinephrin and insulin- stimulated cells (dotted bard).
  • Results are shown as relative OcaB (left panel) or cd20 (right panel) expression level in function of age (months) for vehicle-treated (white bar) and pioglitazone-treated (grey bar) animals. * indicates a significant difference compared to vehicle-treated animals (p ⁇ 0.05).
  • mRNA expression of OcaB and cd20 in omental (visceral - left-sided panels) and subcutaneous (right-sided panels) WAT from obese men aged 23 years ⁇ 1 year (white bar), 40 years ⁇ 1 year (dotted bar) and 59 years ⁇ 1 year (grey bar). Results are shown as relative mRNA levels in function of type of fat and age. n 10 per group. * indicates a significant difference compared to the younger group.
  • Fig. 8 illustrates the expression of OcaB (R148.3) mRNA in C. elegans.
  • b Lipid accumulation is increased in nematodes with OcaB (R148.3) knockdown. Lipids were stained by Oil Red O (top panels) and Nile Red colorations (lower panels) in adult worms at same age.
  • Picture shown are representative of 10 pictures randomly taken in 25 worms of each genotype (sham-treated, left-sided panels; OcaB knockdown, right-sided panels), c. Oil Red O staining of wild-type (L4440, left-sided panels) and OcaB knockdown (R148.3, right-sided panels) worms during their life cycle. Picture shown are representative of 10 pictures randomly taken in 25 worms of each genotype.
  • FIG. 9 illustrates that the impact of R148.3 on lifespan depends on the presence of daf-2 (insulin receptor) and age-1 (PI3K) but not on that of daf-16 (FOXO).
  • daf-2 insulin receptor
  • PI3K age-1
  • Daf-16 daf-16
  • Fig. 10 illustrates a proposed mechanism of action of OcaB.
  • OcaB protects WAT from fat accumulation and insulin resistance. In aging, despite this increase in OcaB, moderate weight gain and insulin resistance develop, suggesting that this mechanism, , is overcomed in obesity.
  • an OcaB-based reagent as a biomarker for insulin resistance and adipogenesis (associated or not with ageing).
  • the use of the ocab gene or the OcaB polypeptide as a therapeutic target for the treatment, prevention and/or alleviations of the symptoms associated with adipogenesis and/or insulin resistance.
  • OcaB-/- embryonic fibroblasts display increased adipogenic potential, whereas ectopic over-expression in 3T3-L1 cells inhibits differentiation into adipocytes.
  • OcaB- autonomous, negative impact of OcaB on adipogenesis is likely mediated through the transcriptional repression of octamer sequences in the promoter of adipogenic genes. Old mice and humans have higher vWAT OcaB mRNA levels than their younger controls. Using C.
  • OcaB is suggested as a transcriptional node linking adipose tissue accumulation and longevity.
  • Adipogenesis This process refers to the cellular proliferation of preadipocytes and/or the differentiation of preadipocytes into adipocytes.
  • Adipocytes also known as lipocytes and fat cells
  • preadipocytes are the cells that primarily compose adipose tissue, specialized in storing energy as fat.
  • preadipocytes are usually considered as undifferentiated fibroblasts that can be stimulated to form adipocytes.
  • a pre-adipocyte is a cell that has committed to the adipocyte lineage (i. e.
  • adipocyte has expressed some early pro-adipocyte genes), but does not: have the mature adipocyte phenotype of intracellular triglyceride accumulation, or expresses adipogenic transcription factors, proteins and enzymes.
  • Agonist This term, as used herein, refers to an agent that mimics or upregulates (e. g., increases, potentiates or supplements) an activity of a compound, e. g. an OcaB protein.
  • An agonist can be a wild-type protein or variant thereof having at least one bioactivity of the wild-type protein.
  • An agonist can also be a compound that upregulates expression of a gene or which increases at least one activity of a protein.
  • An agonist can also be a compound which increases the interaction of a polypeptide with another molecule, e. g. a binding partner.
  • a biological sample is a sample of an individual's bodily fluid, cells or tissues.
  • the biological sample preferably comprises an adipocyte from a white adipose tissue.
  • the biological sample can be used without prior modification in the diagnostic assays described herein.
  • the biological sample can be treated (mechanically, enzymatically, etc.) prior to the assay to optimize the measurement of the OcaB-based reagent.
  • impaired glucose tolerance is a pre- diabetic state of dysglycemia that is associated with insulin resistance and increased risk of cardiovascular pathology. IGT may precede type 2 diabetes mellitus by many years. IGT is also a risk factor for mortality.
  • impaired glucose tolerance is defined as two-hour glucose levels of 140 to 199 mg per dL (7.8 to 1 1.0 mmol) on the 75-g oral glucose tolerance test. An individual is said to be under the condition of IGT when he/she has an intermediately raised glucose level after 2 hours, but less than would qualify for type 2 diabetes mellitus.
  • the fasting glucose may be either normal or mildly elevated.
  • Insulin resistance is a condition in which body cells become less sensitive to the glucose-lowering effects of insulin. Insulin resistance in muscle and fat cells reduces glucose uptake (and so local storage of glucose as glycogen and triglycerides, respectively), whereas insulin resistance in liver cells results in reduced glycogen synthesis and storage and a failure to suppress glucose production and release into the blood. Insulin resistance normally refers to reduced glucose-lowering effects of insulin. However, other functions of insulin can also be affected. For example, insulin resistance in fat cells reduces the normal effects of insulin on lipids and results in reduced uptake of circulating lipids and increased hydrolysis of stored triglycerides. Increased mobilization of stored lipids in these cells elevates free fatty acids in the blood plasma.
  • glucose intolerance also referred to as a prediabetic state
  • glucose intolerance solely concerns in an individual and refers to circumstances where even an increase in insulin secretion by the pancreas does not lower the glucose concentration to a normal level.
  • An individual that is glucose intolerant has thus an elevated insulin level as well as an elevated glucose level. Glucose intolerance can easily be measured with an oral glucose tolerance test.
  • the OcaB-based reagent is a biological entity that is derived from the OcaB polypeptide or its encoding polynucleotide.
  • the OcaB- based reagent may be derived from various sources, such as, for example human (GenBank Accession No. NP_006226.2), mouse (GenBank Accession No. NP_035266) and C. elegans (GenBank Accession No. NP_871687.1 or NP_497667.1).
  • the expression and activity of OcaB is decreased during adipogenesis and/or the development of insulin resistance. This modulation in expression and consequently activity is observed in non-immune cells, particularly in cells from the white adipose tissue.
  • the OcaB-based reagent refers to polypeptide derived from OcaB as well as polynucleotides encoding them which are found in non-immune cells (such as adipocytes of the white adipose tissue).
  • Polynucleotides encoding OcaB In the assay provided herewith, a full length nucleotide sequence encoding the OcaB polypeptide or a fragment thereof can be used.
  • a "fragment" of a OcaB-encoding nucleotide sequence that encodes a biologically active portion (e.g. that retains OcaB specific transcription modulation activity) of OcaB protein will encode at least 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 225, 250 or 255 contiguous amino acids, or up to the total number of amino acids present in a full-length OcaB polypeptide. Fragments of the OcaB-encoding nucleotide sequence that are useful as specific hybridization probes and/or as specific PCR primers generally need not encode a biologically active portion of the OcaB polypeptide.
  • Nucleic acid molecules that are variants of the OcaB-encoding nucleotide sequences disclosed herein can also be used. "Variants" of OcaB nucleotide sequences include those sequences that encode OcaB proteins disclosed herein but that differ conservatively because of the degeneracy of the genetic code. These naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the OcaB proteins.
  • PCR polymerase chain reaction
  • nucleotide sequence variants of the invention will have at least 45%, 55%, 65%, 75%, 85%, 95%, or 98% identity to a particular nucleotide sequence disclosed herein.
  • a variant OcaB-encoding nucleotide sequence will encode an OcaB protein that has an amino acid sequence having at least 45%, 55%, 65%, 75%, 85%, 95%, or 98% identity to the amino acid sequence of OcaB protein disclosed herein. It will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of OcaB proteins may exist within a population (e.g., the human population).
  • Such genetic polymorphism in ocab gene may exist among individuals within a population due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations in ocab sequence that are the result of natural allelic variation and that do not alter the functional activity of OcaB proteins are intended to be used herein.
  • allelic variants of OcaB sequences that may exist in the population
  • changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded OcaB proteins, without altering the biological activity of the OcaB proteins.
  • Such mutations can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein.
  • Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR- mediated mutagenesis. Such variant nucleotide sequences are also encompassed.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • the promoter of the ocab gene operably linked to a reporter gene.
  • the reporter gene can encoded a protein that can be detected in the reaction vessel.
  • the reporter gene can be, for example, the ocab gene itself or any other gene encoding a protein that can be detected in the reaction vessel (for example the green fluorescent protein or the ⁇ -galactosidase protein).
  • the OcaB -reagent maybe the full-length OcaB polypeptide or a biologically active fragment of the OcaB polypeptide that retains its characteristic transcription modulation activity.
  • “Fragments” or “biologically active portions” of the OcaB polypeptide include polypeptide fragments comprising amino acid sequences sufficiently identical to or derived from the amino acid sequence of the OcaB polypeptide and exhibiting at least one activity of the OcaB polypeptide, but which include fewer amino acids than the full-length OcaB polypeptide.
  • biologically active portions comprise a domain or motif with at least one activity (such as lipase activity) of the OcaB polypeptide.
  • a biologically active portion of the OcaB polypeptide can be a polypeptide that is, for example, 10, 25, 50, 100, 150, 200 or 250 or more amino acids in length. Such biologically active portions can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native OcaB polypeptide.
  • the OcaB polypeptide is a transcription factor that binds to regulatory sequence(s) of various genes and modulate their expression. As shown herein, the OcaB polypeptide downregulates the expression of at least on gene associated with adipose tissue metabolism and insulin resistance, such as, for example, aP2, LPL, PPARy, Glut4, ATGL, Adiponectin, Leptin, C/ ⁇ , Perilipin and HSL. In an embodiment, the OcaB polypeptide can dowregulate the expression of more than one of the genes listed below. In order to mediate its transcription factor activity, OcaB can bind directly or indirectly (e.g.
  • OcaB in the form of a complex with other binding partners
  • OcaB can be associated with at least one binding partners such as, for example, Oct-1 , Oct- 2, RXR and PPARy.
  • OcaB can form a complex with more than one of its binding partner.
  • variants proteins or polypeptides having an amino acid sequence that is at least about 45%, 55%, 65%, preferably about 75%, 85%, 95%, or 98% identical to the OcaB polypeptide. Such variants generally retain the biological activity of the OcaB polypeptide. Variants include, but are not limited to, fragments and polypeptides that differ in amino acid sequence due to natural allelic variation or mutagenesis. The methods described herein can also rely on a OcaB polypeptide chimeric or fusion proteins. As used herein, the "chimeric protein” or “fusion protein” comprises the OcaB polypeptide operably linked to a non- OcaB polypeptide.
  • non-OcaB polypeptide is intended to refer to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially identical to the OcaB polypeptide, e.g., a protein that is different from the OcaB polypeptide and which is derived from the same or a different organism.
  • the OcaB polypeptide can correspond to all or a portion of the OcaB polypeptide.
  • the non-OcaB polypeptide can be fused to the N-terminus or C-terminus of the OcaB polypeptide.
  • compositions comprising: a pharmaceutically effective amount or therapeutically effective amount. These expressions refer to an amount (dose) effective in treating a patient. It is also to be understood herein that a "pharmaceutically effective amount” may be interpreted as an amount giving a desired therapeutic effect, either taken in one dose or in any dosage or route, taken alone or in combination with other therapeutic agents.
  • compositions that retain the biological effectiveness and properties of the therapeutic agent described herein. They are formed from suitable non-toxic organic or inorganic acids or organic or inorganic bases.
  • Sample acid-addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfamic acid, phosphoric acid and nitric acid, and those derived from organic acids such as p-toluenesulfonic acid, salicylic acid, methanesulfonic acid, oxalic acid, succinic acid, citric acid, malic acid, lactic acid, fumaric acid, and the like.
  • Sample base-addition salts include those derived from ammonium, potassium, sodium and, quaternary ammonium hydroxides, such as e.g., tetramethylammonium hydroxide.
  • the chemical modification of an agent into a salt is a well known technique which is used in attempting to improve properties involving physical or chemical stability, e.g., hygroscopicity, flowability or solubility of compounds.
  • the reaction vessel where the agent is combined with the OcaB- based reagent, can be an in vitro or in vivo environment.
  • the contact between the agent and the OcaB -based reagent must be made under conditions suitable and for a period of time that will enable the agent to interact with the OcaB-based reagent and possible modify at least one of its parameters.
  • Suitable in vitro environments can include, for example, a cell-free environment where a OcaB polypeptide, biologically active variant thereof or a fusion protein comprising the OcaB polypeptide is combined in a reaction media comprising the appropriate reagents to enable the assessment of the transcription factor activity of the OcaB polypeptide or variants thereof (buffers, substrates, additives, etc.).
  • Symptoms associates with adipogenesis and insulin resistance include, but are not limited to accelerated atherosclerosis, coronary heart disease, diabetes, dyslipidemia, hypertension, chronic inflammation and/or endothelial dysfunction.
  • pulmonary diseases such as abnormal pulmonary function, obstructive sleep apnea and hypoventilation syndrome
  • non-alcoholic fatty liver disease such as steatosis, steatohypatitis and cirrhosis
  • gall bladder disease such as gynecologic abnormalities (such as abnormal menses, infertility and polycystic ovarian syndrome), osteoarthritis, gout, idiopathic intracranial hypertension, stroke, cataracts, severe pancreatitis, cancer (such as breast, uterus, cervix, colon, esophagus, pancreas, kidney and prostate as well as phlebitis.
  • Diagnostic and screening methods are designed to capture the relationship between OcaB and insulin resistance, glucose intolerance and/or adipogenesis to generate valuable information about the individual that is being tested or the agent that is being screened.
  • a biological sample of an individual or an agent to be screened is combined in a reaction vessel with an OcaB-based reagent.
  • the reaction vessel can be any type of container comprising an OcaB-based reagent that can accommodate the measurement of an OcaB-based reagent parameter.
  • a suitable in vitro environment for the screening assay described herewith can be a cultured cell. Such cell should be able to maintain viability in culture.
  • the cultured cell(s) should (i) express a polynucleotide encoding OcaB or biologically active variant thereof (ii) express a OcaB-encoding polynucleotide or variant thereof or related chimeric protein and/or (iii) comprise the OcaB promoter region. In some instances, it may be advisable that the cell may also be able to respond to insulin's actions. If a primary cell culture is used, the cell may be isolated or in a tissue-like structure, for example, as part of an intact islets. In some embodiments, the cell that is being used is not a blood cell such as a lymphocyte B. A further suitable environment is a non-human model, such as an animal model). If the characterization of the agent occurs in a non-human model, then the model (such as a rodent or a worm) is administered with the agent. Various dosage and modes of administration maybe used to fully characterize the agent's ability to increase insulin secretion.
  • a measurement or value of a parameter of the OcaB-based reagent is made. This assessment may be made directly in the reaction vessel (by using a probe) or on a sample of such reaction vessel.
  • the measurement of the parameter of the OcaB-based reagent can be made either at the DNA level, the RNA level and/or the polypeptide level.
  • the measuring step can rely on the addition of a quantifier specific to the parameter to be assessed to the reaction vessel or a sample thereof.
  • the quantifier can specifically bind to a parameter of a OcaB-based reagent that is being assessed, such as, for example, a nucleotide product encoding OcaB or a OcaB polypeptide.
  • the amount of the quantifier that specifically bound (or that did not bind) to the OcaB-based reagent can be determined to provide a measurement of the parameter of the OcaB-based reagent.
  • the quantifier can be modified by a parameter of the OcaB-based reagent, such as, for example, the OcaB transcription factor activity.
  • the amount of modified (or unmodified) quantifier will be determine to provide a measurement of the parameter of the OcaB-based reagent.
  • the signal of the quantifier can be provided by a label that is either directly or indirectly linked to a quantifier.
  • the parameter that is measured can be the polypeptide transcription factor activity, the polypeptide quantity and/or stability.
  • the parameter can be the level of expression or stability of the OcaB-encoding nucleotide. Even though a single parameter is required to enable the characterization of the individual or the agent, it is also provided that more than one parameter of the OcaB-based reagent may be measured.
  • the transcription activity of the promoter associated with the OcaB gene can be assessed. This assessment can be made, for example, by placing a reporter vector (such as a luciferase reporter based assay) in the presence of the OcaB polypeptide (inside or outside a cell).
  • a reporter vector such as a luciferase reporter based assay
  • Such reporter vectors can includes, but are not limited to, the promoter region of the ocab gene (or fragment thereof) operably linked to a nucleotide encoding a reporter polypeptide (such as, for example, OcaB, ⁇ -galactosidase, green-fluorescent protein, yellow-fluorescent protein, etc.).
  • the promotion of transcription from the promoter of the OcaB gene is measured indirectly by measuring the transcription of the reporter polypeptide.
  • the quantifier is the reporter polypeptide and the signal associated to this quantifier that is being measured will vary upon the reporter polypeptide used.
  • the stability and/or the expression level of the OcaB-encoding nucleotide can be assessed by quantifying the amount of a OcaB-encoding nucleotide (for example using qPCR) or the stability of such nucleotide.
  • the expression of a nucleic acid encoding OcaB in a cell or tissue sample is monitored directly by hybridization to the nucleic acids specific for OcaB.
  • cell lines or tissues can be exposed to the agent to be tested under appropriate conditions and time, and total RNA or mRNA isolated, optionally amplified, and quantified. If the measurement of the parameter is performed at the polypeptide level, an assessment of OcaB biological activity can be performed.
  • OcaB is a transcription factor that dowregulates and/or inhibits at least one adipogenesis-related gene expression. As such, one of OcaB's biological activity is to bind to other transcription regulators (also referred to as binding partners) as well as to bind to its target sequences.
  • the reaction mixture can include, e. g. a co- factor, a substrate or other binding partner or potentially interacting fragment thereof.
  • exemplary binding partners include Oct-1 , Oct-2, SRC-1 , RXR and PPARy, or interacting fragments thereof.
  • the binding partner is a direct binding partner.
  • This type of assay can be accomplished, for example, by coupling one of the components, with a label such that binding of the labeled component to the other can be determined by detecting the labeled compound in a complex.
  • a component can be labeled with 125 l, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • a component can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • Competition assays can also be used to evaluate a physical interaction between a test compound and a target.
  • OcaB's activity can be indirectly measured by quantifying the expression levels of its target genes whose expression is modulated by the presence and activity of OcaB.
  • OcaB is usually considered a transcriptional activator of immunoglobulin genes.
  • OcaB is considered to repress the transcription of genes, therefore it is expected that the expression of its target genes is downregulated in the presence of OcaB. Therefore, OcaB's activity is negatively associated with the expression of its target genes.
  • Such targets include, but are not limited to, aP2, LPL, PPARv, Glut4, ATGL, adiponectin, leptin, C/EBPa, perilipin, PEPCK, resistin, PELP1 , E2F1 , HSL, SREBPI c and C/ ⁇ .
  • its targets gene are aP2, LPL, PPARy, Glut4, ATGL, Adiponectin, Leptin, C/EBPa, Perilipin and/or HSL.
  • OcaB's activity is measured indirectly by quantifying the expression of PPARv.
  • Cell-free screening assays usually involve preparing a reaction mixture of the target protein and the test compound under conditions and for a time sufficient to allow the two components to interact and bind, thus forming a complex that can be removed and/or detected.
  • the interaction between two molecules can also be detected, e. g. , using a fluorescence assay in which at least one molecule is fluorescently labeled.
  • a fluorescence assay in which at least one molecule is fluorescently labeled.
  • FET or FRET for fluorescence resonance energy transfer fluorescence energy transfer
  • a fluorophore label on the first "donor” molecule is selected such that its emitted fluorescent energy will be absorbed by a fluorescent label on a second "acceptor” molecule, which in turn is able to fluoresce due to the absorbed energy.
  • the "donor" protein molecule may simply utilize the natural fluorescent energy of tryptophan residues. Labels are chosen that emit different wavelengths of light, such that the "acceptor” molecule label may be differentiated from that of the "donor". Since the efficiency of energy transfer between the labels is related to the distance separating the molecules, the spatial relationship between the molecules can be assessed. In a situation in which binding occurs between the molecules, the fluorescent emission of the "acceptor" molecule label in the assay should be maximal. A FET binding event can be conveniently measured through standard fluorometric detection means well known in the art (e. g. , using a fluorimeter).
  • FP fluorescence polarization
  • FP fluorescence polarization
  • a binding interaction is detected by a change in molecular size of the labeled component. The size change alters the tumbling rate of the component in solution and is detected as a change in FP.
  • the measuring step can rely on the use of real-time Biomolecular Interaction Analysis (BIA). "Surface plasmon resonance" or "BIA” detects biospecific interactions in real time, without labeling any of the interactants (e. g., BIAcore).
  • BIOA Biomolecular Interaction Analysis
  • the OcaB-reagent is anchored onto a solid phase.
  • the OcaB-based reagent-related complexes anchored on the solid phase can be detected at the end of the reaction, e. g. , the binding reaction.
  • the OcaB-based reagent can be anchored onto a solid surface, and the test compound, (which is not anchored), can be labeled, either directly or indirectly, with detectable labels discussed herein. Examples of such solid phase include microtiter plates, test tubes, array slides, beads and micro-centrifuge tubes.
  • a OcaB chimeric protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • the vessels are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of OcaB binding or activity determined using standard techniques.
  • the non-immobilized component (agent or biological agent) is added to the coated surface containing the anchored component. After the reaction is complete, unreacted components are removed (e. g. by washing) under conditions such that any complexes formed will remain immobilized on the solid surface.
  • the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the previously non-immobilized component is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface, e.g., using a labeled antibody specific for the immobilized component (the antibody, in turn, can be directly labeled or indirectly labeled with, e.g., a labeled anti-lg antibody).
  • this assay is performed utilizing antibodies specific to OcaB or target molecules but which do not interfere with binding of the OcaB to its target molecule.
  • Such antibodies can be derivatized to the surface, and unbound target or the OcaB-based reagent trapped on the surface by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the OcaB-based reagent or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the OcaB-based reagent or target molecule.
  • cell free assays can be conducted in a liquid phase.
  • the reaction products are separated from unreacted components, by any of a number of standard techniques, including but not limited to: differential centrifugation; chromatography (gel filtration chromatography, ion-exchange chromatography) and/or electrophoresis.
  • resins and chromatographic techniques are known to one skilled in the art.
  • fluorescence energy transfer may also be conveniently utilized, as described herein, to detect binding without further purification of the complex from solution.
  • a reaction mixture containing the OcaB-based reagent and the binding partner is prepared, under conditions and for a time sufficient, to allow the two products to form complex.
  • the reaction mixture can be provided in the presence and absence of the test agent.
  • the test agent can be initially included in the reaction mixture, or can be added at a time subsequent to the addition of the target and its cellular or extracellular binding partner. Control reaction mixtures are incubated without the test agent or with vehicle. The formation of any complexes between the target product and the cellular or extracellular binding partner is then detected.
  • the formation of a complex in the reaction mixture containing the test compound, but not in the control reaction, indicates that the test agent facilitates the interaction of the OcaB-based reagent and the interactive binding partner.
  • Heterogeneous assays involve anchoring either the OcaB-based reagent or the binding partner onto a solid phase, and detecting complexes anchored on the solid phase at the end of the reaction.
  • homogeneous assays the entire reaction is carried out in a liquid phase.
  • the order of addition of reactants can be varied to obtain different information about the agents being tested. For example, test agents that interfere with the interaction between the OcaB-based reagent and the binding partners, e. g. , by competition, can be identified by conducting the reaction in the presence of the test substance. Alternatively, test agents that facilitates preformed complexes, can be tested by adding the test compound to the reaction mixture prior to complexes have been formed.
  • the various formats are briefly described below.
  • either the OcaB-based reagent or the binding partner is anchored onto a solid surface (e. g. a microtiter plate), while the non- anchored species is labeled, either directly or indirectly.
  • the anchored species can be immobilized by non-covalent or covalent attachments.
  • an immobilized antibody specific for the species to be anchored can be used to anchor the species to the solid surface.
  • the partner of the immobilized species is exposed to the coated surface with or without the agent. After the reaction is complete, unreacted components are removed (e. g. by washing) and any complexes formed will remain immobilized on the solid surface.
  • the detection of label immobilized on the surface indicates that complexes were formed.
  • an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, can be directly labeled or indirectly labeled with, e. g. , a labeled anti-lg antibody).
  • agents that enable complex formation or that promote the stability of preformed complexes can be detected.
  • the reaction can be conducted in a liquid phase in the presence or absence of the agent, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
  • test compounds that enable complex or that promote the stability of preformed complexes can be identified.
  • a homogeneous assay can be used.
  • a preformed complex of the OcaB-based reagent and the interactive cellular or extracellular binding partner product is prepared in that either the target products or their binding partners are labeled, but the signal generated by the label is quenched due to complex formation.
  • agent that favors the formation of the complex will result in the generation of a signal below the control value. In this way, agents that promote OcaB-binding partner interaction can be identified.
  • the OcaB-based reagent can be used as "bait proteins" in a two- hybrid assay or three-hybrid assay, to identify other proteins, which bind to or interact with OcaB binding proteins and are involved in OcaB activity.
  • binding partners can be activators or inhibitors of signals or transcriptional control.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a OcaB binding partner is fused to a gene encoding the DNA binding domain of a known transcription factor (e. g. GAL-4).
  • a known transcription factor e. g. GAL-4
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein ("prey” or “sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the OcaB can be the fused to the activator domain. If the "bait” and the “prey” proteins are able to interact, in vivo, forming a OcaB dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e. g. lacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor.
  • a reporter gene e. g. lacZ
  • the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the OcaB.
  • the two- hybrid assay is used to monitor an interaction between two components, The two hybrid assay can also e conducted in the presence of an agent to be screened, and the assay is used to determine whether the agent enhances or diminishes the interaction between the components.
  • the assay for selecting compounds which interact with OcaB can be a cell-based assay.
  • Useful assays include assays in which a marker of adipocyte differentiation, a fat or lipid parameter is measured.
  • the cell-based assay can include contacting a cell expressing a OcaB-based reagent with a test compound and determining the ability of the test compound to modulate (e. g. stimulate or inhibit) an activity of a OcaB, and/or determine the ability of the agent to modulate expression of a OcaB, e. g. by detecting OcaB-encoding nucleic acids (e. g., mRNA or cDNA) or related proteins in the cell.
  • OcaB-encoding nucleic acids e. g., mRNA or cDNA
  • Determining the ability of the agent to modulate OcaB activity can be accomplished, for example, by determining the ability of the OcaB to bind to or interact with the test molecule, and by determining the ability of the test molecule to modulate adipogenesis.
  • Cell-based systems can be used to identify compounds that decrease expression and/or activity and/or effect of a OcaB. Such cells can be recombinant or non-recombinant, such as cell lines that express the ocab gene. In some embodiments, the cells can be recombinant or non-recombinant cells which express a OcaB-binding partner. Exemplary systems include mammalian or yeast cells that express a OcaB (for example from a recombinant nucleic acid).
  • cells are exposed to agents suspected of increasing expression and/or activity of a OcaB. After exposure, the cells are assayed, for example, for OcaB expression or activity.
  • a cell can be from a stable cell line or a primary culture obtained from an organism (for example an organism treated with the agent). ln addition to cell-based and in vitro assay systems, non-human organisms, e. g. transgenic non-human organisms or a model organism, can also be used.
  • a transgenic organism is one in which a heterologous DNA sequence is chromosomally integrated into the germ cells of the animal. A transgenic organism will also have the transgene integrated into the chromosomes of its somatic cells.
  • Organisms of any species including, but not limited to: yeast, worms, flies, fish, reptiles, birds, mammals (e. g. mice, rats, rabbits, guinea pigs, pigs, micro-pigs, and goats), and non-human primates (e. g. baboons, monkeys, chimpanzees) may be used in the methods described herein.
  • mammals e. g. mice, rats, rabbits, guinea pigs, pigs, micro-pigs, and goats
  • non-human primates e. g. baboons, monkeys, chimpanzees
  • a transgenic cell or animal used in the methods of the invention can include a transgene that encodes, e. g. , an OcaB.
  • the transgene can encode a protein that is normally exogenous to the transgenic cell or animal, including a human protein, e. g., a human OcaB or one of its biding partner.
  • the transgene can be linked to a heterologous or a native promoter. Methods of making transgenic cells and animals are known in the art.
  • the specific activity of OcaB may be assayed in a cell-free system, a cell line or a cell population that has been exposed to the agent to be tested and compared to an unexposed control cell-free system, cell line or cell population.
  • the specific activity of an OcaB-activating reagent can also be assessed using OcaB-deficient systems (OcaB knockout cells or animals).
  • the value of the parameter of the OcaB-based reagent is compared to a control value to determine if OcaB is modulated in the individual or the effect of the agent on OcaB expression or activity.
  • the comparison can be made by an individual.
  • the comparison can be made in a comparison module.
  • Such comparison module may comprise a processor and a memory card to perform an application.
  • the processor may access the memory to retrieve data.
  • the processor may be any device that can perform operations on data. Examples are a central processing unit (CPU), a front-end processor, a microprocessor, a graphics processing unit (PPU/VPU), a physics processing unit (PPU), a digital signal processor and a network processor.
  • the application is coupled to the processor and configured to determine the effect of the agent on the parameter of the OcaB-based reagent with respect to the control value. An output of this comparison may be transmitted to a display device.
  • the memory accessible by the processor, receives and stores data, such as measured parameters of the OcaB-based reagent or any other information generated or used.
  • the memory may be a main memory (such as a high speed Random Access Memory or RAM) or an auxiliary storage unit (such as a hard disk, a floppy disk or a magnetic tape drive).
  • the memory may be any other type of memory (such as a Read-Only Memory or ROM) or optical storage media (such as a videodisc or a compact disc).
  • the characterization can be made by an individual. In another embodiment, the characterization can be made with a processor and a memory card to perform an application.
  • the processor may access the memory to retrieve data.
  • the processor may be any device that can perform operations on data. Examples are a central processing unit (CPU), a front-end processor, a microprocessor, a graphics processing unit (PPU/VPU), a physics processing unit (PPU), a digital signal processor and a network processor.
  • the application is coupled to the processor and configured to characterize the individual or the agent being screened. An output of this characterization may be transmitted to a display device.
  • the memory accessible by the processor, receives and stores data, such as measured parameters of the OcaB- based reagent or any other information generated or used.
  • the memory may be a main memory (such as a high speed Random Access Memory or RAM) or an auxiliary storage unit (such as a hard disk, a floppy disk or a magnetic tape drive).
  • the memory may be any other type of memory (such as a Read-Only Memory or ROM) or optical storage media (such as a videodisc or a compact disc).
  • the diagnostic methods described herein can be used to determine an individual's susceptibility to develop adipogenesis and/or insulin resistance.
  • the premise behind this diagnostic method is that OcaB activity or expression is downregulated prior to the onset of adipogenesis and/or insulin resistance.
  • OcaB activity or expression is downregulated prior to the onset of adipogenesis and/or insulin resistance.
  • a downregulation of OcaB is observed in the individual, it can be linked to a susceptibility to develop adipogenesis and/or insulin resistance.
  • a value for a parameter of the OcaB-based reagent is compared to a control value.
  • Such control value can be, for example, a parameter of the OcaB-based reagent in a biological sample of a control individual (or a group of control individuals) lacking susceptibility to develop adipogenesis and/or insulin resistance. Such control individual is considered responsive to insulin and/or does not show adipogenesis.
  • the control value can also be a pre-determined value associated with a lack of susceptibility to develop adipogenesis and/or insulin resistance.
  • the susceptibility of the individual to develop insulin resistance and/or adipogenesis is characterized.
  • the individual is characterized as susceptible to develop insulin resistance and/or adipogenesis if the value of the OcaB-based reagent parameter is lower than the control value.
  • the individual is characterized not being susceptible to develop insulin resistance and/or adipogenesis if the value of the OcaB-based reagent parameter is equal to or higher than the control value.
  • the diagnostic methods described herein can be used to determine the presence of insulin resistance in an individual.
  • the premise behind this diagnostic method is that OcaB activity or expression is downregulated during insulin resistance.
  • a value for a parameter of the OcaB-based reagent is compared to a control value.
  • control value can be, for example, a parameter of the OcaB-based reagent in a biological sample of a control individual (or a group of control individuals) that is responsive to insulin (e.g. does not show insulin resistance).
  • the control value can also be a pre-determined value associated with a lack of insulin resistance.
  • the presence of insulin resistance can be determined.
  • the individual is characterized being resistant to insulin if the value of the OcaB-based reagent parameter is lower than the control value.
  • the individual is characterized as being responsive to insulin (or lacking insulin resistance) when the value of the OcaB-based reagent parameter is equal to or higher than the control value.
  • the diagnostic methods described herein can be used to determine the effectiveness of a therapy for preventing, treating or alleviation the symptoms of insulin resistance and/or adipogenesis.
  • the premise behind this diagnostic method is that OcaB activity or expression is downregulated during insulin resistance and/or adipogenesis and that the upregulation of OcaB restores insulin responsiveness and limits adipogenesis.
  • an assessment of the modulation of OcaB activity or expression is made and can be linked to treatment efficiency.
  • a value for a parameter of the OcaB-based reagent is compared to a control value.
  • control value can be, for example, the parameter of the OcaB-based reagent in a biological sample from the same individual but obtained during an earlier phase of the treatment.
  • control value can also be the parameter of the OcaB-based reagent in the individual prior to treatment.
  • control value can also be derived from another individual treated with a placebo (e.g a control agent that does not have the ability to prevent, treat and/or alleviate the symptoms of adipogenesis and/or insulin resistance).
  • the control value can be a pre-determined value associated with a lack of ability to prevent, treat and/or alleviate the symptoms of adipogenesis and/or insulin resistance. Once the comparison has been made, the effectiveness of the therapy can be determined.
  • the treatment is characterized as not being efficient if the value of the OcaB-based reagent parameter is lower than or equal to the control value. On the other hand, the treatment is characterized as being efficient when the value of the OcaB-based reagent parameter is higher than the control value.
  • the screening methods described herein can be used to determine an agent's ability to prevent, treat or alleviate the symptoms of adipogenesis and/or insulin resistance.
  • the premise behind this screening method is that OcaB activity or expression is downregulated during insulin resistance and/or adipogenesis. As such, by assessing if a downregulation of OcaB's activity or expression made by the agent, it can be linked to its ability to prevent, treat or alleviate the symptoms of adipogenesis and/or insulin resistance.
  • control value may be the parameter of the OcaB- based reagent in the absence of the agent.
  • the parameter of the OcaB-reagent can be measured prior to the combination of the agent with the OcaB-based reagent or in two replicates of the same reaction vessel where one of the screening system does not comprise the agent.
  • the control value can also be the parameter of the OcaB-based reagent in the presence of a control agent that is known not to increase insulin secretion.
  • control agent may be, for example, a pharmaceutically inert excipient.
  • the control value can also be the parameter of the OcaB-based reagent obtained from a reaction vessel comprising cells or tissues from a healthy subject that is responsive to insulin and/or does not show adipogenesis.
  • the control value can also be a pre-determined value associated with a lack insulin resistance and/or adipogenesis.
  • the ability of the agent is determined based on the comparison of the value of the parameter of the OcaB-based reagent with respect to the control value.
  • the agent is characterized as being able to prevent, treat or alleviate the symptoms of insulin resistance and/or adipogenesis when the value of the parameter of the OcaB-based reagent is higher than the control value.
  • the agent is characterized as lacking the ability to prevent, treat or alleviate the symptoms of insulin resistance and/or adipogenesis when the measurement of the parameter of the OcaB-based reagent is lower than or equal to the control value.
  • the present application also provides diagnostic and screening systems for performing the characterizations and methods described herein. These systems comprise a reaction vessel for combining the biological sample (diagnostic system) or the agent (screening system), a processor in a computer system, a memory accessible by the processor and an application coupled to the processor.
  • the application or group of applications is(are) configured for receiving a test value of a level of an OcaB-based reagent in the presence of the agent; comparing the test value to a control value and/or characterizing the individual and/or agent in function of this comparison.
  • Such characterization takes into account that an increased test value with respect to the control value is associated with a lack of adipogenesis and/or insulin resistance in the individual or with an ability for a screened agent to treat, prevent or alleviate the symptoms of adipogenesis and/or insulin resistance.
  • Such characterization also takes into account that a decreased test value with respect to the control value is associated with adipogenesis and/or insulin resistance in the individual or with a lack of ability, for a screened agent, to treat, prevent or alleviate the symptoms of adipogenesis and/or insulin resistance.
  • the present application also provides a software product embodied on a computer readable medium.
  • This software product comprises instructions for characterizing the individual or the agent according to the methods described herein.
  • the software product comprises a receiving module for receiving a test value of a level of and OcaB- based reagent in the presence of the biological sampled or the agent; a comparison module receiving input from the measuring module for determining if the test value is lower than, equal to or higher than a control value; a characterization module receiving input from the comparison module for performing the characterization based on the comparison. .
  • Such characterization takes into account that an increased test value with respect to the control value is associated with a lack of adipogenesis and/or insulin resistance in the individual or with an ability for a screened agent to treat, prevent or alleviate the symptoms of adipogenesis and/or insulin resistance. Such characterization also takes into account that a decreased test value with respect to the control value is associated with adipogenesis and/or insulin resistance in the individual or with a lack of ability, for a screened agent, to treat, prevent or alleviate the symptoms of adipogenesis and/or insulin resistance.
  • the comparison module and characterization module may each comprise a processor, a memory accessible by the processor to perform an application.
  • an application found in the computer system of the screening system is used in the comparison module.
  • a measuring module extracts/receives information from the reaction vessel with respect to the level of the OcaB-based reagent.
  • the receiving module is coupled to a comparison module which receives the value(s) of the level of the OcaB-based reagent and determines if this value is lower than, equal to or higher than a control value.
  • the comparison module can be coupled to a characterization module.
  • an application found in the computer system of the screening system is used in the characterization module.
  • the comparison module is coupled to the characterization module which receives the comparison and performs the characterization based on this comparison.
  • the receiving module, comparison module and characterization module are organized into a single discrete system.
  • each module is organized into different discrete system.
  • at least two modules are organized into a single discrete system.
  • Therapeutic applications also provides methods and agents useful in the prevention, treatment or the alleviation of symptoms of adipogenesis, insulin resistance and/or glucose intolerance in an individual in need thereof.
  • an effective amount of an agent or a pharmaceutically acceptable salt thereof that agonizes OcaB is administered via any of the usual and acceptable methods known in the art, either singly or in combination.
  • the intake of the agent upregulates the expression or activity of OcaB (either directly or indirectly) to prevent, treat or alleviate the symptoms of adipogenesis, insulin resistance and/or glucose intolerance in the individual.
  • genetics means such as a nucleic acid encoding OcaB
  • the OcaB polypeptide can be directly administered to the individual to augment OcaB activity.
  • small molecules such as thiazolidinediones or other PPARgamma-activating agent (e.g. pioglitazone)
  • the agent is formulated to facilitate its transport to the nucleus where it can mediate some of its therapeutic actions.
  • the agent can further be formulated to be targeted preferably or solely to adipocytes (preferably from the white adipose tissue).
  • the method can be effectively practiced as continuous or prophylactic treatment.
  • the agent can be administered via various administration routes and in the form of solid, liquid or gaseous dosages, including tablets and suspensions.
  • the administration can be conducted in a single unit dosage form with continuous therapy or in a single dose therapy ad libitum.
  • the agent can also be in the form of an oil emulsion or dispersion in conjunction with a lipophilic salt such as pamoic acid, or in the form of a biodegradable sustained-release composition for subcutaneous or intramuscular administration.
  • the dose of the agent depends on a number of factors, such as, e.g., the manner of administration, the age and the body weight of the subject, and the condition of the subject to be treated, and ultimately will be decided by the attending physician or veterinarian.
  • Useful agents may be administered with a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form.
  • Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer such compositions to patients. Any appropriate route of administration may be employed, for example, anal, intraarterial, intravenous, perenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, oral administration or aerosol administration.
  • Therapeutic formulations may be in the form of liquid solutions or suspension; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
  • Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes.
  • Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds.
  • Other potentially useful parenteral delivery systems for agonists of the invention include ethylenevinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes.
  • Formulations for inhalation may contain excipients, (e.g. lactose) or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
  • excipients e.g. lactose
  • aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate
  • glycocholate and deoxycholate may be oily solutions for administration in the form of nasal drops, or as a gel.
  • Useful pharmaceutical carriers for the preparation of the agent can be solids, liquids or gases; thus, the compositions can take the form of tablets, pills, capsules, suppositories, powders, enterically coated or other protected formulations (e.g. binding on ion-exchange resins or packaging in lipid-protein vesicles), sustained release formulations, solutions, suspensions, elixirs, aerosols, and the like.
  • the carrier can be selected from the various oils including those of petroleum, animal, vegetable or synthetic origin, e.g., peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • formulations for intravenous administration comprise sterile aqueous solutions of the active ingredient(s) (e.g. comprising the agent) which are prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile.
  • active ingredient(s) e.g. comprising the agent
  • Suitable pharmaceutical excipients include starch, cellulose, talc, glucose, lactose, gelatin, malt, rice, flour, chalk, silica, magnesium stearate, sodium stearate, glycerol monostearate, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, and the like.
  • the compositions may be subjected to conventional pharmaceutical additives such as preservatives, stabilizing agents, wetting or emulsifying agents, salts for adjusting osmotic pressure, buffers and the like.
  • Suitable pharmaceutical carriers and their formulation are described in Remington's Pharmaceutical Sciences by E. W. Martin. Such compositions will, in any event, contain an effective amount of the active compound together with a suitable carrier so as to prepare the proper dosage form for proper administration to the recipient.
  • the agents that can be administered for the prevention, treatment or alleviations of symptoms of insulin resistance or adipogenesis include, but are not limited to, small molecules, peptides, antibodies, nucleic acids, analogs thereof, multimers thereof, fragments thereof, derivatives thereof and combinations thereof.
  • the agent is a nucleic acid encoding a OcaB polypeptide (or variant thereof) and capable of upregulating the expression or activity of OcaB. This also includes agents that are capable or increasing OcaB activity or levels by inhibiting proteins (or pathways) that normally blunt OcaB mRNA or protein expression and/or activity.
  • nucleic acids can be inserted into any of a number of well-known vectors for their introduction in target cells (such as adipocytes) and individuals as described herein.
  • the nucleic acids are introduced into cells, ex vivo or in vivo, through the interaction of the vector and the target cell.
  • Oligonucleotide refers to naturally-occurring species or synthetic species formed from naturally-occurring subunits or their close homologs. The term may also refer to moieties that function similarly to oligonucleotides, but have non- naturally-occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages. Exemplary among these are phosphorothioate and other sulfur containing species which are known in the art.
  • At least one of the phosphodiester bonds of the oligonucleotide has been substituted with a structure that functions to enhance the ability of the compositions to penetrate into the region of cells where the RNA whose activity is to be modulated is located. It is preferred that such substitutions comprise phosphorothioate bonds, methyl phosphonate bonds, or short chain alkyl or cycloalkyl structures.
  • the phosphodiester bonds are substituted with structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in the practice of the invention.
  • Oligonucleotides may also include species that include at least some modified base forms. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the furanosyl portions of the nucleotide subunits may also be affected. Examples of such modifications are 2'-0-alkyl- and 2'-halogen-substituted nucleotides. Some non-limiting examples of modifications at the 2' position of sugar moieties which are useful in the present invention include OH, SH, SCH 3 , F, OCH 3 , OCN, 0(CH 2 ), NH 2 and 0(CH 2 )nCH 3 , where n is from 1 to about 10. Such oligonucleotides are functionally interchangeable with natural oligonucleotides or synthesized oligonucleotides, which have one or more differences from the natural structure.
  • expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of genetic means to the targeted individual, organ, tissue (such as WAT) or cell population (such as adipocytes).
  • WAT organ, tissue
  • adipocytes cell population
  • Methods which are well known to those skilled in the art can be used to construct recombinant vectors which will express nucleic acid sequence that is complementary to the nucleic acid sequence encoding OcaB.
  • Non-viral vector delivery systems include DNA plasmids, naked nucleic acid, and nucleic acid complexed with a delivery vehicle such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • RNA or DNA based viral systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro and the modified cells then administered to patients (ex vivo).
  • Conventional viral based systems for the delivery of nucleic acids could include retroviral, lentiviral, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer.
  • Viral vectors are currently the most efficient and versatile method of gene transfer in target cells and tissues. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Adenoviral based systems are typically used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained.
  • This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures.
  • AAV Adeno-associated virus
  • numerous viral vector approaches are currently available for gene transfer in clinical trials, with retroviral vectors by far the most frequently used system. All of these viral vectors utilize approaches that involve complementation of defective vectors by genes inserted into helper cell lines to generate the transducing agent.
  • pLASN and MFG-S are examples are retroviral vectors that have been used in clinical trials.
  • rAAV Recombinant adeno-associated virus vectors
  • All vectors are derived from a plasmid that retains only the AAV 145 bp inverted terminal repeats flanking the transgene expression cassette. Efficient gene transfer and stable transgene delivery due to integration into the genomes of the transduced cell are key features for this vector system.
  • Ad vectors Replication-deficient recombinant adenoviral vectors (Ad) are predominantly used in transient expression gene therapy; because they can be produced at high titer and they readily infect a number of different cell types. Most adenovirus vectors are engineered such that a transgene replaces the Ad E1 a, E1 b, and E3 genes; subsequently the replication defective vector is propagated in human 293 cells that supply the deleted gene function in trans. Ad vectors can transduce multiple types of tissues in vivo, including non-dividing, differentiated cells such as those found in the liver, kidney and muscle tissues. Conventional Ad vectors have a large carrying capacity.
  • the gene therapy vector be delivered with a high degree of specificity to a particular tissue type.
  • a viral vector is typically modified to have specificity for a given cell type by expressing a ligand as a fusion protein with a viral coat protein on the viruses outer surface.
  • the ligand is chosen to have affinity for a receptor known to be present on the cell type of interest.
  • Gene therapy vectors can be delivered in vivo by administration to an individual subject, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application.
  • vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lymphocytes, bone marrow aspirates, and tissue biopsy) or universal donor hematopoietic stem cells, followed by re-implantation of the cells into the subject, usually after selection for cells which have incorporated the vector.
  • EXAMPLE I Material and methods Materials and oligonuclotides. All chemicals, except when specified, were purchased from Sigma (Oakville, ON). The oligonucleotide sequences used for various experiments are set forth in the following Table.
  • T3L1 cells freshly isolated pre-adipocytes from mouse adipose tissue and MEFs were grown in Dulbecco's modified Eagle's medium high glucose with 10% fetal bovine serum supplemented with 4 mM and 2 mM, respectively, of glutamine in a 5% C0 2 environment.
  • Cells were differentiated, two days after confluence (DO), in the same medium complemented with 10 ⁇ g/ml insulin, 0.25 mM 3-isobutyl-1-methyl- xanthine and 1 ⁇ dexamethasone. After two days (D2), medium was supplemented with only 10 ⁇ g/ml insulin and replaced every two days until terminal differentiation (D10).
  • Quantitative real-time PCR assays were performed as described in Miard et al. All studies were approved by the institutional ethics committees. Data are presented as mean ⁇ S.E.M. Statistical differences were analyzed by ANOVA and Fisher's t test (ad hoc) when appropriate. A p value ⁇ 0.05 was considered significant.
  • Retroviral infection 293T cells were transfected with either pBABE or pBabe-OcaB using lipofectamine (Invitrogen). After 48 hours of transfection, the medium containing retroviruses was collected, filtered, treated with polybrene (1 ⁇ g/mL) and transferred to 3T3-L1 target cells. Infected cells were selected with puromycin (2.5 pg/mL) for 7 days.
  • mice Male and female C57BL/6 mice (aged 4, 12, and 24 months, kindly provided by NIA, USA), and 2 mo old, male ob/ob, db/db and high-fat, high- sucrose-fed mice (Jackson) were cared for and handled in conformance with the Canadian Guide for the Care and Use of Laboratory Animals, and protocols were approved by our institutional animal care committee. Mice were sacrificed by ketamine- xylazine injection one week after their arrival. In all experiments, adipose tissue samples were immediately harvested and snap frozen in liquid nitrogen.
  • Quantitative PCR Expression of selected genes was measured by quantitative realtime PCR on Rotorgene 3000TM (Corbett Research) using different sets of primers as described in Table 1 . All reactions were performed in duplicate and data were corrected by the expression of a housekeeping gene whose expression remained unchanged upon obesity or aging.
  • Nuclear protein extraction For nuclear extracts, cells were homogenized in ice-cold buffer A (10 mM HEPES pH 7.9, 10 mM KCI, 2 mM MgCI 2 , 0.1 mM EDTA, 1 mM DTT, and diluted 1 : 1000 Protease Inhibitor Cocktail (PIC)). The homogenates were centrifuged 1 min at 1000 g at 4°C to eliminate unbroken tissues. After 20 min on ice, 0.1 volume of 10% NP40 was added and the supernatants were vortexed for 30 sec. The supernatants were then centrifuged for 1 min at 7500 g.
  • PIC Protease Inhibitor Cocktail
  • the nuclear pellet was suspended in 50 ⁇ in ice-cold buffer B (20 mM HEPES pH 7.9, 420 mM NaCI, 1 .5 mM MgCI 2 , 0.1 mM EDTA, 1 mM DTT, 1 : 1000 PIC and 25% glycerol), incubated for 30 min at 4°C with high shaking, and centrifuged for 15 min at 16,000 g at 4°C. The supernatants were collected and protein concentrations were determined with the Bradford assay. Immunofluorescence. Cells were fixed in methanol and subjected to incubations with primary (DAPI and OcaB) and secondary antibodies as previously in Picard et al.
  • Luciferase reporter assays This assay was performed exactly as described in Picard et al. Plasmid used were J3-TK-Luc (Miard et al.) and PEPCK-TK-Luc, pCMV-PPARy (Miard et al.) and pEV-OBF-1 (OcaB).
  • OcaB-/- mice were purchased from Jackson Laboratories. Lipolysis assays and glucose tolerance tests were performed exactly as described in Picard et al.
  • Coimmunoprecipitation assay Cells were lysed in IP buffer (150 mM NaCI, 1 % NP40, 50 mM Tris pH 8.0, 1 :1000 PIC), and an aliquot was taken as input. Cells lysates were precleared with protein A-sepharose beads (GEhealthcare) for 1 hour at 4°C and then centrifuged 5 min at 2300 g. Supernatants were immunoprecipitated with adequate antibody overnight at 4°C, and mouse IgG were used as negative control. Immunoprecipitates were washed once with IP buffer, twice with WB (0.25M KCI in PBS) and then subjected to SDS-PAGE electrophoresis.
  • IP buffer 150 mM NaCI, 1 % NP40, 50 mM Tris pH 8.0, 1 :1000 PIC
  • Cells lysates were precleared with protein A-sepharose beads (GEhealthcare) for 1 hour at 4°C and then
  • Electro mobility shift assay (EMSA). Nuclear extracts were incubated in binding buffer (ZHENG (10mM Tris pH 7.9, 40 mM KCI, 10% glycerol, 0,05% NP40, 1 mM DTT, 1 ⁇ g/ ⁇ L poly (dl:dC)) for 15 min at room temperature. Then, a radiolabeled double- stranded oligonucleotide probe (0.5 ⁇ ) was added for 10 min, and complexes were subjected to electrophoresis on a polyacrylamide gel.
  • binding buffer ZHENG (10mM Tris pH 7.9, 40 mM KCI, 10% glycerol, 0,05% NP40, 1 mM DTT, 1 ⁇ g/ ⁇ L poly (dl:dC)
  • RNAi bacteria were cultured 16 hours in LB containing 100 ⁇ g/mL ampicillin and seeded on RNAi NGM agar plates containing 1 mM isopropylthiogalactoside (IPTG) and 100 ⁇ g/mL carbenicillin. Plates are incubated at room temperature overnight to induce dsRNA expression. Eggs from a synchronous egg-laying were placed on RNAi-agar plates. Hatching day is considered as day 1 of the experiment. Animals were kept at 20°C and scored every day by gentle prodding with a platinum wire to test viability. Animals that died of drying on the edges of the plate, vulva bursting or bagging were excluded from the experiment.
  • IPTG isopropylthiogalactoside
  • Worms were transferred to another plate every other day during the reproductive period, and every week during past-reproductive period.
  • L4440 was used as the empty plasmid used to clone sequences for RNAi treatment. Lifespan assays were performed at 20°C. All strains were maintained as described by Herman et al. at 20 °C. The other following C. elegans strains were used: N2 bristol (wild-type), daf-2(e1370)lll, age-1 (hx546)ll, daf- 16(mu86)l.
  • the promoter region of R148.3 (1.2 kb) was cloned upstream of the GFP gene in an expression plasmid (pFX-EGFP), which was then injected directly into approximately 100 worms.
  • pFX-EGFP expression plasmid
  • a plasmid (pRF4 [rol- 6]) was co-injected as a selection tool to screen worms with successful genetic transformation, as worms with rol-6 are easily noticeable because of their swirling phenotype. Nile Red and Oil Red O staining were performed as described in O'Rourke et al.
  • Pioglitazone treatment Male C57BL/6 mice of 4, 12 and 24 mo old were treated by IP injection of vehicle (DMSO) or pioglitazone (10 mg/kg/d) for 7 days and then sacrificed by ketamine-xylazine injection. In all experiments, adipose tissue samples were immediately harvested and snap frozen in liquid nitrogen.
  • DMSO vehicle
  • pioglitazone 10 mg/kg/d
  • OcaB mRNA levels in scWAT were restored one year after gastric bypass (biliopancreatic derivation) surgery in the same obese patients.
  • OcaB fibroblasts
  • Fig. 3a OcaB -/- MEFs
  • Fig. 3b preadipocytes
  • Fig. 3d and 3e accumulated more lipids and expressed higher mRNA levels of PPARy and its downstream targets aP2, CD36 and adiponectin
  • Position 0 is starting codon ATG.
  • Capital letters indicate nucleotides that are similar to the consensus sequence.
  • Murine sequences are shown. Putative binding regions were also found for the equivalent human genes.
  • the consensus sequence found -45 to -38 of the LPL gene was previously described in Morin et al.
  • a luciferase assay was designed to determine the ability of OcaB to repress expression of at least two different promoters (J3 and PEPCK) associated with PPARD . As shown in Fig 4e, the addition of OcaB did repress expression from these two promoters.
  • Miard S, Dombrowski L, Carter S, Boivin L, Picard F Aging alters PPARgamma in rodent and human adipose tissue by modulating the balance in steroid receptor coactivator-1 .
  • Aging Cell 2009; 8:449-459 Morin, C.L., Schlaepfer, I.R., and Eckel, R.H.: Tumor necrosis factor-a eliminates binding of NF-Y and an octamer-binding protein to the lipoprotein lipase promoter in 3T3-L1 adipocytes. J Clin Invest 1995; 95: 1684

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Abstract

L'invention montre que l'expression de la protéine OCaB est modulée, dans des adipocytes, durant une adipogenèse, le développement d'une insulino-résistance, l'intolérance au glucose et le vieillissement. L'application fournit ainsi des méthodes de caractérisation du risque d'un individu à développer une adipogenèse, une insulino-résistance et/ou une intolérance au glucose, des méthodes de diagnostic d'une insulino-résistance et/ou d'une intolérance au glucose, des méthodes de caractérisation de l'efficacité d'un agent dans le traitement, la prévention ou le soulagement des symptômes de l'adipogenèse, de l'insulino-résistance et/ou de l'intolérance au glucose, des essais de dépistage pour identifier des agents utiles dans le traitement, la prévention ou le soulagement des symptômes de l'adipogenèse, de l'insulino-résistance et/ou de l'intolérance au glucose sur la base de l'évaluation d'un paramètre d'un réactif à base de la protéine OCaB.
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M. JANKOVIC: "OcaB regulates transitional B cell selection", INTERNATIONAL IMMUNOLOGY, vol. 15, no. 9, 1 September 2003 (2003-09-01), pages 1099-1104, XP55076667, DOI: 10.1093/intimm/dxg109 *
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