WO2004047855A2 - Proteines impliquees dans la regulation de l'homeostasie de l'energie - Google Patents

Proteines impliquees dans la regulation de l'homeostasie de l'energie Download PDF

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WO2004047855A2
WO2004047855A2 PCT/EP2003/013377 EP0313377W WO2004047855A2 WO 2004047855 A2 WO2004047855 A2 WO 2004047855A2 EP 0313377 W EP0313377 W EP 0313377W WO 2004047855 A2 WO2004047855 A2 WO 2004047855A2
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nucleic acid
polypeptide
acid molecule
amph
npc1
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WO2004047855A3 (fr
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Martin Meise
Karsten Eulenberg
Tri Nguyen
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Develogen Aktiengesellschaft Fur Entwicklungsbiologische Forschung
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

  • This invention relates to the use of Hormone receptor-like in 46 (Gadfly Accession Number CG11823; referred to as Hr46), Amphiphysin (Gadfly Accession Number CG8604; referred to as Amph), Gadfly Accession Number CG6364 (uridine kinase; referred to as CG6364), Niemann-Pick disease, type C1 (Gadfly Accession Number CG5722; referred to as NPC1), Gadfly Accession Number CG5261 (dihydrolipoamide S-actyltransferase; referred to as CG5261), Gadfly Accession Number CG6903 (referred to as CG6903), Gadfly Accession Number CG8895 (referred to as CG8895), or patched (Gadfly Accession Number CG2411 ; referred to as ptc) homologous proteins, to the use of nucleic acid sequences encoding these, and to
  • Obesity is one of the most prevalent metabolic disorders in the world. It is still a poorly understood human disease that becomes as a major health problem more and more relevant for western society. Obesity is defined as a body weight more than 20% in excess of the ideal body weight, frequently resulting in a significant impairment of health. It is associated with an increased risk for cardiovascular disease, hypertension, diabetes, hyperlipidaemia and an increased mortality rate. Besides severe risks of illness, individuals suffering from obesity are often isolated socially.
  • Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors, and can be caused by different reasons such as non-insulin dependent diabetes, increase in triglycerides, increase in carbohydrate bound energy and low energy expenditure. As such, it is a complex disorder that must be addressed on several fronts to achieve lasting positive clinical outcome. Since obesity is not to be considered as a single disorder but as a heterogeneous group of conditions with (potential) multiple causes, it is also characterized by elevated fasting plasma insulin and an exaggerated insulin response to oral glucose intake (Kolterman O.G. et al., (1980) J. Clin. Invest 65: 1272-1284). A clear involvement of obesity in type 2 diabetes mellitus can be confirmed (Kopelman P.G., (2000) Nature 404: 635-643).
  • Pancreatic beta-cells secrete insulin in response to blood glucose levels. Insulin amongst other hormones plays a key role in the regulation of the fuel metabolism. Insulin leads to the storage of glycogen and triglycerides and to the synthesis of proteins. The entry of glucose into muscles and adipose cells is stimulated by insulin. In patients who suffer from diabetes mellitus type I or LADA (latent autoimmue diabetes in adults (Pozzilli & Di Mario, 2001, Diabetes Care. 8: 1460-1467) beta-cells are being destroyed due to autoimmune attack. The amount of insulin produced by the remaining pancreatic islet cells is too low, resulting in elevated blood glucose levels (hyperglycemia).
  • liver and muscle cells loose their ability to respond to normal blood insulin levels (insulin resistance).
  • Insulin resistance In diabetes type II liver and muscle cells loose their ability to respond to normal blood insulin levels (insulin resistance).
  • High blood glucose levels and also high blood lipid levels) in turn lead to an impairment of beta- cell function and to an increase in Deta-cell apoptosis.
  • Diabetes is a very disabling disease, because today's common anti-diabetic drugs do not control blood sugar levels well enough to completely prevent the occurrence of high and low blood sugar levels. Out of range blood sugar levels are toxic and cause long-term complications like for example renopathy, retinopathy, neuropathy and peripheral vascular disease. There is also a host of related conditions, such as obesity, hypertension, heart disease and hyperlipidemia , for which persons with diabetes are substantially at risk.
  • the technical problem underlying the present invention was to provide for means and methods for modulating (pathological) metabolic conditions influencing body-weight regulation and/or energy homeostatic circuits.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to novel functions of proteins and nucleic acids encoding these in body-weight regulation, energy homeostasis, metabolism, and obesity. Further new compositions are provided that are useful in diagnosis, treatment, and prognosis of metabolic diseases and disorders as described.
  • the present invention discloses that Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous proteins (herein referred to as "proteins of the invention” or "a protein of the invention”) are regulating the energy homeostasis and fat metabolism, especially the metabolism and storage of triglycerides, and polynucleotides, which identify and encode the proteins disclosed in this invention.
  • the invention also relates to vectors, host cells, and recombinant methods for producing the polypeptides and polynucleotides of the invention.
  • the invention also relates to the use of these compounds and effectors/modulators thereof, e.g.
  • antibodies biologically active nucleic acids, such as antisense molecules, RNAi molecules or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynulceotides or polypeptides, in the diagnosis, study, prevention, and treatment of metabolic diseases or dysfunctions, including obesity, diabetes mellitus and/or metabolic syndrome, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • metabolic diseases or dysfunctions including obesity, diabetes mellitus and/or metabolic syndrome, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • RORA, RORB, and RORC are members of the NR1 subfamily of nuclear hormone receptors that can bind as a monomer or as a homodimer to hormone response elements upstream of several genes to enhance the expression of those genes.
  • the specific functions of these proteins are not known, but RORA and RORB have been shown to interact with NM23-2, a nucleoside diphosphate kinase involved in organogenesis and differentiation, as well as with NM23-1, the product of a tumor metastasis suppressor candidate gene.
  • RORC reactive oxygen species
  • RORA farnesoid RI
  • RORB-/- mice display a duck-like gait, transient male incapability to sexually reproduce, and a severely disorganized retina that suffers from postnatal degeneration.
  • Adult RORB-/- mice are blind, yet their circadian activity rhythm is still entrained by light-dark cycles (Andre E. et al., (1998) EMBO J 17: 3867-3877).
  • RORC is induced during adipocyte differentiation in D1 and 3T3-L1 cells and functions as an active transcription factor, suggesting a role for RORC in the regulation of gene expression during this differentiation process (Austin S. et al., (1998) Cell Growth Differ 9: 267-276). RORC has critical functions in T cell repertoire selection and lymphoid organogenesis (Sun Z. et al., (2000) Science 288: 2369-2373).
  • Amphiphysins are involved in clathrin-mediated endocytosis, actin function, and signalling pathways. They are highly concentrated in nerve terminals, where they may act as linkers between the clathrin coat and dynamin in the endocytosis of synaptic vesicles. Such recycling of synaptic vesicles is necessary for neurotransmission to continue, following neurotransmitter release.
  • Amphiphysin family members share a similar three-domain organisation: the N-terminal domain contains six heptad repeats, which are predicted to form a coiled-coiled structure, thought to be involved in the dimerisation of amphiphysin molecules (BAR domain, see below), the central region binds the heavy chain of clathrin and the clathrin adaptor protein AP-2, through distinct sites, and the C-terminal domain contains a Src-homology-3 (SH3) domain that binds the GTPase dynamin and the inositol-5'-phosphatase synaptojanin-1.
  • BAR domain heptad repeats
  • SH3 Src-homology-3
  • Amphiphysin isoforms 1 and 2 form a heterodimer that binds to the the AP2 adaptor in clathrin coated vesicles. Both isoforms have SH3 domains which bind dynamin's proline rich domain (PRD). Amphl and Amph2 also bind clathrin, synaptojanin, and endophilin (see, for example, Lichte, B. et al., (1992) EMBO J. 11: 2521-2530; David C. et al., (1996) Proc Natl Acad Sci U S A 93: 331-335).
  • Clathrin-mediated endocytosis at the synapse is constitutively blocked by tonic phosphorylation of dynamin, amphiphysin and synaptojanin, which prevents their interaction with the plasma-membrane coat complex of AP2 and clathrin.
  • a rise in Ca 2+ levels to submicromolar concentrations activates the calmodulin-dependent phosphatase, calcineurin, which dephosphorylates the proteins and thereby permits assembly of clathrin-coated buds and their subsequent 'pinching off to form clathrin-coated vesicles.
  • Stiff-man syndrome is a rare disease of the central nervous system characterized by progressive rigidity of the body musculature with superimposed painful spasms. Autoantibodies from patients with the stiff-man syndrome show a dominant autoepitope located in the C-terminal region of AMPH1, which contains an SH3 domain.
  • the tissue distribution of AMPH land its association with neurotransmitter vesicles make the gene a candidate for involvement in such diverse heritable disorders as those of the nervous system, certain endocrine tissues (such as the adrenal medulla, pituitary gland or endocrine pancreas), or male fertility (see, for example, David, C. et al., (1994) FEBS Lett. 351: 73-79; De Camilli, P.
  • amphiphysin 1 knockout mice lack of amphiphysin 1 causes a parallel dramatic reduction of amphiphysin 2 selectively in brain.
  • Cell-free assembly of endocytic protein scaffolds is defective in mutant brain extracts.
  • Knockout mice exhibit defects in synaptic vesicle recycling that are unmasked by stimulation and suggest impairments at multiple stages of the cycle. These defects correlate with increased mortality due to rare irreversible seizures and with major learning deficits, suggesting a critical role of amphiphysin for higher brain functions (Di Paolo G. et al., (2002) Neuron 33: 789-804).
  • Uridine-cytidine kinases have important roles for the phosphorylation of nucleoside analogs that are being investigated for possible use in chemotherapy of cancer.
  • UCK1 and UCK2 Two human UCKs, UCK1 and UCK2, were shown to catalyze the phosphorylation of Urd and Cyd. The enzymes did not phosphorylate deoxyribonucleosides or purine ribonucleosides.
  • UCK1 mRNA was detected as two isoforms of approximately 1.8 and approximately 2.7 kb. The 2.7-kb band was ubiquitously expressed in the investigated tissues. The band of approximately 1.8 kb was present in skeletal muscle, heart, liver, and kidney.
  • Uridine triphosphate (UTP) content of heart and diaphragm muscle was decreased in fasted and streptozotocin-diabetic rats and Uracil nucleotide synthesis by the salvage pathway is decreased in experimental diabetes and fasting (Gertz B. J. and Haugaard E. S., (1979) Metabolism 28: 358-362).
  • NPC Nieman ⁇ -Pick type C
  • NPC1 The fatal damage in NPC1 disease is neurodegeneration starting from early life. Patients with Niemann-Pick disease type C usually appear normal for 1 or 2 years and sometimes even longer. They gradually develop neurologic abnormalities, which are initially manifested by ataxia, grand mal seizures, and loss of previously learned speech. Cholestatic jaundice occurs in some patients. Foamy Niemann-Pick cells are found in the bone marrow. Death usually occurs at age 5 to 15. Some patients have vertical ophthalmoplegia. In ocular histopathologic studies of a girl who died at age 11 years, lipid deposits were noted (Palmer M.
  • NPC1 in brain is predominantly a glial protein present in astrocytic processes closely associated with nerve terminals, the earliest site of degeneration in NPC.
  • the pyruvate dehydrogenase complex catalyzes the overall conversion of pyruvate to acetyl-CoA and C0 2 . It contains multiple copies of three enzymatic components: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2) and lipoamide dehydrogenase (E3).
  • E1 pyruvate dehydrogenase
  • the dihydrolipoamide acetyltransferase contains two covalently-bound piolyl cofactors.
  • the pyruvate dehydrogenase complex contains 20 to 30 ⁇ 2 ⁇ 2 tetramers of E1 plus 6 homodimers of E3 plus 60 copies of E2 and is localised in the mitochondrial matrix.
  • the mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids.
  • the dihydrolipoyl acetyltransferase (E2) and the dihydrolipoyl dehydrogenase-binding protein (E3BP) are multidomain proteins that form the oligomeric core of the complex.
  • One or more of their three lipoyl domains selectively bind each PDK (pyruvate dehydrogenase kinase) and PDP1 (pyruvate dehydrogenase phosphatase 1). These adaptive interactions predominantly influence the catalytic efficiencies and effector control of these regulatory enzymes.
  • PDK pyruvate dehydrogenase kinase
  • PDP1 pyruvate dehydrogenase phosphatase 1
  • fatty acids are the preferred source of acetyl-CoA and NADH
  • feedback inactivation of PDC is accomplished by the activity of certain kinase isoforms being stimulated upon preferentially binding a lipoyl domain containing a reductively acetylated lipoyl group.
  • PDC activity is increased in Ca 2+ -sensitive tissues by elevating PDP1 activity via the Ca 2+ -dependent binding of PDP1 to a lipoyl domain of E2.
  • the irrecoverable loss of glucose carbons is restricted by minimizing PDC activity due to high kinase activity that results from the overexpression of specific kinase isoforms.
  • Overexpression of the same PDK isoforms deleteriously hinders glucose consumption in unregulated diabetes (Roche T. E. et al., (2001 ) Prog Nucleic Acid Res Mol Biol 70: 33-75).
  • the sugar transporters belong to a superfamily of membrane proteins responsible for the binding and transport of various carbohydrates, organic alcohols, and acids in a wide range of prokaryotic and eukaryotic organisms. These integral membrane proteins are predicted to comprise twelve membrane spanning domains. It is likely that the transporters have evolved from an ancient protein present in living organisms before the divergence into prokaryotes and eukaryotes. In mammals, these proteins are expressed in a number of organs.
  • Reticulon also know as neuroendocrine-specific protein (NSP), is a protein of unknown function which associates with the endoplasmic reticulum. This family represents the C-terminal end of the three reticulon isoforms and their homologues.
  • Reticulon 1 (Rtn1) is thought to be involved in neuroendocrine secretion or in membrane trafficking in neuroendocrine cells (InterPro).
  • Nogo A receptor (Rtn4 receptor) inhibits neuronal regeneration after CNS or spinal cord injury through a signaling cascade probably including Rho, Rho kinase or cAMP and PKA. In wildtype it is important for preservation of the wiring of the CNS (Woolf C. J.
  • a 66-residue lumenal/extracellular domain of Nogo inhibits axonal extension and collapses dorsal root ganglion growth cones.
  • Reticulon 1 and 3 are not expressed by oligodendrocytes, and the 66-residue lumenal/extracellular domains from Reticulon 1, 2 and 3 do not inhibit axonal regeneration.
  • the transmembrane protein patched is a receptor for the morphogene Sonic Hedgehog.
  • this protein associates with the smoothened protein to transduce hedgehog signals, leading to the activation of wingless, decapentaplegic, and patched itself. It participates in cell interactions that establish pattern within the segment and imaginal disks during development.
  • the mouse homolog may play a role in epidermal development. This protein is involved in the intracellular trafficking of cholesterol, and may play a role in vesicular trafficking in glia, a process that may be crucial for maintaining the structural functional integrity of nerve terminals (InterPro).
  • the absence of Shh expression is required for the early development of the endocrine and exocrine pancreas.
  • Hh signaling might not be restricted to patterning in early pancreas development but also continues to signal in differentiated beta-cells of the endocrine pancreas in regulating insulin production. Thus, defective Hh signaling in the pancreas should be considered as a potential factor in the pathogenesis of type 2 diabetes (Thomas M. K. et al., (2000) Diabetes 49: 2039-2047).
  • the PATCHED (PTC) gene encodes a Sonic hedgehog (Shh) receptor and a tumor suppressor protein that is defective in basal cell nevus syndrome (BCNS). Functions of PTC were investigated by inactivating the mouse gene. Mice homozygous for the ptc mutation died during embryogenesis and were found to have open and overgrown neural tubes. Re appears to be essential for repression of genes that are locally activated by Shh. Mice heterozygous for the ptc mutation were larger than normal, and a subset of them developed hindlimb defects or cerebellar medulloblastomas (Goodrich L. V. et al., (1997) Science 277: 1109-1113).
  • Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds.
  • homologous nucleic acids encoding the human RAR-related orphan receptor A, B and C, human amphiphysin, human bridging integrator 1 (amphiphysin II), human uridine-cytidine kinase 1 (UCK1), human uridine monophosphate kinase (UMPK), human Niemann-Pick disease, type C1 (NPC1), human NPC1-like 1, human dihydrolipoamide S-acetyltransferase, human pyruvate dehydrogenase complex lipoyl-containing component X, human hypothetical protein FLJ32731, human reticulon 1 , 2, 3, and 4, human patched homolog, or human patched homolog 2 proteins as described in Table 1.
  • the invention particularly relates to a nucleic acid molecule encoding a polypeptide contributing to regulating the energy homeostasis and the metabolism of triglycerides, wherein said nucleic acid molecule comprises
  • nucleotide sequence of Drosophila Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc human Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous nucleic acids, particularly nucleic acids as described in Table 1 , and/or a sequence complementary thereto,
  • NPC1 NPC1, CG5261, CG6903, CG8895, or ptc homologous protein, particular a protein as described in Table 1,
  • (f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of at least 15 bases, preferably at least 20 bases, more preferably at least 25 bases and most preferably at least 50 bases.
  • the invention is based on the finding that Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous proteins and the polynucleotides encoding therefore, are involved in the regulation of triglyceride storage and therefore energy homeostasis.
  • compositions comprising Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous polypeptides and polynucleotides as well as modulators/effectors thereof for the diagnosis, study, prevention, or treatment of metabolic diseases or dysfunctions, including obesity, diabetes mellitus and/or metabolic syndrome, as well as related disorders such as eating disorder, cachexia, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones or liver fibrosis.
  • the present invention relates to genes with novel functions in body-weight regulation, energy homeostasis, metabolism, and obesity, functional fragments of said genes, polypeptides encoded by said genes or functional fragments thereof, and modulators/effectors thereof, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • modulators/effectors thereof e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules, or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • model organisms such as the fly Drosophila melanogaster
  • the ability to manipulate and screen the genomes of model organisms provides a powerful tool to analyze biological and biochemical processes that have direct relevance to more complex vertebrate organisms due to significant evolutionary conservation of genes, cellular processes, and pathways (see, for example, Adams M.D. et al., (2000)
  • a forward genetic screen was performed in fly displaying a mutant phenotype due to misexpression of a known gene (see, St Johnston D., (2002) Nat Rev Genet 3: 176-188; Rorth P., (1996) Proc Natl Acad Sci U S A 93: 12418-12422).
  • this invention we have used a genetic screen to identify mutations that cause changes in the body weight, which are reflected by a significant change of triglyceride levels.
  • Triglycerides are the most efficient storage for energy in cells.
  • genes with a function in energy homeostasis several thousand proprietary and publicly available EP-lines were tested for their triglyceride content after a prolonged feeding period (see Examples and Figures for more detail). Lines with significantly changed triglyceride content were selected as positive candidates for further analysis.
  • the change of triglyceride content due to the loss of a gene function suggests gene activities in energy homeostasis in a dose dependent manner that controls the amount of energy stored as triglycerides.
  • the content of triglycerides of a pool of flies with the same genotype was analyzed after prolonged feeding using a triglyceride assay.
  • Male flies homozygous, hemizygous, or heterozygous for the integration of vectors for Drosophila EP-lines were analyzed in an assay measuring the triglyceride contents of these flies, illustrated in more detail in the Examples section.
  • the results of the triglyceride content analysis are shown in Figures 1, 4, 8, 12, 15, 19, 22, and 25, respectively.
  • Genomic DNA sequences were isolated that are localized adjacent to the EP vector integration. Using those isolated genomic sequences public databases like Berkeley Drosophila Genome Project (GadFly; see also FlyBase (1999) Nucleic Acids Research 27: 85-88) were screened thereby identifying the integration sites of the vectors, and the corresponding genes, described in more detail in the Examples section. The molecular organization of the genes is shown in Figures 2, 5, 9, 13, 16, 20, 23, and 26, respectively.
  • the Drosophila genes and proteins encoded thereby with functions in the regulation of triglyceride metabolism were further analysed in publicly available sequence databases (see Examples for more detail) and mammalian homologs were identified.
  • the function of the mammalian homologs in energy homeostasis was further validated in this invention by analyzing the expression of the transcripts in different tissues and by analyzing the role in adipocyte differentiation.
  • Expression profiling studies confirm the particular relevance of the protein(s) of the invention as regulators of energy metabolism in mammals. Further, we show that the proteins of the invention are regulated by fasting and by genetically induced obesity.
  • mice carrying gene knockouts in the leptin pathway (for example, ob (leptin) or db (leptin receptor) mice) to study the expression of the proteins of the invention.
  • leptin pathway for example, ob (leptin) or db (leptin receptor) mice
  • mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al., (1998) Mol. Cell. 2: 559-569).
  • Microarrays are analytical tools routinely used in bioanalysis.
  • a microarray has molecules distributed over, and stably associated with, the surface of a solid support.
  • the term "microarray” refers to an arrangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as monitoring gene expression, drug discovery, gene sequencing, gene mapping, bacterial identification, and combinatorial chemistry.
  • One area in particular in which microarrays find use is in gene expression analysis (see Example 6).
  • array technology can be used to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (see for example, Brennan T.M., (1995) U.S. Patent No. US5474796;
  • Oligonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microarray.
  • the microarray can be used in transcript imaging techniques, which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microarray may also be used to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease. In particular, this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents, which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • amphiphysin As determined by microarray analysis, amphiphysin (AMPH), uridine monophosphate kinase (UMPK), dihydrolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase complex, component X (PDHX), hypothetical protein FLJ32371, reticulon 2 (RTN2), and reticulon 3 (RTN3) show differential expression in human primary adipocytes and/or a human adipocyte cell line.
  • AMPPH, UMPK, DLAT, PDHX, FLJ32371, RTN2, and RTN3 are suitable for the manufacture of a pharmaceutical composition and a medicament for the treatment of conditions related to human metabolism, such as obesity, diabetes, and/or metabolic syndrome.
  • the invention also encompasses polynucleotides that encode the proteins of the invention and homologous proteins. Accordingly, any nucleic acid sequence, which encodes the amino acid sequences of the proteins of the invention and homologous proteins, can be used to generate recombinant molecules that express the proteins of the invention and homologous proteins.
  • the invention encompasses a nucleic acid encoding Drosophila Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc, or human Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologs, preferably a human homologous protein as described in Table 1; referred to herein as the proteins of the invention.
  • nucleotide sequences encoding the proteins may be produced.
  • the invention contemplates each and every possible variation of nucleotide sequence that can be made by selecting combinations based on possible codon choices.
  • polynucleotide sequences that are capable of hybridizing to the claimed nucleotide sequences, and in particular, those of the polynucleotide encoding the proteins of the invention, under various conditions of stringency.
  • Hybridization conditions are based on the melting temperature (Tm) of the nucleic acid binding complex or probe, as taught in Wahl G.M. et al., (1987: Methods Enzymol. 152: 399-407) and Kimmel A.R. (1987; Methods Enzymol. 152: 507-511), and may be used at a defined stringency.
  • hybridization under stringent conditions means that after washing for 1 h with 1 x SSC and 0.1% SDS at 50°C, preferably at 55°C, more preferably at 62°C and most preferably at 68°C, particularly for 1 h in 0.2 x SSC and 0.1% SDS at 50°C, preferably at 55°C, more preferably at 62°C, and most preferably at 68°C, a positive hybridization signal is observed.
  • Altered nucleic acid sequences encoding the proteins which are encompassed by the invention include deletions, insertions or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent protein.
  • the encoded proteins may also contain deletions, insertions or substitutions of amino acid residues, which produce a silent change and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological activity of the protein is retained.
  • the invention relates to peptide fragments of the proteins or derivatives thereof such as cyclic peptides, retro-inverso peptides or peptide mimetics having a length of at least 4, preferably at least 6 and up to 50 amino acids.
  • an 'allele' or 'allelic sequence' is an alternative form of the gene, which may result from at least one mutation in the nucleic acid sequence. Alleles may result in altered mRNAs or polypeptides whose structures or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes, which give rise to alleles, are generally ascribed to natural deletions, additions or substitutions of nucleotides. Each of these types of changes may occur alone or in combination with the others, one or more times in a given sequence.
  • nucleic acid sequences encoding the proteins of the invention and homologous proteins may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method which may be employed, 'restriction-site' PCR uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar G. et al., (1993) PCR Methods Applic. 2: 318-322).
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia T. et al., (1988) Nucleic Acids Res. 16: 8186).
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom M. et al., (1991) PCR Methods Applic. 1: 111-119). Another method which may be used to retrieve unknown sequences is that of Parker J.D. et al., (1991) Nucleic Acids Res. 19: 3055-3060. Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • nucleotide sequences encoding the proteins or functional equivalents may be inserted into appropriate expression vectors, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • appropriate expression vectors i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • Methods which are well known to those skilled in the art, may be used to construct expression vectors containing sequences encoding the proteins and the appropriate transcriptional and translational control elements.
  • Regulatory elements include for example a promoter, an initiation codon, a stop codon, a mRNA stability regulatory element, and a polyadenylation signal.
  • a polynucleotide can be assured by (i) constitutive promoters such as the Cytomegalovirus (CMV) promoter/enhancer region, (ii) tissue specific promoters such as the insulin promoter (see, Soria et al., (2000) Diabetes 49: 157), SOX2 gene promoter (see Li et al., (1998) Curr. Biol. 8: 971-974), Msi-1 promoter (see Sakakibara et al., (1997) J. Neuroscience 17: 8300-8312), alpha-cardia myosin heavy chain promoter or human atrial natriuretic factor promoter (Klug et al., (1996) J. clin.
  • constitutive promoters such as the Cytomegalovirus (CMV) promoter/enhancer region
  • tissue specific promoters such as the insulin promoter (see, Soria et al., (2000) Diabetes 49: 157), SOX2 gene promoter (see Li et al
  • Expression vectors can also contain a selection agent or marker gene that confers antibiotic resistance such as the neomycin, hygromycin or puromycin resistance genes.
  • selection agent or marker gene confers antibiotic resistance such as the neomycin, hygromycin or puromycin resistance genes.
  • natural, modified or recombinant nucleic acid sequences encoding the proteins of the invention and homologous proteins may be ligated to a heterologous sequence to encode a fusion protein.
  • a variety of expression vector/host systems may be utilized to contain and express sequences encoding the proteins or fusion proteins. These include, but are not limited to, micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, adenovirus, adeno-associated virus, lentiverus, retrovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or PBR322 plasmids); or animal cell systems.
  • micro-organisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus, aden
  • polynucleotide sequences of the invention in a sample can be detected by DNA-DNA and/or DNA-RNA hybridization and/or amplification using probes or portions or fragments of said polynucleotides.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequences specific for the gene to detect transformants containing DNA or RNA encoding the corresponding protein.
  • Oligonucleotides' or 'oligomers' refer to a nucleic acid sequence of at least about 10 nucleotides and as many as about 60 nucleotides, preferably about 15 to 30 nucleotides, and more preferably about 20-25 nucleotides, which can be used as a probe or amplimer.
  • Means for producing labeled hybridization or PCR probes for detecting polynucleotide sequences include oligo-labeling, nick translation, end-labeling of RNA probes, PCR amplification using a labeled nucleotide, or enzymatic synthesis. These procedures may be conducted using a variety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp., (Cleveland, Ohio).
  • Suitable reporter molecules or labels include radionuclides, enzymes, fluorescent, chemiluminescent or chromogenic agents as well as substrates, co-factors, inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding a protein of the invention may be cultured under conditions suitable for the expression and recovery of said protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides, which encode the protein may be designed to contain signal sequences, which direct secretion of the protein through a prokaryotic or eukaryotic cell membrane.
  • Other recombinant constructions may be used to join sequences encoding the protein to nucleotide sequence encoding a polypeptide domain, which will facilitate purification of soluble proteins.
  • Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAG extension/affinity purification system (Immunex Corp., Seattle, Wash.)
  • metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAG extension/affinity purification system Immunex Corp., Seattle, Wash.
  • cleavable linker sequences such as those specific for Factor XA or Enterokinase (Invitrogen, San Diego, Calif.) between the purification domain and the desired protein may be used to facilitate purification.
  • nucleic acids and proteins of the invention and effector molecules thereof are useful in diagnostic and therapeutic applications implicated, for example but not limited to, in metabolic disorders such as obesity as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • nucleic acids and proteins of the invention are, for example but not limited to, the following: (i) protein therapy, (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker, (v) gene therapy (gene delivery/gene ablation), (vi) research tools, and (vii) tissue regeneration in vitro and in vivo
  • nucleic acids and proteins of the invention and effectors thereof are useful in diagnostic and therapeutic applications implicated in various applications as described below.
  • cDNAs encoding the proteins of the invention and particularly their human homologues may be useful in gene therapy, and the proteins of the invention and particularly their human homologues may be useful when administered to a subject in need thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from, for example, but not limited to, in metabolic disorders as described above.
  • nucleic acids of the invention or fragments thereof may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acids or the proteins are to be assessed.
  • Further antibodies that bind immunospecifically to the substances of the invention may be used in therapeutic or diagnostic methods.
  • antibodies which are specific for the proteins of the invention and homologous proteins, may be used directly as an effector, e.g. an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express the protein.
  • the antibodies may be generated using methods that are well known in the art.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric single chain, Fab fragments, and fragments produced by a Fab expression library.
  • Neutralising antibodies i.e., those which inhibit dimer formation are especially preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others, may be immunized by injection with the protein or any fragment or oligopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response. It is preferred that the peptides, fragments or oligopeptides used to induce antibodies to the protein have an amino acid sequence consisting of at least five amino acids, and more preferably at least 10 amino acids.
  • Monoclonal antibodies to the proteins may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (Kohler G. and Milstein C, (1975) Nature 256: 495-497; Kozbor D. et al. (1985) J. Immunol. Methods 81: 31-42; Cote R.J. et al., (1983) Proc. Natl. Acad. Sci. 80: 2026-2030; Cole S.P. etal., (1984) Mol. Cell Biochem. 62: 109-120).
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries (Kang A.S. et al., (1991) Proc. Natl. Acad. Sci. 88: 11120-11123). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi R. et al., (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. and Milstein C, (1991) Nature 349: 293-299).
  • Antibody fragments which contain specific binding sites for the proteins may also be generated.
  • fragments include, but are not limited to, the F(ab') 2 fragments which can be produced by Pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of F(ab') 2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W.D. et al., (1989) Science 246: 1275-1281).
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding and immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between the protein and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reacive to two non-interfering protein epitopes are preferred, but a competitive binding assay may also be employed (Maddox D.E. et al., (1983) J. Exp. Med. 158: 1211-1216).
  • the polynucleotides or fragments thereof or nucleic acid effector molecules such as aptamers, antisense molecules, RNAi molecules or ribozymes may be used for therapeutic urposes.
  • aptamers i.e. nucleic acid molecules, which are capable of binding to a protein of the invention and modulating its activity may be generated by a screening and selection procedure involving the use of combinatorial nucleic acid libraries.
  • antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA.
  • cells may be transformed with sequences complementary to poly-nucleotides encoding the proteins of the invention and homologous proteins.
  • antisense molecules may be used to modulate protein activity or to achieve regulation of gene function.
  • sense or antisense oligomers or larger fragments can be designed from various locations along the coding or control regions of sequences encoding the proteins.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods, which are well known to those skilled in the art, can be used to construct recombinant vectors, which will express antisense molecules complementary to the polynucleotides of the genes encoding the proteins of the invention and homologous proteins. These techniques are described both in Sambrook et al. (supra) and in Ausubel et al. (supra).
  • Genes encoding the proteins of the invention and homologous proteins can be turned off by transforming a cell or tissue with expression vectors, which express high levels of polynucleotides that encode the proteins of the invention and homologous proteins or fragments thereof.
  • Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector and even longer if appropriate replication elements are part of the vector system.
  • antisense molecules e.g. DNA, RNA or nucleic acid analogues such as PNA
  • PNA nucleic acid analogues
  • Oligonucleotides derived from the transcription initiation site e.g., between positions -10 and +10 from the start site, are preferred.
  • inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing is useful because it cause inhibition of the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors or regulatory molecules.
  • the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Examples, which may be used, include engineered hammerhead motif ribozyme molecules that can be specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding the proteins of the invention and homologous proteins.
  • Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites which include the following sequences: GUA, GUU, and GUC.
  • RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Nucleic acid effector molecules e.g. antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences. Such DNA sequences may be incorporated into a variety of vectors with suitable RNA polymerase promoters such as T7 or SP6.
  • these cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half-life.
  • flanking sequences at the 5' and/or 3' ends of the molecule or modifications in the nucleobase, sugar and/or phosphate moieties, e.g. the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • An additional embodiment of the invention relates to the administration of a pharmaceutical composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of the nucleic acids and the proteins of the invention and homologous nucleic acids or proteins, antibodies to the proteins of the invention and homologous proteins, mimetics, agonists, antagonists or inhibitors of the proteins of the invention and homologous proteins or nucleic acids.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.
  • the pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art. For any compounds, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of preadipocyte cell lines or in animal models, usually mice, rabbits, dogs or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example the nucleic acids or the proteins of the invention or fragments thereof, or antibodies, which is sufficient for treating a specific condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50. Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration. The exact dosage will be determined by the practitioner, in light of factors related to the subject that requires treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect.
  • Factors which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination ⁇ ), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • antibodies which specifically bind to the proteins may be used for the diagnosis of conditions or diseases characterized by or associated with over- or underexpression of the proteins of the invention and homologous proteins or in assays to monitor patients being treated with the proteins of the invention and homologous proteins, or effectors thereof, e.g. agonists, antagonists, or inhibitors.
  • Diagnostic assays include methods which utilize the antibody and a label to detect the protein in human body fluids or extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by joining them, either covalently or non-covalently, with a reporter molecule.
  • reporter molecules which are known in the art may be used several of which are described above.
  • a variety of protocols including ELISA, RIA, and FACS for measuring proteins are known in the art and provide a basis for diagnosing altered or abnormal levels of gene expression.
  • Normal or standard values for gene expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibodies to the protein under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of protein expressed in control and disease, samples e.g. from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • the polynucleotides specific for the proteins of the invention and homologous proteins may be used for diagnostic purposes.
  • the polynucleotides, which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which gene expression may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess gene expression, and to monitor regulation of protein levels during therapeutic intervention.
  • hybridization with probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding the proteins of the invention and homologous proteins or closely related molecules may be used to identify nucleic acid sequences which encode the respective protein.
  • the hybridization probes of the subject invention may be DNA or RNA and are preferably derived from the nucleotide sequence of the polynucleotide encoding the proteins of the invention or from a genomic sequence including promoter, enhancer elements, and introns of the naturally occurring gene.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, radionuclides such as 32 P or 35 S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • reporter groups for example, radionuclides such as 32 P or 35 S or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences specific for the proteins of the invention and homologous nucleic acids may be used for the diagnosis of conditions or diseases, which are associated with the expression of the proteins. Examples of such conditions or diseases include, but are not limited to, metabolic diseases and disorders, including obesity and diabetes. Polynucleotide sequences specific for the proteins of the invention and homologous proteins may also be used to monitor the progress of patients receiving treatment for metabolic diseases and disorders, including obesity and diabetes. The polynucleotide sequences may be used qualitative or quantitative assays, e.g. in Southern or Northern analysis, dot blot or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered gene expression.
  • nucleotide sequences specific for the proteins of the invention and homologous nucleic acids may be useful in assays that detect activation or induction of various metabolic diseases such as obesity as well as related disorders such as eating disorder, cachexia, diabetes mellitus, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.
  • the nucleotide sequences may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value.
  • the presence of altered levels of nucleotide sequences encoding the proteins of the invention and homologous proteins in the sample indicates the presence of the associated disease.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence or a fragment thereof, which is specific for the nucleic acids encoding the proteins of the invention and homologous nucleic acids, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease.
  • Deviation between standard and subject values is used to establish the presence of disease. Once disease is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that, which is observed in the normal patient. The results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an unusual amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression of the metabolic diseases and disorders.
  • oligonucleotides designed from the sequences encoding the proteins of the invention and homologous proteins may involve the use of PCR.
  • Such oligomers may be chemically synthesized, generated enzymatically or produced from a recombinant source.
  • Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5'.fwdarw.3') and another with antisense (3'.rarw.5'), employed under optimized conditions for identification of a specific gene or condition.
  • the same two oligomers, nested sets of oligomers or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantification of closely related DNA or RNA sequences.
  • the nucleic acid sequences may also be used to generate hybridization probes, which are useful for mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques.
  • Such techniques include FISH, FACS or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.
  • FISH as described in Verma et al.
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier or affected individuals. An analysis of polymorphisms, e.g. single nucleotide polymorphisms may be carried out. Further, in situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms or parts thereof, by physical mapping.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequences of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc. among normal, carrier or affected individuals.
  • the proteins of the invention can be used for screening libraries of compounds in any of a variety of drug screening techniques.
  • the protein or fragment thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly.
  • the formation of binding complexes, between the proteins of the invention and the agent tested, may be measured.
  • Agents can also be identified, which either directly or indirectly, influence the activity of the proteins of the invention.
  • binding of a fluorescently labeled peptide derived from a protein of the invention to the interacting protein could be detected by a change in polarisation.
  • binding partners which can be either the full length proteins as well as one binding partner as the full length protein and the other just represented as a peptide are fluorescently labeled
  • binding could be detected by fluorescence energy transfer (FRET) from one fluorophore to the other.
  • FRET fluorescence energy transfer
  • the interaction of the proteins of the invention with cellular proteins could be the basis for a cell-based screening assay, in which both proteins are fluorescently labeled and interaction of both proteins is detected by analysing cotranslocation of both proteins with a cellular imaging reader, as has been developed for example, but not exclusively, by Cellomics or EvotecOAI.
  • the two or more binding partners can be different proteins with one being the protein of the invention, or in case of dimerization and/or oligomerization the protein of the invention itself.
  • Proteins of the invention for which one target mechanism of interest, but not the only one, would be such protein/protein interactions are Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous proteins.
  • luciferase reporter assays are commercially available, e.g. from BD Bioscience, Promega, and Boehringer Mannheim.
  • Other reporter genes can be also used to detect eukaryotic gene expression, like chloramphenicol acetyltransferase (CAT), beta-galactosidase (beta-Gal), or human placental alkaline phosphatase (SEAP).
  • CAT chloramphenicol acetyltransferase
  • beta-Gal beta-galactosidase
  • SEAP human placental alkaline phosphatase
  • agent as used herein describes any molecule, e.g. protein or pharmaceutical, with the capability of altering or mimicking the physiological function of one or more of the proteins of the invention.
  • Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
  • the candidate agents often comprise carbocyclic or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, nucleic acids and derivatives, structural analogs or combinations thereof.
  • Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries.
  • pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.
  • the screening assay is a binding assay
  • one or more of the molecules may be joined to a label, where the label can directly or indirectly provide a detectable signal.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564.
  • large numbers of different small test compounds e.g. aptamers, peptides, low-molecular weight compounds etc.
  • the test compounds are reacted with the proteins or fragments thereof, and washed. Bound proteins are then detected by methods well known in the art. Purified proteins can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the nucleic acids encoding the proteins of the invention can be used to generate transgenic animals or site-specific gene modifications in cell lines. These transgenic non-human animals are useful in the study of the function and regulation of the proteins of the invention in vivo.
  • Transgenic animals particularly mammalian transgenic animals, can serve as a model system for the investigation of many developmental and cellular processes common to humans.
  • a variety of non-human models of metabolic disorders can be used to test effectors/modulators of the proteins of the invention.
  • Misexpression for example, overexpression or lack of expression
  • such assays use mouse models of insulin resistance and/or diabetes, such as mice carrying gene knockouts in the leptin pathway (for example, ob (leptin) or db (leptin receptor) mice).
  • leptin pathway for example, ob (leptin) or db (leptin receptor) mice.
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning et al., 1998, supra).
  • Susceptible wild type mice for example C57BI/6) show similiar symptoms if fed a high fat diet.
  • mice could be used to test whether administration of a candidate effector/modulator alters for example lipid accumulation in the liver, in plasma, or adipose tissues using standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • Transgenic animals may be made through homologous recombination in non-human embryonic stem cells, where the normal locus of the gene encoding a protein of the invention is altered.
  • a nucleic acid construct encoding a protein of the invention is injected into oocytes and is randomly integrated into the genome.
  • Vectors for stable integration include plasmids, retroviruses and other animal viruses, yeast artificial chromosomes (YACs), and the like.
  • the modified cells or animal are useful in the study of the function and regulation of the proteins of the invention. For example, a series of small deletions and/or substitutions may be made in the gene that encodes a protein of the invention to determine the role of particular domains of the protein, functions in pancreatic differentiation, etc.
  • variants of the genes of the invention like specific constructs of interest include anti-sense molecules, which will block the expression of the proteins of the invention, or expression of dominant negative mutations.
  • a detectable marker such as for example lac-Z or luciferase may be introduced in the locus of a gene of the invention, where up regulation of expression of the genes of the invention will result in an easily detected change in phenotype.
  • genes of the invention or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development.
  • proteins of the invention in cells in which they are not normally produced, one can induce changes in cell behavior.
  • DNA constructs for homologous recombination will comprise at least portions of the genes of the invention with the desired genetic modification, and will include regions of homology to the target locus. DNA constructs for random integration do not need to contain regions of homology to mediate recombination. Conveniently, markers for positive and negative selection are included. DNA constructs for random integration will consist of the nucleic acids encoding the proteins of the invention, a regulatory element (promoter), an intron and a poly-adenylation signal. Methods for generating cells having targeted gene modifications through homologous recombination are known in the art. For non-human embryonic stem (ES) cells, an ES cell line may be employed, or embryonic cells may be obtained freshly from a host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an appropriate fibroblast-feeder layer and are grown in the presence of leukemia inhibiting factor (LIF).
  • LIF leukemia inhibiting factor
  • non-human ES or embryonic cells or somatic pluripotent stem cells When non-human ES or embryonic cells or somatic pluripotent stem cells have been transfected, they may be used to produce transgenic animals. After transfection, the cells are plated onto a feeder layer in an appropriate medium. Cells containing the construct may be selected by employing a selective medium. After sufficient time for colonies to grow, they are picked and analyzed for the occurrence of homologous recombination or integration of the construct. Those colonies that are positive may then be used for embryo transfection and morula aggregation. Briefly, morulae are obtained from 4 to 6 week old superovulated females, the Zona Pellucida is removed and the morulae are put into small depressions of a tissue culture dish.
  • the ES cells are trypsinized, and the modified cells are placed into the depression closely to the morulae.
  • the aggregates are transfered into the uterine horns of pseudopregnant females.
  • Females are then allowed to go to term.
  • Chimeric offsprings can be readily detected by a change in coat color and are subsequently screened for the transmission of the mutation into the next generation (F1 -generation).
  • Offspring of the F1- generation are screened for the presence of the modified gene and males and females having the modification are mated to produce homozygous progeny. If the gene alterations cause lethality at some point in development, tissues or organs can be maintained as allogenic or congenic grafts or transplants, or in vitro culture.
  • the transgenic animals may be any non-human mammal, such as laboratory animal, domestic animals, etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and others.
  • the transgenic animals may be used in functional studies, drug screening, and other applications and are useful in the study of the function and regulation of the proteins of the invention in vivo.
  • the invention also relates to a kit comprising at least one of
  • the kit may be used for diagnostic or therapeutic purposes or for screening applications as described above.
  • the kit may further contain user instructions.
  • Figure 1 shows the triglyceride and glycogen content of a Drosophila Hr46 (GadFly Accession Number CG11823) mutant. Shown is the change of triglyceride content of HD-EP(2)25108 flies caused by integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG)' column 2). Also shown is the change of glycogen content of HD-EP(2)25108 flies caused by integration of the P-vector into the annotated transcription unit (column 5) in comparison to controls (referred to as 'control (glycogen)' column 4).
  • FIG. 3 shows the expression of the Hr46 (GadFly Accession Number
  • CG11823 homologs in mammalian (mouse) tissues.
  • Figure 3A shows the real-time PCR analysis of RAR-related orphan receptor alpha (Rora) expression in wild-type mouse tissues.
  • Figure 3B shows the real-time PCR analysis of Rora expression in different mouse models.
  • Figure 3C shows the real-time PCR analysis of Rora expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Figure 3D shows the real-time PCR analysis of Rora expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 3E shows the real-time PCR analysis of RAR-related orphan receptor beta (Rorb) expression in wild-type mouse tissues.
  • Figure 3F shows the real-time PCR analysis of Rorb expression in different mouse models.
  • Figure 3G shows the real-time PCR analysis of Rorb expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Figure 3H shows the real-time PCR analysis of Rorb expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 31 shows the real-time PCR analysis of RAR-related orphan receptor gamma (Rorc) expression in wild-type mouse tissues.
  • Figure 3J shows the real-time PCR analysis of Rorc expression in different mouse models.
  • Figure 3K shows the real-time PCR analysis of Rorc expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Figure 3L shows the real-time PCR analysis of Rorc expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 4 shows the triglyceride and glycogen content of a Drosophila Amph (GadFly Accession Number CG8604) mutant.
  • Figure 5 shows the molecular organization of the mutated Amph gene locus.
  • FIG. 6 shows the expression of the Amph (GadFly Accession Number
  • CG8604 homologs in mammalian (mouse) tissues.
  • Figure 6A shows the real-time PCR analysis of amphiphysin (Amph) expression in wild-type mouse tissues.
  • Figure 6B shows the real-time PCR analysis of Amph expression in different mouse models.
  • Figure 6C shows the real-time PCR analysis of bridging integrator 1 (Bin1) expression in wild-type mouse tissues.
  • Figure 6D shows the real-time PCR analysis of Bin1 expression in different mouse models.
  • Figure 6E shows the real-time PCR analysis of Bin1 expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Figure 6F shows the real-time PCR analysis of Bin1 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 7 shows the expression of a human Amph homolog in mammalian (human) tissue. Shown is the microarray analysis of amphiphysin (AMPH) expression in a human adipocyte cell line, during the differentiation from preadipocytes to mature adipocytes.
  • AMPH amphiphysin
  • Figure 8 shows the triglyceride content of a Drosophila CG6364 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(3)31088 flies caused by integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG) 1 column 2). Also shown is the change of glycogen content of HD-EP(3)31088 flies caused by integration of the P-vector into the annotated transcription unit (column 5) in comparison to controls (referred to as 'control (glycogen)' column 4).
  • Figure 9 shows the molecular organization of the mutated CG6364 (Gadfly Accession Number) gene locus.
  • Figure 10 shows the expression of the uridine kinase (GadFly Accession
  • Figure 10A shows the real-time PCR analysis of uridine monophosphate kinase (Umpk) expression in wild-type mouse tissues.
  • Figure 10B shows the real-time PCR analysis of Umpk expression in different mouse models.
  • Figure 10C shows the real-time PCR analysis of Umpk expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Figure 10D shows the real-time PCR analysis of Umpk expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 10E shows the real-time PCR analysis of uridine-cytidine kinase 2
  • Figure 10F shows the real-time PCR analysis of Uck2 expression in different mouse models.
  • Figure 10G shows the real-time PCR analysis of Uck2 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 11 shows the expression of human uridine kinase (GadFly Accession Number CG6364) homologs in mammalian (human) tissue.
  • Figure 11 A shows the microarray analysis of uridine monophosphate kinase (UMPK) expression in human abdominal derived primary adipocyte cells, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 11 B shows the microarray analysis of UMPK expression in a human adipocyte cell line, during the differentiation from preadipocytes to mature adipocytes.
  • Figure 12 shows the triglyceride content of a Drosophila NPC1 (GadFly Accession Number CG5722) mutant.
  • UMPK uridine monophosphate kinase
  • Figure 13 shows the molecular organization of the mutated NPC1 gene locus.
  • Figure 14 shows the expression of the NPC1 (GadFly Accession Number
  • CG5722 homolog in mammalian (mouse) tissues.
  • Figure 14A shows the real-time PCR analysis of Niemann Pick type C1
  • Npc1 expression in wild-type mouse tissues.
  • Figure 14B shows the real-time PCR analysis of Npc1 expression in different mouse models.
  • Figure 14C shows the real-time PCR analysis of Npc1 expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Figure 14D shows the real-time PCR analysis of Npc1 expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 15 shows the triglyceride content of a Drosophila CG5261 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(2)25938/CyO flies caused by heterozygous integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG)' column 2).
  • Figure 16 shows the molecular organization of the mutated CG5261 (Gadfly Accession Number) gene locus.
  • Figure 17 shows the expression of the GadFly Accession Number CG5261 homolog in mammalian (mouse) tissues.
  • Figure 17A shows the real-time PCR analysis of dihydrolipoamide S- acetyltransferase (Dlat) expression in wild-type mouse tissues.
  • Figure 17B shows the real-time PCR analysis of Dlat expression in different mouse models and of Dlat expression in mice fed with a high fat diet compared to mice fed with a control diet.
  • Dlat dihydrolipoamide S- acetyltransferase
  • Figure 17C shows the real-time PCR analysis of Dlat expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.
  • Figure 18 shows the expression of human CG5261 (GadFly Accession
  • Figure 18A shows the microarray analysis of dihydrolipoamide S- acetyltransferase (DLAT) expression in human abdominal derived primary adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • DLAT dihydrolipoamide S- acetyltransferase
  • Figure 18B shows the microarray analysis of DLAT expression in a human adipocyte cell line during the differentiation from preadipocytes to mature adipocytes.
  • Figure 18C shows the microarray analysis of pyruvate dehydrogenase complex, component X (PDHX) expression in human abdominal derived primary adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • PDHX pyruvate dehydrogenase complex, component X
  • Figure 18D shows the microarray analysis of PDHX expression in a human adipocyte cell line during the differentiation from preadipocytes to mature adipocytes.
  • Figure 19 shows the triglyceride content of a Drosophila CG6903 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(X)10662 flies caused by integration of the P-vector into the annotated transcription unit (column 2) in comparison to controls containing all flies of the EP collection ('EP-control', column 1).
  • Figure 20 shows the molecular organization of the mutated CG6903 (Gadfly Accession Number) gene locus.
  • Figure 21 shows the expression of human CG6903 (GadFly Accession
  • Figure 22 shows the triglyceride content of a Drosophila CG8895 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(2)25095 flies caused by integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG)' column 2).
  • 'HD-control (TG)', column 1 controls containing more than 2000 fly lines of the proprietary EP collection
  • 'WT-control (TG)' column 2 wildtype controls determined in more than 80 independent assays
  • Figure 23 shows the molecular organization of the mutated CG8895 (Gadfly Accession Number) gene locus.
  • Figure 24 shows the expression of human CG8895 (GadFly Accession Number) homologs in mammalian (human) tissue.
  • Figure 24A shows the microarray analysis of reticulon 2 (RTN2) expression in human abdominal derived primary adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • RTN2 reticulon 2
  • Figure 24B shows the microarray analysis of RTN2 expression in a human adipocyte cell line during the differentiation from preadipocytes to mature adipocytes.
  • Figure 24C shows the microarray analysis of reticulon 3 (RTN3) expression in human abdominal derived primary adipocyte cells during the differentiation from preadipocytes to mature adipocytes.
  • Figure 24D shows the microarray analysis of RTN3 expression in a human adipocyte cell line during the differentiation from preadipocytes to mature adipocytes.
  • RTN3 reticulon 3
  • Figure 25 shows the triglyceride content of a Drosophila ptc (GadFly Accession Number CG2411) mutant. Shown is the change of triglyceride content of HD-EP(2)20701 flies caused by integration of the P-vector into the annotated transcription unit (column 3) in comparison to controls containing more than 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1) and wildtype controls determined in more than 80 independent assays (referred to as 'WT-control (TG)' column 2). Also shown is the change of glycogen content of HD-EP(2)20701 flies caused by integration of the P-vector into the annotated transcription unit (column 5) in comparison to controls (referred to as 'control (glycogen)' column 4).
  • Figure 26 shows the molecular organization of the mutated ptc (Gadfly Accession Number CG2411) gene locus.
  • Example 1 Measurement of triglyceride and glycogen content in Drosophila
  • Mutant flies are obtained from a fly mutation stock collection. The flies are grown under standard conditions known to those skilled in the art. In the course of the experiment, additional feedings with bakers yeast (Saccharomyces cerevisiae) are provided for the EP-lines HD-EP(2)25108, HD-EP(2)21926, HD-EP(3)31088, HD-EP(2)26176, HD-EP(2)25938, HD-EP (X)10662, HD-EP(2)25095, or HD-EP(2)20701.
  • bakers yeast Sacharomyces cerevisiae
  • ESM energy storage metabolites
  • the triglyceride content of the flies extract was determined using Sigma Triglyceride (INT 336-10 or -20) assay by measuring changes in the optical density according to the manufacturer's protocol, and the glycogen content of the flies extract was determined using Roche (Starch UV-method Cat. No. 0207748) assay by measuring changes in the optical density according to the manufacturer's protocol.
  • Roche Starch UV-method Cat. No. 0207748
  • protein content of the same extract was measured using BIO-RAD DC Protein Assay according to the manufacturer's protocol.
  • the average triglyceride level ( ⁇ g triglyceride/ ⁇ g protein) of all flies of the EP collection (referred to as 'EP-control') is shown as 100% in the first column in
  • FIG 19 The average triglyceride level ( ⁇ g triglyceride/ ⁇ g protein) of 2108 flies of the proprietary EP collection (referred to as 'HD-control (TG)') is shown as 100% in the first columns in Figures 1, 4, 8, 12, 15, 22, and 25.
  • the average triglyceride level ( ⁇ g triglyceride/ ⁇ g protein) of wildtype OreR flies was determined in 84 independent assays (referred to as 'WT-control (TG)') is shown as 102% in the second columns in Figures 1 , 4, 8, 12, 15, 22, and 25.
  • the average glycogen level ( ⁇ g glycogen/ ⁇ g protein) of an internal assay control consisting of two different wildtype strains and an inconspicuous EP-line of the HD stock collection (referred to as 'control (glycogen)') is shown as 100% in the fourth columns in Figures 1 , 4, 8, and 25. Standard deviations of the measurements are shown as thin bars.
  • HD-EP(2)25108 homozygous flies (column 3 in Figure 1, ⁇ D-25108 (TG)'), HD-EP(2)25938 heterozygous flies, containing a CyO balancer chromosome (column 3 in Figure 15, ⁇ D-25938/CyO (TG)'), HD-EP(X)10662 hemizygous flies (column 2 in Figure 19, 'HD-EP10662 (TG)'), HD-EP(2)25095 homozygous flies (column 3 in Figure 22, 'HD-26176 (TG)'), and HD-EP(2) 20701 homozygous flies (column 3 in Figure 25, ⁇ D-20701 (TG)') show constantly a lower triglyceride content ( ⁇ g triglyceride/ ⁇ g protein) than the controls.
  • HD-EP(2)21926 homozygous flies (column 3 in Figure 4, ⁇ D-21926 (TG)'), HD-EP(3)31088 homozygous flies (column 3 in Figure 8, 'HD-31088 (TG)'), and HD-EP(2)26176 homozygous flies (column 3 in Figure 12, 'HD-26176 (TG)') show constantly a higher triglyceride content ( ⁇ g triglyceride/ ⁇ g protein) than the controls.
  • HD-EP(2)25108 homozygous flies (column 5 in Figure 1, 'HD-25108 (glycogen)'), HD-EP(2)21926 homozygous flies (column 5 in Figure 4, 'HD-21926 (glycogen)'), and HD-EP(2)20701 homozygous flies (column 5 in Figure 25, 'HD-20701 (glycogen)') constantly show a lower glycogen content ( ⁇ g glycogen/ ⁇ g protein) than the controls.
  • HD-EP(3)31088 homozygous flies constantly show a higher glycogen content ( ⁇ g glycogen/ ⁇ g protein) than the controls (column 5 in Figure 8, ⁇ D-31088 (glycogen)').
  • Example 2 Identification of Drosophila genes responsible for changes in triglyceride and glycogen contents
  • Nucleic acids encoding the proteins of the present invention were identified using a plasmid-rescue technique.
  • Genomic DNA sequences were isolated that are localized adjacent to the EP vector (herein HD-EP(2)25108, HD-EP(2) 21926, HD-EP(3)31088, HD-EP(2)26176, HD-EP(2)25938, HD-EP(X)10662, HD-EP(2)25095, or HD-EP(2)20701) integration.
  • public databases like Berkeley Drosophila Genome Project (GadFly) were screened, thereby identifying the integration sites of the vectors, and the corresponding genes. The molecular organization of these gene loci is shown in Figures Figures 2, 5, 9, 13, 16, 20, 23, and 26.
  • genomic DNA sequence is represented by the assembly as a dotted black line in the middle that includes the integration sites of the vectors for lines HD-EP(2)25108,
  • Numbers represent the coordinates of the genomic DNA.
  • the upper parts of the figures represent the sense strand "+”, the lower parts represent the antisense strand "-”.
  • the insertion sites of the P-elements in the Drosophila lines are shown as triangles or boxes in the "P-elements +" or/and "P-elements
  • genomic DNA sequence is represented by the assembly as a horizontal black scaled double-headed arrow that includes the integration sites of the vectors for lines HD-EP(2)25108, HD-EP(2)25938, HD-EP(2)25095, or HD-EP(2)20701.
  • Ticks represent the length in basepairs of the genomic DNA (1000 base pairs ( Figure 16) or 10000 base pairs ( Figures 2, 23, and 26) per tick).
  • the part of the figure above the double-headed arrow represents the sense strand, the part below the double-headed arrow represents the antisense strand.
  • the grey arrows in the upper part and/or lower part of the figures represent BAC clones
  • the black arrows in the topmost part of the figures represent the sections of the chromosomes.
  • the insertion sites of the P-elements in the Drosophila lines are shown as triangles and are labeled.
  • the cDNA sequences of the predicted genes are shown as dark grey bars (exons), linked by dark grey lines (introns), and are labeled (see also key at the bottom of the figures).
  • the HD-EP(2)25108 vector is homozygous viable integrated into the transcription unit of the transcribed DNA sequence (EST) GH09429, which overlaps with the cDNA of gene Hr46, in antisense orientation.
  • the chromosomal localization site of integration of the vector of HD-EP(2)25108 is at gene locus 2R, 46F5-6.
  • Figure 2 shows the molecular organization of this gene locus.
  • the insertion site of the P-element in Drosophila line HD-EP(2)25108 is shown as arrow and is labeled.
  • the transcrips of the predicted Drosophila gene Hr46 are labeled.
  • numbers represent the coordinates of the genomic DNA, starting at position 5264000 on chromosome 2R, ending at position 5297000.
  • the insertion site of the P-element in Drosophila line HD-EP(2)25108 is shown in the "P Elements +" line and is labeled.
  • the gene Hr46 is labeled (referred to as "CG11823 Hr46").
  • the HD-EP(2)21926 vector is homozygous viable integrated 107 base pairs downstream of the transcriptional start site of Amph and 310 base pairs downstream of the translational start site of Amph in sense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(2)21926 is at gene locus 2R, 49B7-8.
  • Figure 5 shows the molecular organization of this gene locus. In the upper part of Figure 5, numbers represent the coordinates of the genomic DNA starting at position 7656709 on chromosome 2R, ending at position 7659834.
  • the insertion site of the P-element in Drosophila line HD-EP(2)21926 is shown in the "P Elements +" line and is labeled.
  • the first exon of gene Amph is labeled (referred to as "amph., exonl").
  • the predicted Amph gene is shown as black bars linked by slim black lines.
  • the integrations site of the P-element is shown as triangle, the direction of the P-element is shown as arrow, and the translation start is labeled as "ATG" in the first exon of the Amph gene.
  • the HD-EP(3)31088 vector is homozygous viable integrated into the cDNA (at base pair 35) of the predicted gene CG6364 in antisense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(3)31088 is at gene locus 3R, 95F2.
  • Figure 9 shows the molecular organization of this gene locus.
  • numbers represent the coordinates of the genomic DNA starting at position 20106031 on chromosome 3R, ending at position 20109156.
  • the insertion site of the P-element in Drosophila line HD-EP(3) 31088 is shown in the "P Elements -" line and is labeled.
  • the gene CG6364 is shown in the "cDNA +" line, and the corresponding transcribed DNA sequences (ESTs) shown as grey bars in the "EST +” line are labeled.
  • the HD-EP(2)26176 vector is homozygous viable integrated 6 base pairs 5' of the cDNA of the predicted gene NPC1 in sense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(2)26176 is at gene locus 2L, 31A2-3 (according to Flybase), 31 B1 (according to GadFly release 3).
  • Figure 13 shows the molecular organization of this gene locus. In Figure 13, numbers represent the coordinates of the genomic DNA starting at position 10205000 on chromosome 2L, ending at position 10215000.
  • the insertion site of the P-element in Drosophila line HD-EP(2)26176 is shown in the "P Elements -" line and is labeled.
  • the gene NPC1 is labeled (referred to as 'NPC1, CG7522').
  • the HD-EP(2)25938 vector is heterozygous viable integrated 2940 base pairs 5' of CG5261-RB in antisense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(2)25938 is at gene locus 2L, 27F1-2 (according to Flybase), 27F6 (according to Gadfly release 3).
  • Figure 16 shows the molecular organization of this gene locus.
  • the insertion site of the P-element in Drosophila line HD-EP(2)25938 is shown as triangle and is labeled.
  • the transcrips of the predicted Drosophila gene CG5261 are labeled.
  • the HD-EP(X)10662 vector is hemizygous viable integrated into the first exon of the cDNA (base pair 80) of the predicted gene CG6903 in antisense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(X)10662 is at gene locus X, 4E1 (according to Flybase), 4D1 (according to Gadfly release 2).
  • Figure 20 shows the molecular organization of this gene locus.
  • numbers represent the coordinates of the genomic DNA starting at position 4727500 on chromosome X, ending at position 4731000.
  • the insertion site of the P-element in Drosophila line HD-EP (X)10662 is shown in the "P Elements -" line and is labeled.
  • the gene CG6903 is shown in the "cDNA +" line, and the corresponding ESTs shown in the "EST +” line are labeled.
  • the HD-EP(2)25095 vector is homozygous viable integrated into an intron of the Drosophila gene CG8895 in antisense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(2)25095 is at gene locus 2L, 25B3-4 (according to Flybase), 25B9-10 (according to Gadfly release 3).
  • Figure 23 shows the molecular organization of this gene locus.
  • the insertion site of the P-element in Drosophila line HD-EP(2)25095 is labeled.
  • the predicted cDNAs of the Drosophila CG8895 (GadFly Accession Number) gene are shown in the middle of the figure.
  • the HD-EP(2)20701 vector is homozygous viable integrated933 base pairs ⁇ prime of the cDNA of the gene ptc in sense orientation.
  • the chromosomal localization site of integration of the vector HD-EP(2)20701 is at gene locus 2R, 44D5-E1.
  • Figure 26 shows the molecular organization of this gene locus.
  • the insertion site of the P-element in Drosophila line HD-EP(2) 20701 is labeled.
  • Predicted cDNAs of the Drosophila gene ptc are shown in the middle of the figure.
  • expression of the cDNAs encoding the proteins of the invention could be affected by integration of the vectors, leading to a change in the amount of triglycerides or energy storage metabolites.
  • Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds.
  • nucleic acids comprising Drosophila Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc, or human Hr46, Amph, CG6364, NPC1, CG5261, CG6903, CG8895, or ptc homologs (in particular the human homolous proteins as described in Table 1).
  • the activation of the genes therefore occurs in the same cells (eye) that overexpress dUCPy. Since the mutant collection contains several thousand strains with different integration sites of the EP-element it is possible to test a large number of genes whether their expression interacts with dUCPy activity. In case a gene acts as an enhancer of UCP activity the eye defect will be worsened; a suppressor will ameliorate the defect.
  • Example 5 Expression of the polypeptides in mammalian (mouse) tissues
  • mice strains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standard model systems in obesity and diabetes research
  • Harlan Winkelmann 33178 Borchen, Germany
  • constant temperature preferrably 22°C
  • 40 per cent humidity a light / dark cycle of preferrably 14 / 10 hours.
  • the mice were fed a standard chow (for example, from ssniff Spezialitaten GmbH, order number ssniff M-Z V1126- 000).
  • wild type mice For the fasting experiment (“fasted wild type mice”), wild type mice were starved for 48 h without food, but only water supplied ad libitum (see, for example, Schnetzler B. et al., 1993, J Clin Invest 92: 272-280, Mizuno T.M. et al., 1996, Proc Natl Acad Sci U S A 93: 3434-3438).
  • wild-type (wt) mice were fed a control diet (preferably Altromin C1057 mod control, 4.5% crude fat) or high fat diet (preferably Altromin C1057mod. high fat, 23.5% crude fat). Animals were sacrificed at an age of 6 to 8 weeks. The animal tissues were isolated according to standard procedures known to those skilled in the art, snap frozen in liquid nitrogen and stored at -80°C until needed.
  • mammalian fibroblast (3T3-L1) cells e.g., Green H. and Kehinde O., 1974, Cell 1: 113-116 were obtained from the American Tissue Culture Collection (ATCC, Hanassas, VA, USA; ATCC- CL 173).
  • 3T3- L1 cells were maintained as fibroblasts and differentiated into adipocytes as described in the prior art (e.g., Qiu Z. et al., 2001, J. Biol. Chem. 276: 11988-11995; Slieker L.J.
  • dO serum-free cells were transferred to serum-free (SF) medium, containing DMEM/HamF12 (3:1; Invitrogen), Fetuin (300 ⁇ g/ml; Sigma, Kunststoff, Germany), transferrin (2 ⁇ g/ml; Sigma), pantothenate (17 ⁇ M; Sigma), biotin (1 ⁇ M; Sigma), and EGF (0.8 nM; Hoffmann-La Roche, Basel, Switzerland).
  • Differentiation was induced by adding dexamethasone (DEX; 1 ⁇ M; Sigma), 3-methyl-isobutyl-1 - methylxanthine (MIX; 0.5 mM; Sigma), and bovine insulin (5 ⁇ g/ml; Invitrogen).
  • DEX dexamethasone
  • MIX 3-methyl-isobutyl-1 - methylxanthine
  • bovine insulin 5 ⁇ g/ml; Invitrogen).
  • d4 Four days after confluence (d4), cells were kept in SF medium, containing bovine insulin (5 ⁇ g/ml) until differentiation was completed. At various time points of the differentiation procedure, beginning with day 0 (day of confluence) and day 2 (hormone addition; for example, dexamethasone and 3-isobutyl-1 -methylxanthine), up to 10 days of differentiation, suitable aliquots of cells were taken every two days.
  • Taqman analysis was performed preferably using the following primer/probe pairs:
  • mouse RAR-related orphan receptor alpha (Rora) sequence (GenBank Accession Number NM__013646): Mouse Rora forward primer (SEQ ID NO: 1): 5'- GGC TGA CGG AAC TGC ATG AT -3'; mouse Rora reverse primer (SEQ ID NO: 2): 5'- GAG TCG GCC TTG CTG CC -3'; mouse Rora Taqman probe (SEQ ID NO: 3): (5/6-FAM)- ACC TCA GCA CCT ATA TGG ATG GGC ACA C -(5/6-TAMRA).
  • Mouse Rorb forward primer (SEQ ID NO: 4): 5'- ACC GGC GGC ACA TAC G -3'
  • mouse Rorb reverse primer (SEQ ID NO: 5): 5'- ACC GGA ATC TAT GCT GTA ATA ACC TT -3'
  • mouse Rorb Taqman probe (SEQ ID NO: 6): (5/6-FAM)- ACG GGC ACG TCA TTG ACC TGC C -(5/6-TAMRA).
  • Mouse Rorc forward primer (SEQ ID NO: 7): 5'- TTT CTG AGG ATG AGA TTG CCC T -3'
  • mouse Rorc reverse primer (SEQ ID NO: 8): 5'- TTG GAG CCC AGG ACG GT -3'
  • mouse Rorc Taqman probe (SEQ ID NO: 9): (5/6- FAM)- ACA CGG CCC TGG TTC TCA TCA ATG C -(5/6-TAMRA).
  • mouse amphiphysin (Amph) sequence GenBank Accession Number XM_1272173:
  • Mouse Amph forward primer (SEQ ID NO: 10): 5'- CGG AGC TTG CAA TCA GTG AG -3'; mouse Amph reverse primer (SEQ ID NO: 11): 5'- AAC AGA AGG GAT GAC CTG AGG A -3'; mouse Amph Taqman probe (SEQ ID NO: 12): (5/6-FAM)- CTC AAC CAG TGG AGC CCG AAG CG -(5/6-TAMRA).
  • mice bridging integrator 1 (Bin1) sequence (GenBank Accession Number NM_009668):
  • Mouse Bin1 forward primer (SEQ ID NO: 13): 5'- TAC CAT CCC CAA GTC CCC AT -3'; mouse Bin1 reverse primer (SEQ ID NO: 14): 5'- TGT GTT TGG GAG GCG GA -3'; mouse Bin1 Taqman probe (SEQ ID NO: 15): (5/6- FAM)- AGC TCC GGA AAG GCC CAC CTG TC -(5/6-TAMRA).
  • mouse Umpk For the amplification of mouse uridine monophosphate kinase (Umpk) sequence (GenBank Accession Number XM_130160): Mouse Umpk forward primer (SEQ ID NO: 16): 5'- CGC TGA CGT GAT CA TCC CT -3'; mouse Umpk reverse primer (SEQ ID NO: 17): 5'- GGT CCC CGT TGA GGA TGT C -3'; mouse Umpk Taqman probe (SEQ ID NO: 18): (5/6-FAM)- TGG CCA TCA ACC TGA TCG TGC AA -(5/6-TAMRA).
  • Mouse Uck2 forward primer (SEQ ID NO: 19): 5'- AAG CGG CAG ACG AAC GG -3'
  • mouse Uck2 reverse primer (SEQ ID NO: 20): 5'- GAC TCT GAC GCC TGC CTC TT -3'
  • mouse Uck2 Taqman probe (SEQ ID NO: 21): (5/6- FAM)- ATC TCA ACG GCT ACA CCC CTT CCC G -(5/6-TAMRA).
  • Mouse Npd forward primer (SEQ ID NO: 22): 5'- TTG ACT GGG TCT CGC CAC A -3'; mouse Npd reverse primer (SEQ ID NO: 23): 5'- GGG TCC ATC ACA GAA GCA TTG -3'; mouse Npd Taqman probe (SEQ ID NO: 24): (5/6- FAM)- CTG CAG ACT CTA CAA CGT CAC TCA CCA GTT CT - (5/6- TAMRA).
  • mice dihydrolipoamide S-acetyltransferase E2 component of pyruvate dehydrogenase complex
  • Dlat mouse dihydrolipoamide S-acetyltransferase sequence
  • Mouse Dlat forward primer (SEQ ID NO: 25): 5'- TCA GCT TTG GCC TGT CTG AA -3'; mouse Dlat reverse primer (SEQ ID NO: 26): 5'- ACC ACA TGA TTT TGC CTT ATA ACT GT -3'; mouse Dlat Taqman probe (SEQ ID NO: 27): (5/6-FAM)- TTC CCG AAG CAA ACT CGT CTT GGA TG -(5/6-TAMRA).
  • RNA-expression is shown on the Y-axis.
  • the tissues tested are given on the X-axis.
  • WAT refers to white adipose tissue
  • BAT refers to brown adipose tissue.
  • the X-axis represents the time axis.
  • dO refers to day 0 (start of the experiment)
  • d2 -
  • d12 refers to day 2 - day 12 of adipocyte differentiation.
  • the function of the proteins of the invention in metabolism was further validated by analyzing the expression of the transcripts in different tissues and by analyzing the role in adipocyte differentiation.
  • mice carrying gene knockouts in the leptin pathway (for example, ob/ob (leptin) or db/db (leptin receptor/ligand) mice) to study the expression of the proteins of the invention.
  • leptin pathway for example, ob/ob (leptin) or db/db (leptin receptor/ligand) mice
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J.C. et al, (1998) Mol. Cell. 2: 559-569).
  • expression of the mRNAs encoding the proteins of the invention was also examined in susceptible wild type mice (for example, C57BI/6) that show symptoms of diabetes, lipid accumulation, and high plasma lipid levels, if fed a high fat diet.
  • RAR-related orphan receptor alpha (Rora) is expressed in several mammalian tissues, showing highest level of expression in muscle, brain, and hypothalamus and higher levels in further tissues, e.g. white adipose tissue (WAT), liver, testis, heart, lung, and kidney. Furthermore Rora is expressed on lower but still robust levels in brown adipose tissue (BAT), pancreas, colon, small intestine, spleen, and bone marrow, as depicted in Figure 3A. We found, for example, that the expression of Rora is down-regulated in the WAT and bone marrow of fasted mice compared to wild type mice.
  • Rora is down- regulated in the bone marrow of genetically induced obese mice (ob/ob) compared to wild type mice, (see Figure 3B).
  • the expression of Rora is up-regulated in the brain, and down-regulated in the liver, colon, small intestine, spleen, and kidney compared to mice fed a control diet, as depicted in Figure 3C.
  • Figure 3D We show in this invention (see Figure 3D) that the Rora mRNA is expressed and up-regulated during the differentiation into mature adipocyctes. Therefore, the Rora protein plays a role in adipogenesis.
  • Rora in metabolic active tissues of wild type mice, as well as the regulation of Rora in different animal models used to study metabolic disorders, shows that this gene plays a central role in energy homeostasis. This result is supported by the regulation during the differentiation from preadipocytes to mature adipocytes. Taqman analysis revealed that the RAR-related orphan receptor beta (Rorb) is expressed predominantly in the hypothalamus and brain. Furthermore Rorb is expressed on lower but still robust levels in white adipose tissue, (WAT), brown adipose tissue (BAT), muscle, colon, heart, lung, and kidney, as depicted in Figure 3E.
  • WAT white adipose tissue
  • BAT brown adipose tissue
  • Rorb is up-regulated in the WAT of fasted mice and down-regulated in the WAT of genetically induced obese mice (ob/ob) compared to wild type mice. Furthermore the expression of Rorb is up-regulated in the spleen and bone marrow of genetically induced obese mice (ob/ob) compared to wild type mice, (see Figure 3F). In wild type mice fed a high fat diet, the expression of Rorb is up-regulated in the BAT and brain, and down-regulated in the WAT and lung compared to mice fed a control diet, as depicted in Figure 3G. We show in this invention (see Figure 3H) that the Rorb mRNA is slightly expressed and down-regulated during the differentiation into mature adipocyctes. Therefore, the Rorb protein plays a role in adipogenesis.
  • Rorb The strong expression of Rorb in brain regions known to be involved in appetite control and the expression in metabolic active tissues of wild type mice, as well as the regulation of Rorb in different animal models used to study metabolic disorders, shows that this gene plays a central role in energy homeostasis. This result is supported by the regulation during the differentiation from preadipocytes to mature adipocytes.
  • RAR-related orphan receptor gamma (Rorc) is expressed in several mammalian tissues, showing highest level of expression in BAT and muscle, and higher levels in further tissues, e.g. WAT, liver, colon, small intestine, heart, lung, and kidney. Furthermore Rorc is expressed on lower but still robust levels in pancreas, hypothalamus, brain, testis, spleen, and bone marrow, as depicted in Figure 31. We found, for example, that the expression of Rorc is up-regulated in the BAT, muscle, pancreas, and heart, and down-regulated in the bone marrow of fasted mice compared to wild type mice.
  • Rorc is up- regulated in the liver and colon and clearly down-regulated in the WAT and bone marrow of genetically induced obese mice (ob/ob) compared to wild type mice (see Figure 3J).
  • the expression of Rorc is up-regulated in the muscle, and down-regulated in the heart and clearly down-regulated in the WAT when compared to mice fed a control diet, as depicted in Figure 3K. Therefore Rorc is down-regulated in genetically as well as in diet induced obesity models.
  • Figure 3L shows in this invention (see Figure 3L) that the Rorc mRNA is expressed and up-regulated during the differentiation into mature adipocyctes. Therefore, the Rorc protein plays a role in adipogenesis.
  • Rorc in metabolic active tissues of wild type mice, as well as the regulation of Rorc in different animal models used to study metabolic disorders, shows that this gene plays a central role in energy homeostasis. This result is supported by the regulation during the differentiation from preadipocytes to mature adipocytes.
  • Amph amphiphysin
  • Bin1 the bridging integrator 1 (Bin1) is expressed predominantly in the hypothalamus and brain. Furthermore Bin1 is expressed on lower but still robust levels in WAT, BAT, muscle, colon, heart, lung, and spleen, as depicted in Figure 6C.
  • the expression of Bin1 is down-regulated in the hypothalamus of fasted mice compared to wild type mice.
  • the expression of Bin1 is up-regulated in the WAT of genetically induced obese mice (ob/ob) compared to wild type mice (see Figure 6D). In wild type mice fed a high fat diet, the expression of Bin1 is up- regulated in the WAT and BAT when compared to mice fed a control diet, as depicted in Figure 6E.
  • Bin1 mRNA is expressed and up-regulated during the differentiation into mature adipocyctes. Therefore, the Bin1 protein plays a role in adipogenesis.
  • uridine monophosphate kinase (Umpk) is expressed in several mammalian tissues, showing highest level of expression in BAT and higher levels in further tissues, e.g. WAT, hypothalamus, brain, testis, and kidney.
  • Umpk is expressed on lower but still robust levels in muscle, liver, colon, small intestine, heart, lung, and spleen of wild type mice as depicted in Figure 10A.
  • the expression of Umpk is up-regulated in the liver and down-regulated in the WAT of genetically induced obese mice (ob/ob) compared to wild type mice.
  • Umpk is down-regulated in the liver of fasted mice compared to wild type mice (see Figure 10B).
  • Umpk in metabolic active tissues (e.g. WAT and liver) of different animal models used to study metabolic disorders, together with the regulated expression during the differentiation from preadipocytes to mature adipocytes, shows that this gene plays a central role in energy homeostasis.
  • Uck2 uridine-cytidine kinase 2
  • Uck2 The regulated expression of Uck2 in metabolic active tissues (e.g. liver) of an animal model used to study metabolic disorders, together with the regulated expression during the differentiation from preadipocytes to mature adipocytes, shows that this gene plays a central role in energy homeostasis.
  • Npd Niemann Pick type C1
  • Npd The expression of Npd in metabolic active tissues of wild type mice, as well as the regulation of Npd in different animal models used to study metabolic disorders, shows that this gene plays a central role in energy homeostasis. This result is supported by the expression during the differentiation from preadipocytes to mature adipocytes.
  • the panel of the wild type mice tissues comprises WAT, BAT, muscle, liver, pancreas, hypothalamus, brain, testis, colon, small intestine, heart, lung, spleen, and kidney
  • the panel of the control diet-mice tissues comprises WAT, BAT, muscle, liver, brain, testis, colon, small intestine, heart, lung, spleen, and kidney.
  • Taqman analysis revealed that dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex) (Dlat) is expressed in several mammalian tissues, showing highest level of expression in brown adipose tissue (BAT), and higher levels in further tissues, e.g.
  • WAT white adipose tissue
  • muscle liver, hypothalamus, brain, testis, colon, small intestine, heart, lung, and kidney.
  • testis colon, small intestine, heart, lung, and kidney.
  • colon small intestine
  • heart heart
  • lung and kidney.
  • Dlat is expressed on lower but still robust levels in spleen, as depicted in Figure 17A.
  • Dlat is down-regulated in the WAT, BAT, liver, and small intestine of fasted mice compared to wild type mice (see Figure 17B). Furthermore the expression of Dlat is down-regulated in the BAT of genetically induced obese mice (ob/ob) compared to wild type mice, as depicted in Figure 17B. We show in this invention (see Figure 17C) that the o Dlat mRNA is expressed and up-regulated during the differentiation into mature adipocyctes.
  • Dlat in metabolic active tissues of wild type mice, as well as the regulation of Dlat in different animal models used to study metabolic 5 disorders, shows that this gene plays a central role in energy homeostasis. This result is supported by the expression and regulation during the differentiation from preadipocytes to mature adipocytes.
  • RNA preparation from human primary adipose tissues and adipocyte cell line was done as described in Example 5.
  • the target preparation, hybridization, and scanning was performed as described in the manufactures manual (see Affymetrix Technical Manual, 2002, obtained from Affymetrix, Santa Clara, o USA).
  • the X-axis represents the time axis, shown are day 0 and day 12 of adipocyte differentiation.
  • the Y-axis represents the flourescent intensity.
  • the expression analysis (using Affymetrix GeneChips) 5 of the amphiphysin (AMPH), uridine monophosphate kinase (UMPK), dihydrolipoamide S-acetyltransferase (DLAT), pyruvate dehydrogenase complex, component X (PDHX), hypothetical protein FLJ32371, reticulon 2 (RTN2), and reticulon 3 (RTN3) genes using human abdominal derived primary adipocyte cells and/or human adipocyte cell line (SGBS) differentiation, clearly shows differential expression of human AMPH, UMPK, DLAT, PDHX, FLJ32371 , RTN2, and RTN3 genes in adipocytes.
  • AMPH amphiphysin
  • UMPK uridine monophosphate kinase
  • the AMPH, DLAT, PDHX, and RTN3 proteins have to be significantly increased and the UMPK, FLJ32371 , and RTN2 proteins have to be significantly decreased in order for the preadipocyctes to differentiate into mature adipocycte. Therefore, the AMPH, DLAT, PDHX, and RTN3 proteins in preadipocyctes have the potential to enhance and UMPK, FLJ32371, and RTN2 in preadipocyctes have the potential to inhibit adipose differentiation.
  • the AMPH, UMPK, DLAT, PDHX, FLJ32371 , RTN2, and RTN3 proteins play an essential role in the regulation of human metabolism, in particular in the regulation of adipogenesis and thus theyt play an essential role in obesity, diabetes, and/or metabolic syndrome.

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Abstract

L'invention concerne de nouvelles utilisations de protéines de régulation de l'homéostasie de l'énergie et de polynucléotides codant lesdites protéines dans le diagnostic, l'étude, la prévention et le traitement de maladies et de troubles métaboliques.
PCT/EP2003/013377 2002-11-27 2003-11-27 Proteines impliquees dans la regulation de l'homeostasie de l'energie WO2004047855A2 (fr)

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