WO2004064856A2

WO2004064856A2 - Proteins involved in the regulation of energy homeostasis

Info

Publication number: WO2004064856A2
Application number: PCT/EP2004/000566
Authority: WO
Inventors: Karsten Eulenberg
Original assignee: Develogen AG
Current assignee: Develogen AG
Priority date: 2003-01-23
Filing date: 2004-01-23
Publication date: 2004-08-05
Anticipated expiration: 2005-07-23
Also published as: WO2004064856A3

Abstract

The present invention discloses novel uses for energy homeostasis regulating proteins and polynucleotides encoding these in the diagnosis, study, prevention, and treatment of metabolic diseases and disorders.

Description

Proteins involved in the regulation of energy homeostasis

Description

This invention relates to the use of Gadfly Accession Number CG7145, Gadfly Accession Number CG9547, Gadfly Accession Number CG5807, Gadfly Accession Number CG8315, or Gadfly Accession Number CG3625 homologous proteins, to the use of nucleic acid sequences encoding these, and to the use of modulators/effectors of the proteins or nucleotides in the diagnosis, study, prevention, and treatment of obesity and/or diabetes and/or metabolic syndrome.

There are several metabolic diseases of human and animal metabolism, e.g., obesity and severe weight loss that relate to energy imbalance where caloric intake versus energy expenditure is imbalanced. 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 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 (Koltermann 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). Triglycerides and glycogen are used as the body's fuel energy storage. Glycogen is a large branched polymer of glucose residues that is mainly stored in liver and muscle cells. Glycogen synthesis and degradation is central to the control of the blood glucose level.

Triglycerides are stored in the cytoplasm of adipocytes. Adipocytes are specialized for the synthesis, storage and mobilization of triglycerides. The glycogen and triglyceride metabolism is highly regulated and their interplay is essential for the energy homeostasis of the body. A high glucose level in the adipocytes results in the synthesis of triglycerides as fuel store. A low intracellular glucose level leads to a release of fatty acids, which can be used as substrates for the beta-oxidation to generate energy. Glycogen levels in cells are more variable than triglyceride levels because the turnover of glycogen is higher. Triglycerides are used as long term energy donors once the glycogen stores run low.

Insulin amongst other hormones plays a key role in the regulation of the fuel metabolism. High blood glucose levels stimulate the secretion of insulin by pancreatic beta-cells. 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 either the amount of insulin produced by the pancreatic islet cells is to low (diabetes type 1 or insulin dependent diabetes mellitus IDDM) or liver and muscle cells loose their ability to respond to normal blood insulin levels (insulin resistance). In the next stage pancreatic cells become unable to produce sufficient amounts of insulin (diabetes type II or non insulin dependent diabetes mellitus NIDDM).

Hyperlipidemia and elevation of free fatty acids correlate clearly with the metabolic syndrome, which is defined as the linkage between several diseases, including obesity an insulin resistance. These often occur in the same patients and are major risk factors for development of type 2 diabetes and cardiovascular disease. It was suggested that the control of lipid levels and glucose levels is required to treat type 2 diabetes, heart disease, and other occurrences of metabolic syndrome (see, for example, Santomauro AT. et al., (1999) Diabetes, 48: 1836-1841 and Lakka H.M. et al., (2002) JAMA 288: 2709-2716).

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 autoimmune 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). In diabetes type 2 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 beta-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.

Apart from the impaired quality of life for the patients, the treatment of diabetes and its long term complications presents an enormous financial burden to our healthcare systems with rising tendency. Thus, for the treatment of, type 1 and type 2 diabetes as well as for latent autoimmune diabetes in adults (LADA) there is a strong need in the art to identify factors that induce regeneration of pancreatic insulin producing beta-cells. These factors could restore normal function of the endocrine pancreas once its function is impaired or event could prevent the development or progression of diabetes type I, diabetes type II, or LADA.

The concept of metabolic syndrome (syndrome x, insulin-resistance syndrome, deadly quartet) was first described 1966 by Camus and reintroduced 1988 by Reaven (Camus JP, 1966, Rev Rhum Mai Osteoartic 33(1): 10-14; Reaven et al. 1988, Diabetes, 37(12): 1595-1607). Today "metabolic syndrome" is commonly defined as clustering of cardiovascular risk factors like hypertension, abdominal obesity, high blood levels of triglycerides and fasting glucose as well as low blood levels of HDL cholesterol. Insulin resistan- ce greatly increases the risk of developing the metabolic syndrome (Reaven, 2002, Circulation 106: 286-288). The metabolic syndrome often precedes the development of type 2 diabetes and cardiovascular disease (Lakka H.M. et al., 2002, supra).

The molecular factors regulating food intake and body weight balance are incompletely understood. Even if several candidate genes have been described which are supposed to influence the homeostatic system(s) that regulate body mass/weight, like leptin or the peroxisome proliferator-activated receptor-gamma co-activator, the distinct molecular mechanisms and/or molecules influencing obesity or body weight/body mass regulations are not known. In addition, several single-gene mutations resulting in obesity have been described in mice, implicating genetic factors in the etiology of obesity (Friedman J. M. and Leibel R. L, (1992) Cell 69(2): 217-220). In the ob mouse a single gene mutation (obese) results in profound obesity, which is accompanied by diabetes (Friedman J. M. et. al., (1991) Genomics 11: 1054-1062 ).

Therefore, 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.

Accordingly, 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.

So far, it has not been described that a protein of the invention or a homologous protein is involved in the regulation of energy homeostasis and body-weight regulation and related disorders, and thus, no functions in metabolic diseases and dysfunctions and other diseases as listed above have been discussed.

Before the present proteins, nucleotide sequences, and methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, vectors, and reagents described as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the cell lines, vectors, and methodologies that are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure. The present invention discloses that CG7145, CG9547, CG5807, CG8315, or CG3625 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, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

Aldehyde dehydrogenase 4 family, member A1 (ALDH4A1) is a NAD⁺-dependent aldehyde dehydrogenase localized in the mitochondrial matrix. Highest expression is found in the liver followed by skeletal muscle, kidney, heart, brain, placenta, lung and pancreas. The homodimeric enzyme catalyzes the irreversible conversion of delta-1-pyrroline-5-carboxylate (P5C), derived either from proline or omithine, to glutamate: 1 -PYRROLINE-5-GARBOXYLATE + NAD⁺ + H₂0 -> L-GLUTAMATE + NADH. This reaction is a necessary step in the pathway interconnecting the urea and tricarboxylic acid cycles. ALDH4A1 deficiency is associated with type II hyperprolinemia (HPII) characterized by the accumulation of delta-1-pyrroline-5-carboxylate (P5G) and proline (Goodman S.I. et al, (1974) Biochem Med 10: 329-336; Valle D. et al., (1979) J. Clin. Invest. 64: 1365-1370). The disorder may be causally related to neurologic manifestations, including seizures and mental retardation (Geraghty M.T. et al., (1998) Hum Mol Genet 7: 1411-1415). Glutaryl-coenzyme A dehydrogenase is a multifunctional enzyme responsible for the dehydrogenation and decarboxylation of glutaryl-CoA to crotonyl-CoA in the degradative pathway of L-lysine, L-hydroxylysine, and L-tryptophan metabolism. The enzyme exists in the mitochondrial matrix as a homotetramer of 45-kD subunits. The enzyme is a member of the acyl-CoA dehydrogenase family.

Glutaric acidemia type I (GA-I) is an autosomal recessive disorder of amino acid metabolism resulting from a deficiency of glutaryl-CoA dehydrogenase (GCDH). Patients accumulate glutaric acid (GA) and 3-OH glutaric acid (3-OHGA) in their blood, urine and CSF. Clinically, GA-I is characterized by macrocephaly, progressive dystonia and dyskinesia. Degeneration of the caudate and putamen of the basal ganglia, widening of the Sylvian fissures, fronto-temporal atrophy and severe spongiform change in the white matter are also commonly observed. Koeller et al. (2002) generated a mouse model of GA-I via targeted deletion of the GCDH gene in embryonic stem cells. The Gcdh -/- mice had a biochemical phenotype very similar to human GA-I patients, including comparable elevations of glutaric acid and 3-OH glutaric acid. The affected mice had a mild motor deficit but did not develop the progressive dystonia seen in human patients. Pathologically, the Gcdh -/- mice had a diffuse spongiform myelinopathy similar to that seen in GA-I patients. However, unlike in human patients, there was no evidence of neuron loss or astrogliosis in the striatum. Subjecting the Gcdh -/- mice to a metabolic stress failed to have any neurologic effect. The lack of similarity in regards to the neurologic phenotype and striatal pathology of GA-I patients, as compared with the Gcdh -/- mice, might be due to intrinsic differences between the striata of mice and men (Koeller D.M. et al., (2002) Hum Mol Genet 11: 347-357).

Lipocalins are a family of cytosolic fatty-acid binding proteins. About 20 proteins have been designated as lipocalins including serum retinol-binding protein, beta-lactoglobulin, alpha-2-globulin, and alpha-1-microglobulin. The amphiphilic lipocalins bind small lipids and facilitate their transport by minimizing solvent contact (Pervaiz S. et al., (1987) FASEB J 1: 209-214). A member of the lipocalin structural superfamily designated human lipocalin-1 is produced by a variety of glands and tissues. It stabilizes the lipid film of human tear fluid and it is suggested to act as a physiological scavenger of potentially harmful lipophilic compounds, in general. Lipocalin-interacting membrane receptor (LIMR) is a 55-kDa protein with nine putative transmembrane domains binding to Lipocalin-1 (Wojnar P. et al., (2001) J Biol Chem 276: 20206-20212).

Chromosome 7 open reading frame 2 (C7orf2 (LMBR1 )) is the human ortholog of the mouse Lrnbrl gene and encodes a putative receptor (Heus H.C. et al., (1999). Genomics 57: 342-351). Lrnbrl expression alters the developing limbs of Hemimelic extra toes (Hx) mice (Clark et al., (2000) Genomics 67: 19-27). The genomic location of the LMBR1 gene as well as the phenotype of Hx mice suggested LMBR1 as a good candidate for acheiropody a disorder characterized by bilateral congenital amputations of the upper and lower extremities and aplasia of the hands and feet that had been mapped by linkage analysis to chromosome 7q36 (Escamilla et al., (2000) Am. J. Hum. Genet. 66: 1995-2000). Disruption of the C7orf2/Lmbr1 genie region is associated with preaxial polydactyly in humans and mice (Horikoshi T. et al., (2003) J Bone Miner Metab 21: 1-4).

Mammalian cells typically contain hundreds of peroxisomes but can increase peroxisome abundance further in response to extracellular stimuli. The PEX11 peroxisomal membrane proteins promote peroxisome division in multiple eukaryotes. Overexpression of PEX11 alpha is sufficient to promote peroxisome division, and a class of chemicals known as peroxisome proliferating agents (PPAs) induce the expression of PEX11 alpha and promote peroxisome division. These observations led to the hypothesis that PPAs induce peroxisome abundance by enhancing PEX11 alpha expression. The phenotypes of PEX11alpha(-/-) mice indicate that this hypothesis remains valid for a novel class of PPAs that act independently of peroxisome proliferator-activated receptor alpha (PPARalpha) but is not valid for the classical PPAs that act as activators of PPARalpha. Furthermore, PEX11alpha(-/-) mice had normal peroxisome abundance and cells lacking both PEXHalpha and PEXHbeta, a second mammalian PEX11 gene, had no greater defect in peroxisome abundance than cells lacking only PEXHbeta (Li X. et al., (2002) Mol Cell Biol 22(23): 8226-8240).

The PEX11 peroxisomal membrane proteins have a direct role in peroxisomal fatty acid oxidation, and that they only affect peroxisome abundance indirectly. PEX11 proteins are unique in their ability to promote peroxisome division, and PEX11 overexpression promotes peroxisome division in the absence of peroxisomal metabolic activity. Mouse cells lacking PEXHbeta display reduced peroxisome abundance, even in the absence of peroxisomal metabolic substrates, and PEX11 beta(-/-) mice are partially deficient in two distinct peroxisomal metabolic pathways, ether lipid synthesis and very long chain fatty acid oxidation. PEX11 proteins might act directly in peroxisome division, and that their loss might have indirect effects on peroxisome metabolism (Li X. and Gould S.J., (2002) J Cell Biol 156(4): 643-651).

Zellweger syndrome is a lethal neurological disorder characterized by severe defects in peroxisomal protein import. The resulting defects in peroxisome metabolism and the accumulation of peroxisomal substrates are thought to cause the other Zellweger syndrome phenotypes, including neuronal migration defects, hypotonia, a developmental delay, and neonatal lethality. These phenotypes are also manifested in mouse models of Zellweger syndrome generated by disruption of the PEX5 or PEX2 gene. Mice lacking peroxisomal membrane protein PEX11 beta display several pathologic features shared by these mouse models of Zellweger syndrome, including neuronal migration defects, enhanced neuronal apoptosis, a developmental delay, hypotonia, and neonatal lethality. However, PEX11 beta deficiency differs significantly from Zellweger syndrome and Zellweger syndrome mice in that it is not characterized by a detectable defect in peroxisomal protein import and displays only mild defects in peroxisomal fatty acid beta-oxidation and peroxisomal ether lipid biosynthesis. These results demonstrate that the neurological pathologic features of Zellweger syndrome can occur without peroxisomal enzyme mislocalization and challenge current models of Zellweger syndrome pathogenesis (Li X. et al., (2002) Mol Cell Biol 22: 4358-4365).

The Saccharomyces cerevisiae peroxisomal membrane protein Pex11p plays a primary role in medium-chain fatty acid oxidation, a process that affects peroxisome number and size in Saccharomyces cerevisiae. (van Roermund C.W. et al., (2000) J Cell Biol 150: 489-498).

Cultured human dermal papilla cells are useful for studying the androgen-dependent growth of hair follicles. The human homolog of FAR-17a, a gene identified from the hamster flank organ as one of the androgen inducible genes, was cloned by degenerative PCR and human dermal papilla cDNA library screening. A novel cDNA clone, designated as AIG1 (androgen-inducible gene 1) was cloned, whose expression was found to be inducible by androgen. AIG1 cDNA consists of 1,398 nucleotides in length, which encodes a protein of 238 amino acids (27 kDa). The deduced protein sequence showed 35% overall homology with FAR-17a. RT-PCR of human dermal papilla cDNA revealed two mRNA transcripts, which differed by 156 nucleotides. This results in an in-frame deletion of 52 amino acids. A computer analysis of hydropathy indicated five hydrophobic domains are present in the large protein sequence, while four hydrophobic portions are in the smaller protein sequence. In a Northern blot analysis, the major 1.5 kb and minor 1.2 kb bands of AIG1 mRNA were detected. AIG1 mRNA was expressed at a relatively high level in the heart, ovary, testis, liver, and kidney. However, they were expressed at a low level in the spleen, prostate, brain, skeletal muscle, pancreas, small intestine, and colon. When dermal sheath cells were stimulated with DHT, the level of AIG1 mRNA expression was increased at 30 ng/ml. The level of expression was higher in males than females (Seo J. et al., (2001) Mol Cells 11(1): 35-40). FAR-17A expression is stimulated by androgens either dihydrotestosterone or dehydroepiandrosterone (DHEA) (Puy LA. et al., (1996) J Invest Dermatol 107: 44-50).

CG7145, CG9547, CG5807, CG8315, or CG3625 homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds. Particulariy preferred are nucleic acids encoding the human CG7145, CG9547, CG5807, CG8315, or CG3625 homologs 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 and glycogen, wherein said nucleic acid molecule comprises (a) the nucleotide sequence of Drosophila CG7145, CG9547, CG5807,

CG8315, or CG3625, human CG7145, CG9547, CG5807, CG8315, or

CG3625 homologous nucleic acids, particularly the nucleic aicds as described in Table 1 , and/or a sequence complementary thereto,

(b) a nucleotide sequence which hybridizes at 50°C in a solution containing 1 x SSC and 0.1 % SDS to a sequence of (a),

(c) a sequence corresponding to the sequences of (a) or (b) within the degeneration of the genetic code,

(d) a sequence which encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to the amino acid sequences of the GG7145, CG9547, GG5807, GG8315, or GG3625 protein, preferably human GG7145, GG9547, CG5807, GG8315, or CG3625 homologous protein, particularly a protein as described in Table 1,

(e) a sequence which differs from the nucleic acid molecule of (a) to (d) by mutation and wherein said mutation causes an alteration, deletion, duplication and/or premature stop in the encoded polypeptide or

(f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of 15-25 bases, preferably 25-35 bases, more preferably 35-50 bases and most preferably at least 50 bases.

The invention is based on the finding that CG7145, CG9547, CG5807, CG8315, or CG3625 homologous proteins and the polynucleotides encoding therefore, are involved in the regulation of triglyceride storage and therefore energy homeostasis. The invention describes the use of compositions comprising CG7145, CG9547, CG5807, CG8315, or CG3625 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, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones or liver fibrosis.

Accordingly, the present invention relates to genes with novel functions in body-weight regulation, energy homeostasis, metabolism, and obesity, fragments of said genes, polypeptides encoded by said genes or fragments thereof, and effectors 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.

The ability to manipulate and screen the genomes of model organisms such as the fly Drosophila melanogaster 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) Science 287: 2185-2195). Identification of novel gene functions in model organisms can directly contribute to the elucidation of correlative pathways in mammals (humans) and of methods of modulating them. A correlation between a pathology model (such as changes in triglyceride levels as indication for metabolic syndrome including obesity) and the modified expression of a fly gene can identify the association of the human ortholog with the particular human disease.

A forward genetic screen may be performed in flies displaying a mutant phenotype due to misexpression of a known gene (see, Johnston Nat Rev Genet 3: 176-188 (2002); Rorth P., (1996) Proc Natl Acad Sci U S A 93: 12418-12422). In this invention, we have used a genetic screen to identify mutations of CG7145, CG9547, CG5807, CG8315, or CG3625 homologous genes that cause changes in the body weight, which are reflected by a significant change of triglyceride levels. Additionally glycogen levels are analysed.

One resource for screening was a Drosophila melanogaster stock collection of EP-lines. The P-vector of this collection has Gal4-UAS-binding sites fused to a basal promoter that can transcribe adjacent genomic Drosophila sequences upon binding of Gal4 to UAS-sites (Brand & Perrimon (1993) Development 118: 401-415; Rorth P., supra). This enables the EP-line collection for overexpression of endogenous flanking gene sequences. In addition, without activation of the UAS-sites, integration of the EP-element into the gene is likely to cause a reduction of gene activity, and allows determining its function by evaluating the loss-of-function phenotype.

Obese people mainly show a significant increase in the content of triglycerides. Triglycerides are the most efficient storage for energy in cells. In order to isolate genes with a function in energy homeostasis, several thousand EP-lines were tested for their triglyceride/glycogen content after a prolonged feeding period (see Examples for more detail). Lines with significantly changed triglyceride/glycogen content were selected as positive candidates for further analysis. The change of triglyceride/glycogen content due to the loss of a gene function suggests gene activities in energy homeostasis in a dose dependent manner that control the amount of energy stored as triglycerides or glycogens.

In this invention, the content of triglycerides and glycogen of a pool of flies with the same genotype was analyzed after feeding for six days using a triglyceride and a glycogen assay. Male flies homozygous for the integration of vectors were analyzed in assays measuring the triglyceride/glycogen contents of these flies (illustrated in more detail in the Examples section). The results of the triglyceride/glycogen content analysis are shown in Figures 1, 5, 9, 13, and 17.

Genomic DNA sequences were isolated that are localized directly 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 site of the vectors, and the corresponding gene, described in more detail in the Examples section. The molecular organization of the gene is shown in Figures 2, 6, 10, 14, and 18.

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 (see Examples for more detail) confirm the particular relevance of the proteins 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. In this invention, we used 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) to study the expression of the proteins of the invention. 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). 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. When the expression of a single gene is examined, arrays are employed to detect the expression of a specific gene or its variants. When an expression profile is examined, 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; Schena M. et al., (1996) Proc. Natl. Acad. Sci. USA 93: 10614-10619; Baldeschwieler et al., (1995) PCT application W09525116; Shalon T.D. and Brown P.O., (1995) PGT application WO9535505; Heller R.A. et al., (1997) Proc. Natl. Acad. Sci. USA 94: 2150-2155; Heller M.J. and Tu E., (1997) U.S. Patent No. US5605662). Various types of microarrays are well known and thoroughly described in Schena M., ed. (1999); DNA Microarrays: A Practical Approach, Oxford University Press, London.

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.

As determined by microarray analysis, aldehyde dehydrogenase 4 family, member A1 (ALDH4A1), glutaryl-Coenzyme A dehydrogenase (GCDH), lipocalin-interacting membrane receptor (LIMR), chromosome 7 open reading frame 2 (C7orf2), peroxisomal biogenesis factor 11 A (PEX11 A), and androgen- induced 1 (AIG1) are strong candidates 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. In a particular embodiment, the invention encompasses a nucleic acid encoding Drosophila GG7145, GG9547, GG5807, GG8315, or CG3625 or human GG7145, GG9547, GG5807, GG8315, or GG3625 homologous proteins, preferably a human homologous protein as described in Table 1, referred to herein as the proteins of the invention. It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of nucleotide sequences encoding the proteins, some bearing minimal homology to the nucleotide sequences of any known and naturally occurring gene, 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.

Also encompassed by the invention are 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 described in Wahl & Berger (1987: Methods Enzymol. 152: 399-407) and Kimmel (1987; Methods Enzymol. 152: 507-511), and may be used at a defined stringency. Preferably, 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 65°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 65°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. Furthermore, 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.

Also included within the scope of the present invention are alleles of the genes encoding the proteins of the invention and homologous proteins. As used herein, 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.

The 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.

In order to express a biologically active protein, the 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. 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. Expression of a polynucleotide can be assured by (i) constitutive promoters such as the Gytomegalovirus (CMV) promoter/enhancer region, (ii) tissue specific promoters such as the insulin promoter (see, Soria B. et al., (2000), Diabetes 49: 157-162), SOX2 gene promoter (see Li M. et al., (1998) Gurr. Biol. 8: 971-974), Msi-1 promoter (see Sakakibara S. and Okano H., (1997) J. Neuroscience 17: 8300-8312), alpha-cardia myosin heavy chain promoter or human atrial natriuretic factor promoter (Klug M.G. et al., (1996) J. Clin. Invest 98: 216-224; Wu J. et al., (1989) J. Biol. Chem. 264: 6472-6479) or (iii) inducible promoters such as the tetracycline inducible system. Expression vectors can also contain a selection agent or marker gene that confers antibiotic resistance such as the neomycin, hygromycin or puromycin resistance genes. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and Ausubel, F.M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

In a further embodiment of the invention, 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.

The presence of polynucleotide sequences of the invention in a sample can be detected by DNA-DNA 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. As used herein '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.

A wide variety of labels and conjugation techniques are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. 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).

The presence of proteins of the invention in a sample can be determined by immunological methods or activity measurement. A variety of protocols for detecting and measuring the expression of proteins, using either polyclonal or monoclonal antibodies specific for the protein or reagents for determining protein activity are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on the protein is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton, R. et al. (1990; Serological Methods, a Laboratory Manual, APS Press, St Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med. 158: 1211-1216).

Suitable reporter molecules or labels, which may be used, 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 or/and the vector used. As will be understood by those of skill in the art, 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.) The inclusion of 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.

Diagnostics and Therapeutics

The data disclosed in this invention show that the nucleic acids and proteins of the invention and modulator/effector molecules thereof are useful in diagnostic and therapeutic applications implicated, for example, but not limited to, metabolic syndrome, obesity or/and diabetes mellitus as well as related disorders, including eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis. Hence, diagnostic and therapeutic uses for the proteins and nucleic acids of the invention are, for example but not limited to, the following: (i) protein therapy, (ii) small molecule drug target, (Hi) 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 (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues).

The nucleic acids and proteins of the invention and effectors thereof are useful in diagnostic and therapeutic applications implicated in various applications as described below. For example, but not limited to, 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. By way of non-limiting example, 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.

The 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 novel substances of the invention may be used in therapeutic or diagnostic methods.

For example, in one aspect, antibodies, which are spe ific for the proteins of the invention and homologous proteins, may be used d rectly as an effector, e.g. an antagonist or indirectly as a targeting or del very 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.

For the production of antibodies, 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. Depending on the host species, 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. et al. (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. et al., (1984) Mol. Cell Biochem. 62: 109-120).

In addition, techniques developed for the production of 'chimeric antibodies', the splicing of mouse antibody genes to human antibody genes to obtain a molecule with appropriate antigen specificity and biological activity can be used (Morrison S.L. et al. (1984) Proc. Natl. Acad. Sci. 81: 6851-6855; Neuberger M.S. et al (1984) Nature 312: 604-608; Takeda S. et al. (1985) Nature 314: 452-454). Alternatively, techniques described for the production of single chain antibodies may be adapted, using methods known in the art, to produce single chain antibodies specific for the proteins of the invention and homologous proteins. 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. USA 86: 3833-3837; Winter G. and Milstein G., (1991) Nature 349: 293-299).

Antibody fragments, which contain specific binding sites for the proteins may also be generated. For example, such 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. Alternatively, 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 254: 1275-1281). Various 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, supra).

In another embodiment of the invention, the polynucleotides or fragments thereof or nucleic acid effector molecules such as antisense molecules, aptamers, RNAi molecules or ribozymes may be used for therapeutic purposes. In one aspect, 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.

In a further aspect, antisense molecules may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding the proteins of the invention and homologous proteins. Thus, antisense molecules may be used to modulate protein activity or to achieve regulation of gene function. Such technology is now well known in the art, and 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.

As mentioned above, modifications of gene expression can be obtained by designing antisense molecules, e.g. DNA, RNA or nucleic acid analogues such as PNA, to the control regions of the genes encoding the proteins of the invention and homologous proteins, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between positions -10 and +10 from the start site, are preferred. Similarly, 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. Recent therapeutic advances using triplex DNA have been described in the literature (Gee J.E. et al. (1994) Gene 149: 109-114; Huber B.E. and Carr B. I., Molecular and Immunologic Approaches, Futura Publishing Co., Mt Kisco, N.Y.). 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. Once identified, short 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. Alternatively, 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. Alternatively, 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. Possible modifications include, but are not limited to, the addition of 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. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of non-traditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, 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. The 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.

In addition to the active ingredients, 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(s), 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 μg, 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.

In another embodiment, antibodies wh ch specifically bind to the proteins may be used for the diagnosis of conditi ons or diseases characterized by or associated with over- or underexpressi on 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. A wide variety of 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.

In another embodiment of the invention, 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.

In one aspect, 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 ³²P or ³⁵S 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.

In a particular aspect, the 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 or dysfunctions, including metabolic syndrome, obesity, or diabetes mellitus as well as related disorders such as eating disorder, cachexia, pancreatitis, 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.

In order to provide a basis for the diagnosis of a disease associated with expression of the proteins of the invention and homologous proteins, 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.

With respect to metabolic diseases such as described above 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.

Additional diagnostic uses for 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 (5prime.fwdarw.3prime) and another with antisense (3prime.rarw.5prime), 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.

In another embodiment of the invention, 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 CM. (1993) Blood Rev. 7: 127-134, and Trask B.J. (1991) Trends Genet. 7: 149-154. FISH (as described in Verma R.S. and Babu A., (1989) Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, N.Y.). The results may be correlated with other physical chromosome mapping techniques and genetic map data. Examples of genetic map data can be found in the 1994 Genome Issue of Science (265: 1981f). Correlation between the location of the gene encoding the proteins of the invention on a physical chromosomal map and a specific disease or predisposition to a specific disease, may help to delimit the region of DNA associated with that genetic disease.

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. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomic region, for example, AT to 11q22-23 (Gatti R.A. et al., (1988) Nature 336: 577-580), 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.

In another embodiment of the invention, the proteins of the invention, their catalytic or immunogenic fragments or oligopeptides thereof, an in vitro model, a genetically altered cell or animal, can be used for screening libraries of compounds in any of a variety of drug screening techniques. One can identify effectors, e.g. receptors, enzymes, proteins, ligands, or substrates that bind to, modulate or mimic the action of one or more of the proteins of the invention. The protein or functional fragment thereof employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellulary. The formation of binding complexes, between the proteins of the invention and the agent tested, may be measured. Agents could also, either directly or indirectly, influence the activity of the proteins of the invention.

In addition activity of the proteins of the invention against their physiological substrate(s) or derivatives thereof could be measured in cell-based assays. Agents may also interfere with posttranslational modifications of the protein, such as phosphorylation and dephosphorylation, famesylation, palmitoylation, acetylation, alkylation, ubiquiti nation, proteolytic processing, subcellular localization and degradation. Moreover, agents could influence the dimerization or oligomerization of the proteins of the invention or, in a heterologous manner, of the proteins of the invention with other proteins, for example, but not exclusively, docking proteins, enzymes, receptors, or translation factors. Agents could also act on the physical interaction of the proteins of this invention with other proteins, which are required for protein function, for example, but not exclusively, their downstream signaling.

Methods for determining protein-protein interaction are well known in the art. For example binding of a fluorescently labeled peptide derived from the interacting protein to the protein of the invention, or vice versa, could be detected by a change in polarisation. In case that both 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. In addition, a variety of commercially available assay principles suitable for detection of protein-protein interaction are well known in the art, for example but not exclusively AlphaScreen (PerkinElmer) or Scintillation Proximity Assays (SPA) by Amersham. Alternatively, 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. In all cases 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 interaction are ALDH4A1 , GCDH, LIMR, C7orf2, PeC11A, PEX11B, PEX11G, AIG-1 and/or MGC 12335.

Assays for determining enzymatic activity of the proteins of the invention are well known in the art. Well known in the art are also a variety of assay formats to measure receptor-ligand binding or receptor downstream signalling.

Genetic reporter systems are widely used to study eukaryotic gene expression and cellular physiology. Applications include the study of receptor activity, transcription factors, intracellular signalling, mRNA processing, and protein folding. For example, the firefly luciferase is used as a reporter because the luciferase assay is very sensitive and rapid. 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).

Of particular interest are screening assays for agents that have a low toxicity for mammalian cells. The term "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. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Where 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, which may be used, provides for high throughput screening of compounds having suitable binding affinity to the protein of interest as described in published PCT application WO84/03564. In this method, as applied to the proteins of the invention large numbers of different small test compounds, e.g. aptamers, peptides, low-molecular weight compounds etc., are provided or synthesized on a solid substrate, such as plastic pins or some other surface. 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. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support. In another embodiment, one may use competitive drug screening assays in which neutralizing antibodies capable of binding a protein of the invention specifically compete with a test compound for binding the ALDH4A1, GCDH, LIMR, C7orf2, PEX11A, PEX11B, PEX11G, AIG-1 and/or MGC12335 proteins. In this manner, the antibodies can be used to detect the presence of any peptide, which shares one or more antigenic determinants with the ALDH4A1, GCDH, LIMR, C7orf2, PEX11A, PEX11B, PEX11G, AIG-1 and/or MGC12335 proteins.

The nucleic acids encoding the protein 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 protein 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 protein of the invention. Misexpression (for example, overexpression or lack of expression) of the protein of the invention, particular feeding conditions, and/or administration of biologically active compounds can create models of metablic disorders.

In one embodiment of the invention, 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). 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, supra). Susceptible wild type mice (for example C57BI/6) show similiar symptoms if fed a high fat diet. In addition to testing the expression of the proteins of the invention in such mouse strains (see Examples section), these 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.

Transgenic animals may be made through homologous recombination in non-human embryonic stem cells, where the normal locus of the gene encoding the protein of the invention is altered. Alternatively, a nucleic acid construct encoding the 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 animals are useful in the study of the function and regulation of the protein of the invention. For example, a series of small deletions and/or substitutions may be made in the gene that encodes the protein of the invention to determine the role of particular domains of the protein, functions in pancreatic differentiation, etc.

Furthermore, variants of the gene of the invention like specific constructs of interest include anti-sense molecules, which will block the expression of the protein 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 the gene of the invention, where up regulation of expression of the gene of the invention will result in an easily detected change in phenotype.

One may also provide for expression of the gene of the invention or variants thereof in cells or tissues where it is not normally expressed or at abnormal times of development. In addition, by providing expression of the protein 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 gene 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 protein 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).

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 trypsini∑ed, and the modified cells are placed into the depression closely to the morulae. On the following day 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 protein of the invention in vivo.

Finally, the invention also relates to a kit comprising at least one of (a) a nucleic acid molecule coding for a protein of the invention or a fragment thereof;

(b) a protein of the invention or a fragment or an isoform thereof;

(c) a vector comprising the nucleic acid of (a);

(d) a host cell comprising the nucleic acid of (a) or the vector of (c); (e) a polypeptide encoded by the nucleic acid of (a);

(f) a fusion polypeptide encoded by the nucleic acid of (a);

(g) an antibody, an aptamer or another modulator/effector of the nucleic acid of (a) or the polypeptide of (b), (e) or (f) and

(h) an anti-sense oligonucleotide of the nucleic acid of (a).

The kit may be used for diagnostic or therapeutic purposes or for screening applications as described above. The kit may further contain user instructions.

The Figures show:

Figyr© 1 shows the content of energy storage triglyceride (TG) of a Drosophila GG7145 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(3)35340 flies caused by integration of the P-vector into the annotated transcription unit (ΗD-EP35340 (TG, 90°C)', column 3) in comparison to controls containing about 2000 fly lines of the proprietary EP collection ('HD-control (TG, 90°G)\ column 1) and wild type controls determined in more than 80 independent assays ('WT-control (TG, 90°C)', column 2). Figure 2 shows the molecular organization of the mutated CG7145 gene locus.

Figure 3 shows the expression of the CG7145 homolog in mammalian

(mouse) tissues.

Figure 3A shows the real-time PCR analysis of aldehyde dehydrogenase 4A1 precursor (mitochondrial delta-1-pyrroline 5-carboxylate dehydrogenase;

1 P5cdh) expression in wild-type mouse tissues. Figure 3B shows the real-time PCR analysis of 1 P5cdh expression in different mouse models.

Figure 30 shows the real-time PCR analysis of 1 P5cdh 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 1P5cdh expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.

Figure 4 shows the expression of the human CG7145 homolog in human tissue. Shown is the microarray analysis of aldehyde dehydrogenase 4 family, member A1 (ALDH4A1) expression in a human adipocyte cell line (SGBS) during the differentiation from preadipocytes to mature adipocytes.

Figure 5 shows the content of energy storage triglyceride (TG) of a Drosophila CG9547 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(2)26478 flies caused by integration of the P-vector into the annotated transcription unit ('HD-EP26478 (TG)', column 3) in comparison to controls containing about 2000 fly lines of the proprietary EP collection ('HD-control (TG)', column 1) and wild type controls determined in more than 80 independent assays (referred to as 'WT-control (TG)', column 2).

Figure 6 shows the molecular organization of the mutated CG9547 gene locus. Figure 7 shows the expression of the CG9547 homolog in mammalian

(mouse) tissues.

Figure 7A shows the real-time PCR analysis of glutaryl-Coenzyme A dehydrogenase (Gcdh) expression in wild-type and control-diet mouse tissues.

Figure 7B shows the real-time PCR analysis of Gcdh expression in different mouse models compared to wild type mice and in mice fed with a high fat diet compared to mice fed with a control diet.

Figure 7C shows the real-time PCR analysis of Gcdh expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.

Figure 8 shows the expression of the human CG9547 homolog in human tissue. Shown is the microarray analysis of glutaryl-Coenzyme A dehydrogenase (GCDH) expression in human abdominal derived primary adipocyte cells (PRIM) and in a human adipocyte cell line (SGBS) during the differentiation from preadipocytes to mature adipocytes.

Figure 9 shows the content of energy storage triglyceride (TG) of Drosophila CG5807 (GadFly Accession Number) mutants. Shown is the change of triglyceride content of HD-EP(3)32040 flies caused by integration of the P-vector into the annotated transcription unit (ΗD-32040 (TG, 90°C)', column 3) in comparison to controls containing about 2000 fly lines of the proprietary EP collection (ΗD-control (TG, 90DC)', column 1) and wild type controls determined in more than 80 independent assays (referred to as 'WT-control (TG, 90°C)\ column 2).

Figur© 10 shows the molecular organization of the mutated CG5807 gene locus.

Figure 11 shows the expression of a CG5807 homolog in mammalian (mouse) tissues.

Figure 11 A shows the real-time PCR analysis of lipocalin-interacting membrane receptor (Limr) expression in wild-type and control-diet mouse tissues.

Figure 11 B shows the real-time PCR analysis of Limr expression in different mouse models compared to wild type mice and in mice fed with a high fat diet compared to mice fed with a control diet. Figure 11C shows the real-time PCR analysis of Limr expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.

Figure 12 shows the expression of the human CG5807 homologs in human tissue. Figure 12A shows the microarray analysis of lipocalin-interacting membrane receptor (LIMR) expression in human abdominal derived primary adipocyte cells (PRIM) and in a human adipocyte cell line (SGBS) during the differentiation from preadipocytes to mature adipocytes.

Figure 12B shows the microarray analysis of chromosome 7 open reading frame 2 (C7orf2) expression in human abdominal derived (AB) and mammary gland derived (MA) primary adipocyte cells during the differentiation from preadipocytes to mature adipocytes.

Figure 13 shows the content of energy storage metabolites (ESM; triglyceride (TG) and glycogen) of a Drosophila CG8315 (GadFly Accession Number) mutant. Shown is the change of triglyceride content of HD-EP(2)21554 flies caused by integration of the P-vector into the annotated transcription unit

(ΗD-EP21554 (TG)', column 3) in comparison to controls containing about

2000 fly lines of the proprietary EP collection ('HD-controI (TG)'), column 1) and wild type 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)21554 flies caused by integration of the P-vector the into the annotated transcription unit ('HD-EP21554 (glycogen)', column 6) in comparison to an internal assay control including two wildtype strains and one HD-line ('internal control (glycogen)' column 5) and a wildtype control containing one wild type strain ('WT-control (glycogen)', column 4). Figure 14 shows the molecular organization of the mutated CG8315 gene locus.

Figure 15 shows the expression of the CG8315 homologs in mammalian (mouse) tissues.

Figure 15A shows the real-time PCR analysis of peroxisomal biogenesis factor 11a (Pex11a) expression in wild-type and control-diet mouse tissues. Figure 15B shows the real-time PCR analysis of Pex11a expression in different mouse models compared to wild type mice and in mice fed with a high fat diet compared to mice fed with a control diet.

Figure 15G shows the real-time PCR analysis of Pex11a expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes. Figure 15D shows the real-time PCR analysis of peroxisomal biogenesis factor 11b (Pex11b) expression in wild-type and control-diet mouse tissues. Figure 15E shows the real-time PCR analysis of Pex11b expression in different mouse models compared to wild type mice and in mice fed with a high fat diet compared to mice fed with a control diet.

Figure 15F shows the real-time PCR analysis of Pex11 b expression during the differentiation of 3T3-L1 cells from preadipocytes to mature adipocytes.

Figure 16 shows the expression of a human CG8315 homolog in human tissue. Shown is the microarray analysis of peroxisomal biogenesis factor 11A (PEX11A) expression in human abdominal derived primary adipocyte cells (PRIM) and in a human adipocyte cell line (SGBS) during the differentiation from preadipocytes to mature adipocytes.

Figure 17 shows the content of energy storage triglyceride (TG) of Drosophila GG3625 (GadFly Accession Number) mutants. Shown is the change of triglyceride content of HD-EP(2)21897 flies caused by integration of the P-vector into the annotated transcription unit (ΗD-21897 (TG; 90°C)', column 6) in comparison to controls containing about 2000 fly lines of the proprietary EP collection (ΗD-control (TG; 90°C)', column 4) and wild type controls determined in more than 80 independent assays (referred to as 'WT-control (TG; 90°C)\ column 5). Also shown is the change of triglyceride content of HD-EP(2)21897 flies at different assay conditions (70°C instead of 90°C) (ΗD-21897 (TG; 70°C)', column 3) in comparison to controls containing about 880 fly lines of the proprietary EP collection ('HD-control (TG; 70°C)', column 1) and wildtype controls determined in 4 independent assays (referred to as 'WT-control (TG; 70°C)', column 2).

Figure 18 shows the molecular organization of the mutated CG3625 gene locus.

Figure 1® shows the expression of a human CG3625 homolog in human tissue. Shown is the microarray analysis of androgen-induced 1 (AIG1) expression in human abdominal derived (AB) and mammary gland derived (MA) primary adipocyte cells during the differentiation from preadipocytes to mature adipocytes

The examples illustrate the invention:

Example 1: Measurement of energy storage metabolites (ESM; triglyceride and/or 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. The average change of triglyceride and/or glycogen (herein referred to as energy storage metabolites, ESM) content of Drosophila containing the EP-vectors in homozygous viable integration was investigated in comparison to control flies grown under the same conditions (see Figures 1, 5, 9, 13, and 17). For determination of triglyceride and glycogen content, flies were incubated for 5 min at 70°C or 90° C in an aqueous buffer using a waterbath, followed by hot extraction. After another 5 min incubation at 70°C or 90°C and mild centrifugation, 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. The glycogen content of the flies extract was determined using the Roche Starch UV-method assay (Cat. No. 0207748) by measuring changes in the optical density according to the manufacturer's protocol. As a reference the protein content of the same extract was measured using BIO-RAD DC Protein Assay according to the manufacturer's protocol. These experiments and assays were repeated several times.

The average triglyceride level (μg triglyceride/μg protein) of 2108 flies of the proprietary EP collection determined at 90°C (referred to as 'HD-control (TG; 90°C)' or 'HD-control (TG)') is shown as 100% in the first columns in Figures 1, 5, 9, and 13, and the fourth column in Figure 17. The average triglyceride level (μg triglyceride/μg protein) of 883 flies of the proprietary EP collection determined at 70°C (referred to as ΗD-control (TG; 70°GY) is shown as 100% in the first column in Figure 17. The average triglyceride level (μg triglyceride/μg protein) of Drosophila wildtype strain Oregon R flies determined in 84 independent assays at 90°C (referred to as 'WT-control (TG; 90°C)' or 'WT-control (TG)') is shown as 102% in the second columns in Figures Figures 1, 5, 9, and 13, and the fifth column in Figure 17. The average triglyceride level (μg triglyceride/μg protein) of Drosophila wildtype strain Oregon R flies determined in 4 independent assays at 70°C (referred to as "WT-control (TG; 70°C)') is shown as 116% in the second column in Figure 17. The average glycogen level (μg glycogen/μg protein) of a wild type control consisting of flies of the wild type strain Oregon R (referred to as 'WT-control (glycogen)') is shown as 100% in the fourth column in Figure 13. The average glycogen level (μg glycogen/μg protein) of an internal assay control consisting of two different wild type strains and an inconspicuous EP-line of the HD-stock collection (referred to as 'internal control (glycogen)') is shown as 100% in the fifth column in Figure 13. Standard deviations of the measurements are shown as thin bars.

HD-EP(3)35340 homozygous flies (column 3 in Figure 1, ΗD-EP35340 (TG, 90°C)') show constantly a lower triglyceride content than the controls. HD-EP (2)26478 homozygous flies (column 3 in Figure 5, ΗD-EP26478 (TG)'), HD-EP (3)32040 homozygous flies (column 3 in Figure 9, ΗD-EP32040 (TG, 90°C)'), HD-EP(2)21554 homozygous flies (column 3 in Figure 13, 'HD-EP21554 (TG)'), and HD-EP(2)1897 homozygous flies (column 3 in Figure 17, ΗD-21897 (TG; 70°C)' and column 6 in Figure 17, 'HD-21897 (TG; 90°C)') show constantly a higher triglyceride content than the controls. Furthermore, HD-EP(2)21554 homozygous flies (column 5 in Figure 13, 'HD-EP21554 (glycogen)') also show a lower glycogen content than the controls.

Therefore, the loss of gene activity is responsible for changes in the metabolism of the energy storage metabolites.

sam le 2: Identification of Drosophila genes associated with energy homeostasis

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 vectors (herein HD-EP(3)35340, HD-EP (2)26478, HD-EP(3)32040, HD-EP(2)21554, or HD-EP(2)21897) integration. Using those isolated genomic sequences 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 2, 6, 10, 14, and 18.

In Figures 2, 10, and 18, 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(3)35340, HD-EP(3)32040, or HD-EP(2)21897. Ticks represent the length in basepairs of the genomic DNA (1000 base pairs per tick). The part of the figure above the double-headed arrow represents the sense strand, the part below the arrow represents the antisense strand. The grey arrows in the upper part of the figures represent BAG 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 (as predicted by the Berkeley Drosophila Genome Project, GadFly and by Magpie) 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(3)35340 vector is homozygous viable integrated 302 base pairs 5' of the shorter transcript variant of CG7145 and into the first intron of the longer transcript variant of CG7145 in antisense orientation. The chromosomal localization site of HD-EP(3)35340 vector integration is at gene locus 3L, 78F4 (Flybase). In Figure 2, the two transcript variants of CG7145 (as predicted by the Berkeley Drosophila Genome Project) are shown as dark gray bars (exons) linked by dark gray lines (introns) in the lower half of the figure and are labeled. The integration site of HD-EP35340 is indicated with a black triangle 5' of the first exon of the predicted transcript variants of the CG7145 gene.

The HD-EP(3)32040 vector is homozygous viable integrated into base pair 35 of cDNA CG5807 in sense orientation (Gadfly release 3). The chromosomal localization site of vector integration is at gene locus 3R, 96A23 (Flybase). In Figure 10, the transcript of CG5807 (as predicted by the Berkeley Drosophila Genome Project) is shown in the lower half of the figure, and is labeled as CG11375BcDNA:GH12663. The integration site of HD-EP(3)32040 is indicated with a black triangle labeled 'HD-EP32040'.

The HD-EP(2)21897 vector is homozygous viable integrated into the first intron of cDNA CG3625 transcript variant CG3625-RA and 6 base pairs 5' of transcript variant CG3625-RB in antisense orientation (Gadfly release 3). The chromosomal localization site of vector integration is at gene locus 2L, 21 B7 (Flybase and Gadfly). In Figure 18, the transcript of CG3625 (as predicted by the Berkeley Drosophila Genome Project) is shown in the lower half of the figure, and is labeled as 'CG3625'. The integration site of the HD-EP(2)21897 vector is indicated as black triangle labeled as 'HD-EP21897'. ln Figures 6 and 14, 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)26478 or HD-EP(2)21554. 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 in the "P-elements +" or "P-elements -" lines. Transcribed DNA sequences (ESTs) are shown as grey bars in the "EST +", "EST -", "IPI +", and/or the "IPI -" lines, and predicted cDNAs are shown as bars in the "cDNA +" and/ or "cDNA -" lines. Predicted exons of the cDNAs are shown as dark grey bars and predicted introns are shown as light grey bars.

The HD-EP(2)26478 vector is homozygous viable integrated into base pair 30 of the CG9547 cDNA transcript CG9547-RA in sense orientation. The chromosomal localization site of integration of the vector of HD-EP(2)26478 is at gene locus 2L, 26D9 (according to Flybase and Gadfly). In Figure 6, numbers represent the coordinates of the genomic DNA (starting at position 6485000 on chromosome 2L, ending at position 6487200). The insertion site of the P-element in Drosophila line HD-EP(2)26478 is labeled. The gene CG9547 (Gadfly Accession Number) shown in the "cDNA -" line is labeled. The corresponding ESTs are shown in the "EST -" line.

The HD-EP(2)21554 vector is homozygous viable integrated into the 3' end of the GG8315 gene in sense orientation. The chromosomal localization site of integration of the vector of HD-EP(2)21554 is at gene locus 2R, 52D9 (according to Flybase and Gadfly). In Figure 14, numbers represent the coordinates of the genomic DNA (starting at position 11054900 on chromosome 2R, ending at position 11056200). The insertion site of the P-element in Drosophila line HD-EP(2)21554 is labeled. The gene CG8315 (Gadfly Accession Number) shown in the "cDNA-" line is labeled. The corresponding ESTs are shown "EST -" line. Therefore, expression of the cDNAs encoding the proteins of the present invention could be affected by integration of the vectors, leading to a change in the amount of energy storage triglycerides and/or glycogen.

Example 3: Identification of human homologous genes and proteins

The Drosophila genes and proteins encoded thereby with functions in the regulation of triglyceride and/or glycogen metabolism were further analysed using the BLAST algorithm searching in publicly available sequence databases and mammalian homologs were identified (see Table 1).

The term "polynucleotide comprising the nucleotide sequence as shown in GenBank Accession number" relates to the expressible gene of the nucleotide sequences deposited under the corresponding GenBank Accession number. The term "GenBank Accession number" relates to NCBi GenBank database entries (Ref.: Benson et al., (2000) Nucleic Acids Res. 28: 15-18). Sequences homologous to Drosphila CG7145, CG9547, CG5807, CG8315, or CG3625 were identified using the publicly available program BLASTP 2.2.3 of the non-redundant protein data base of the National Center for Biotechnology Information (NCBI) (see, Altschul S.F. et al., (1997) Nucleic Acids Res. 25: 3389-3402).

Table 1: Human homologs of the Drosophila (Dm) genes

CG7145, CG9547, CG5807, CG8315, or CG3625 homologous proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds. Particularly preferred are nucleic acids as described in Table 1.

The mouse homologous cDNAs encoding the polypeptides of the invention were identified as GenBank Accession Number XM_204153 (for the mouse homolog of ALDH4A1), GenBank Accession Number NM_029098 (for the mouse homolog of LIMR), GenBank Accession Numbers NMJ320295 and XM_110554 (for the mouse homologs of C7orf2), GenBank Accession Number NM_011068 (for the mouse homolog of PEX11A), GenBank Accession Number NM_011069 (for the mouse homolog of PEX11 B), GenBank Accession Number NM_026951 (for the mouse homolog of PEX11G), GenBank Accession Number NM_008097 (for the mouse homolog of GCDH), GenBank Accession Number NM_025446 (Mus musculus 1500031019Rik, for the mouse homolog of AIG-1), and GenBank Accession Number AK029025 (for the mouse homolog of MGC12335).

Example 4: Expression of the polypeptides in mammalian (mouse) tissues

To analyse the expression of the polypeptides disclosed in this invention in mammalian tissues, several mouse strains (preferably mice strains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db which are standard model systems in obesity and diabetes research) were purchased from Harlan Winkelmann (33178 Borchen, Germany) and maintained under constant temperature (preferably 22°C), 40 per cent humidity and a light / dark cycle of preferably 14 / 10 hours. The mice were fed a standard chow (for example, from ssniff Spezialitaten GmbH, order number ssniff M-Z V1126- 000). 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 USA 93: 3434-3438). In a further experiment 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°G until needed.

For analyzing the role of the proteins disclosed in this invention in the in vitro differentiation of mammalian cell culture cells for the conversion of preadipocytes to adipocytes, 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. et al., (1998) BBRC 251: 225-229). In brief, cells were plated in DMEM/10% FCS (Invitrogen, Karlsruhe, Germany) at 50,000 cells/well in duplicates in 6-well plastic dishes and cultured in a humidified atmosphere of 5% C0₂ at 37°C. At confluence (defined as day 0: dO) cells were transferred to serum-free (SF) medium, containing DMEM/HamF12 (3:1; Invitrogen), fetuin (300 μg/ml; Sigma, Munich, Germany), transferrin (2 μg/ml; Sigma), pantothenate (17μM; Sigma), Biotin (1μM; Sigma), and EGF (0.8nM; Hoffmann-La Roche, Basel, Switzerland). Differentiation was induced by adding dexamethasone (DEX; 1μM; Sigma), 3-methyl-isobutyl-1-methylxanthine (MIX; 0.5mM; Sigma), and bovine insulin (5μg/ml; Invitrogen). Four days after confluence (d4), cells were kept in SF medium, containing bovine insulin (5μg/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 12 days of differentiation, suitable aliquots of cells were taken every two days.

RNA was isolated from mouse tissues or cell culture cells using Trizol Reagent (for example, from Invitrogen, Karlsruhe, Germany) and further purified with the RNeasy Kit (for example, from Qiagen, Germany) in combination with a DNase-treatment according to the instructions of the manufacturers and as known to those skilled in the art. Total RNA was reverse transcribed (preferably using Superscript II RNaseH^" Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany) and subjected to Taqrnan analysis preferably using the Taqman 2xPCR Master Mix (from Applied Biosystems, Weiterstadt, Germany; the Mix contains according to the Manufacturer for example AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP, passive reference Rox and optimized buffer components) on a GeneAmp 5700 Sequence Detection System (from Applied Biosystems, Weiterstadt, Germany). Taqman analysis was performed preferably using the following primer/probe pairs:

For the amplification of mouse protein similar to aldehyde dehydrogenase 4A1 precursor (mitochondrial delta-1-pyrroline 5-carboxylate dehydrogenase; 1P5cdh) sequence (GenBank Accession Number XM_204153):

Mouse 1P5cdh forward primer (SEQ ID NO: 1): 5'- GCA TTA AGA AGT GGT TGG AGC AT -3'; mouse 1P5cdh reverse primer (SEQ ID NO: 2): 5'- CTT GCT CTC AAT GAT ACA TGG TTC C -3'; mouse 1P5cdh Taqman probe (SEQ ID NO: 3): (5/6-FAM)- CAC GTT CCT CGC CCA GCC TCA -(5/6- TAMRA).

For the amplification of mouse glutaryl-Coenzyme A dehydrogenase (Gcdh) sequence (GenBank Accession Number NM_008097):

Mouse Gcdh forward primer (SEQ ID NO:4): 5'- CCC AGG TGG CAT GGA GAC -3'; mouse Gcdh reverse primer (SEQ ID NO: 5): 5'- TTG GTC CCA CTG AGG GTG TAG -3'; mouse Gcdh Taqman probe (SEQ ID NO: 6): (5/6- FAM)- AGC TCG CCA CAA TCC ATC AAA CCA GA -(5/6-TAMRA).

For the amplification of mouse lipocalin-interacting membrane receptor (RIKEN cDNA 1110013E13 gene; Limr) sequence (GenBank Accession Number NM_029098): Mouse Limr forward primer (SEQ ID NO: 7): 5'- CTC CAT GAT GAG CAA GGA GGT -3'; mouse Limr reverse primer (SEQ ID NO: 8): 5'- TGA GGG AGG GAT TGA GGC -3'; mouse Limr Taqman probe (SEQ ID NO: 9): (5/6- FAM)- CTG CTC TGG TTG GGA GGC AAC TAG TAG AT -(5/6-TAMRA).

For the amplification of mouse peroxisomal biogenesis factor 11a (Pex11a) sequence (GenBank Accession Number NM_011068):

Mouse Pex11a forward primer (SEQ ID NO:10): 5'- GCA ACG TGT TCC ATG CCA -3'; mouse Pex11a reverse primer (SEQ ID NO: 11): 5'- CGC GAG GTC AGC GGC -3'; mouse Pex11a Taqman probe (SEQ ID NO: 12): (5/6-FAM)- CCA GGC AAC TGA GCA GAG CAT CCA -(5/6-TAMRA).

For the amplification of mouse peroxisomal biogenesis factor 11b (Pex11 b) sequence (GenBank Accession Number NM_011069): Mouse Pex11b forward primer (SEQ ID NO: 13): 5'- CCT CTC TTG CTG GAT GTG CTC -3'; mouse Pex11b reverse primer (SEQ ID NO: 14): 5'- CCA CAA TCC CAG GGC CA -3'; mouse Pex11b Taqman probe (SEQ ID NO: 15): (5/6-FAM)- CAC TGG ACA AAC TAG GCC TCT GGC GTT -(5/6- TAMRA).

In the figures the relative RNA-expression is shown on the Y-axis. In Figures 3A-C, 7A-B, 11A-B, 15A-B, and 15D-E, the tissues tested are given on the X-axis. "WAT" refers to white adipose tissue, "BAT" refers to brown adipose tissue.

In Figures 7A-B, 11A-B, 15A-B, and 15D-E, 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, and the panel of the control diet-mice tissues comprises WAT, BAT, muscle, liver, brain, testis, colon, small intestine, heart, lung, spleen, and kidney. In Figures 3D, 7C, 11C, 15C, and 15F, 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.

For this purpose, mouse models of insulin resistance and/or diabetes were used, such as 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. 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).

Further, 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.

Expression profiling studies confirm the particular relevance of the proteins of the present invention as regulators of energy metabolism in mammals.

Taqman analysis revealed that the mouse protein similar to aldehyde dehydrogenase 4A1 precursor (mitochondrial delta-1-pyrroline 5-carboxylate dehydrogenase; 1P5cdh) is expressed in several mammalian tissues, showing highest level of expression in liver and kidney and higher levels in further tissues, e.g. WAT, BAT, muscle, small intestine, and heart. Furthermore 1P5cdh is expressed on a lower but still robust levels in the lung, colon, hypothalamus, testis, spleen, brain, and pancreas of wild type mice as depicted in Figure 3A. We found, for example, that the expression of 1P5cdh is up-regulated in the brain and kidney, and down-regulated in the BAT of ob/ob mice compared to wildtype mice (see Figure 3B). We also found, that the expression of 1P5cdh is strongly up-regulated in the brain of fasted mice compared to wild type mice. In wild type mice fed a high fat diet, the expression of 1 P5cdh is up-regulated in the muscle and down-regulated in the colon compared to mice fed a control diet, as depicted in Figure 3G. We further show in this invention (see Figure 3D) that the 1P5cdh mRNA is expressed and transiently up-regulated during the differentiation into mature adipocyctes. The expression of 1P5cdh in metabolic active tissues of wild type mice, as well as the regulation in different animal models used to study metabolic disorders, suggests 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 glutaryl-Coenzyme A dehydrogenase (Gcdh) is expressed in several mammalian tissues, showing highest level of expression in liver and kidney and higher levels in further tissues, e.g. WAT, BAT, muscle, hypothalamus, brain, testis, colon, small intestine, heart, lung, and spleen of wild type and control-diet mice. Furthermore Gcdh is expressed on lower but still robust levels in the pancreas of wild type mice as depicted in Figure 7A. We found, for example, that the expression of Gcdh is down-regulated in the WAT of ob/ob mice compared to wild type mice (see Figure 7B). In wild type mice fed a high fat diet, the expression of Gcdh is up-regulated in the muscle and down-regulated in the lung compared to mice fed a control diet, as depicted in Figure 7B. We further show in this invention (see Figure 7C) that the Gcdh mRNA is expressed and slightly up-regulated during the differentiation into mature adipocyctes. Therefore, the Gcdh protein might play an essential role in adipogenesis. The expression of Gcdh in metabolic active tissues and the regulated expression in tissues of animal models used to study metabolic disorders, together with the expression during the differentiation from preadipocytes to mature adipocytes, suggests that this gene plays a central role in energy homeostasis.

Taqman analysis revealed highest level of expression of mouse lipocalin- interacting membrane receptor (RIKEN cDNA 1110013E13 gene; Limr) in the testis of wild type and control diet mice. Furthermore Limr is expressed on lower but still robust levels in WAT, BAT, muscle, liver, hypothalamus, brain, colon, small intestine, heart, lung, spleen, and kidney of wild type and/or control diet mice as depicted in Figure 11 A. We found, for example, that in wild type mice fed a high fat diet, the expression of Limr is up- regulated in muscle compared to mice fed a control diet, as depicted in Figure 11B. We further show in this invention (see Figure 11C) that the Limr mRNA is expressed during the differentiation into mature adipocyctes.

Taqman analysis revealed that peroxisomal biogenesis factor 11a (Pex11a) is expressed in several mammalian tissues, showing highest level of expression in BAT, and higher levels in further tissues, e.g. WAT, liver, testis, kidney, small intestine, heart, lung, colon, brain, muscle, and hypothalamus. Furthermore Pex11a is expressed on lower but still robust levels in the pancreas and spleen of wild type and control-diet mice as depicted in Figure 15A. We found, for example, that the expression of Pex11a is up-regulated in the liver of ob/ob mice (see Figure 15B). In wild type mice fed a high fat diet, the expression of Pex11a is up-regulated in muscle and liver compared to mice fed a control diet, as depicted in Figure 15B. We show in this invention (see Figure 15C) that the Pex11a mRNA is expressed and up-regulated during the differentiation into mature adipocyctes. Therefore, the Pex11a protein might play an essential role in adipogenesis. The expression of Pex11a in metabolic active tissues and the up-regulation in liver of genetically and diet induced obese mice, together with the regulation during the differentiation from preadipocytes to mature adipocytes, suggests that this gene plays a central role in energy homeostasis.

Taqman analysis revealed that peroxisomal biogenesis factor 11b (Pex11b) is expressed in several mammalian tissues, showing highest level of expression in testis, and higher levels in further tissues, e.g. WAT, BAT, muscle, liver, hypothalamus, brain, colon, small intestine, heart, lung, spleen, and kidney. Furthermore Pex11b is expressed on a lower but still robust levels in pancreas of wild type and control-diet mice as depicted in Figure 15D. We found, for example, that the expression of Pex11 b is slightly down-regulated in spleen of ob/ob mice compared to wild type mice (see Figure 15E). We show in this invention (see Figure 15F) that the Pex11b mRNA is expressed during the differentiation into mature adipocyctes.

RNA preparation from human primary adipose tissues was done as described in Example 4. The target preparation, hybridization, and scanning was performed as described in the manufactures manual (see Affymetrix Technical Manual, 2002, obtained from Affmetrix, Santa Clara, USA). ln Figures 4, 8, 12, 16, and 19 the X-axis represents the time axis, shown are day 0 and day 12 of adipocyte differentiation. The Y-axis represents the fluorescent intensity. The expression analysis (using Affymetrix GeneChips) of the aldehyde dehydrogenase 4 family, member A1 (ALDH4A1 ), glutaryl- Coenzyme A dehydrogenase (GCDH), lipocalin-interacting membrane receptor (LIMR), chromosome 7 open reading frame 2 (C7orf2), peroxisomal biogenesis factor 11A (PEX11A), and androgen-induced 1 (AIG1) genes using human abdominal and/or mammary derived primary adipocytes and human adipocyte cell line (SGBS) differentiation, clearly shows differential expression of human ALDH4A1, GCDH, LIMR, C7orf2, PEX11A, and AIG1 genes in adipocytes. Several independent experiments were done. The experiments further show that the ALDH4A1, GGDH, PEX11A, and AIG1 transcripts are most abundant at day 12 compared to day 0 during differentiation (see Figures 4, 8, 16, and 19). The experiments further show that the LIMR and C7orf2 transcripts are most abundant at day 0 compared to day 12 during differentiation (see Figure 12).

Thus, the ALDH4A1, GCDH, PEX11A, and AIG1 proteins have to be significantly increased in order for the preadipocyctes to differentiate into mature adipocycte, and the LIMR and C7orf2 proteins have to be significantly decreased in order for the preadipocyctes to differentiate into mature adipocycte. Therefore, ALDH4A1, GCDH, PEX11A, and AIG1 in preadipocyctes have the potential to enhance adipose differentiation, and LIMR and C7orf2 in preadipocyctes have the potential to inhibit adipose differentiation.

Therefore, ALDH4A1, GCDH, LIMR, C7orf2, PEX11 , and AIG1 proteins might play an essential role in the regulation of human metabolism, in particular in the regulation of adipogenesis and thus it might play an essential role in obesity, diabetes, and/or metabolic syndrome. For the purpose of the present invention, it will be understood by the person having average skill in the art that any combination of any feature mentioned throughout the specification is explicitly disclosed herewith.

Claims

1. A pharmaceutical composition comprising a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous protein or/and a functional fragment thereof, a nucleic acid molecule encoding a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous protein or/and a functional fragment or/and a modulator/effector of said nucleic acid molecule or said protein, preferably together with pharmaceutically acceptable carriers, diluents or/and additives.

2. The composition of claim 1, wherein the nucleic acid molecule is a vertebrate or insect CG7145, CG9547, CG5807, CG8315, or CG3625 nucleic acid, particularly encoding a human protein as described in Table 1 , or/and a nucleic acid molecule which is complementary thereto or/and a functional fragment thereof or/and a variant thereof.

3. The composition of claim 1 or 2, wherein said nucleic acid molecule is selected from the group consisting of (a) a nucleic acid molecule encoding a polypeptide as shown in

Table 1 or/and an isoform, fragment or variant of said polypeptide; (b) a nucleic acid molecule which comprises or is the nucleic acid molecule as shown in Table 1; (c) a nucleic acid molecule being degenerate as a result of the genetic code to the nucleic acid sequences as defined in (a) or

(b),

(d) a nucleic acid molecule that hybridizes at 50°C in a solution containing 1 x SSC and 0.1% SDS to a nucleic acid molecule as defined in claim 2 or as defined in (a) to (c) and/or a nucleic acid molecule which is complementary thereto;

(e) a nucleic acid molecule that encodes a polypeptide which is at least 85%, preferably at least 90%, more preferably at least 95%, more preferably at least 98% and up to 99,6% identical to the human CG7145, CG9547, CG5807, CG8315, or CG3625 homologous protein, preferably as described in Table 1 or as defined in claim 2 or to a polypeptide as defined in (a); (f) a nucleic acid molecule that differs from the nucleic acid molecule of (a) to (e) by mutation and wherein said mutation causes an alteration, deletion, duplication or premature stop in the encoded polypeptide.

4. The composition of any one of claims 1-3, wherein the nucleic acid molecule is a DNA molecule, particularly a cDNA or a genomic DNA.

5. The composition of any one of claims 1-4, wherein said nucleic acid encodes a polypeptide contributing to regulating the energy homeostasis and/or the metabolism of triglycerides.

6. The composition of any one of claims 1 -5, wherein said nucleic acid molecule is a recombinant nucleic acid molecule.

7. The composition of any one of claims 1-6, wherein the nucleic acid molecule is a vector, particularly an expression vector.

8. The composition of any one of claims 1 -5, wherein the polypeptide is a recombinant polypeptide.

9. The composition of claim 8, wherein said recombinant polypeptide is a fusion polypeptide.

10. The composition of any one of claims 1-7, wherein said nucleic acid molecule is selected from hybridization probes, primers and anti-sense oligonucleotides.

11. The composition of any one of claims 1-10 which is a diagnostic composition.

12. The composition of any one of claims 1-10 which is a therapeutic 5 composition.

13. The composition of any one of claims 1-12 for the manufacture of an agent for detecting or/and verifying, for the treatment, alleviation or/and prevention of metabolic diseases or dysfunctions, including metabolic o syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis in cells, cell masses, organs and/or subjects.

5 14. The composition of any one of claims 1-13 for application in vivo.

15. The composition of any one of claims 1-13 for application in vitro.

16. Use of a nucleic acid molecule encoding a CG7145, CG9547, CG5807, o CG8315, or CG3625 homologous protein or/and and isoform, a functional fragment or/and a variant thereof, in particular a nucleic acid molecule as described in Table 1, particularly of a nucleic acid molecule according to claim 3 (a), (b), or (c), or/and a polypeptide encoded thereby or/and a functional fragment or/and a variant of said nucleic 5 acid molecule or said polypeptide or/and a modulator/effector of said nucleic acid molecule or said polypeptide for the manufacture of a medicament for treatment of obesity, diabetes, or/and metabolic syndrome for controlling the function of a gene and/or a gene product which is influenced and/or modified by a CG7145, CG9547, CG5807, o CG8315, or CG3625 homologous polypeptide, particularly by a polypeptide according to claim 3.

17. Use of the nucleic acid molecule encoding a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous protein or/and an isoform, a functional fragment or/and a variant thereof, in particular a nucleic acid molecule as described in Table 1, particularly of a nucleic acid molecule according to claim 3 (a), (b), or (c), or/and a polypeptide encoded thereby or/and a functional fragment or/and a variant of said nucleic acid molecule or said polypeptide, or/and a modulator/effector of said nucleic acid molecule or said polypeptide for identifying substances capable of interacting with a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous polypeptide, particularly with a polypeptide according to claim 3.

18. A non-human transgenic animal exhibiting a modified expression of a CG7145, CG9547, GG5807, CG8315, or CG3625 homologous polypeptide, particularly of a polypeptide according to claim 3.

19. The animal of claim 18, wherein the expression of the CG7145, CG9547, CG5807, CG8315, or CG3625 homologous polypeptide, particularly of a polypeptide according to claim 3, is increased and/or reduced.

20. A recombinant host cell exhibiting a modified expression of a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous polypeptide, particularly of a polypeptide according to claim 3.

21. The cell of claim 20 which is a human cell.

22. A method of identifying a (poly)peptide involved in the regulation of energy homeostasis or/and metabolism of triglycerides in a mammal comprising the steps of (a) contacting a collection of (poly)peptides with a CG7145,

CG9547, CG5807, CG8315, or CG3625 homologous polypeptide, particularly with a polypeptide according to claim 3, or a functional fragment thereof under conditions that allow binding of said (poly)peptides;

(b) removing (poly)peptides which do not bind and

(c) identifying (poly)peptides that bind to said CG7145, CG9547, CG5807, CG8315, or CG3625 homologous polypeptide.

23. A method of screening for an agent which modulates the interaction of a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous polypeptide, particularly of a polypeptide according to claim 3, with a binding target/agent, comprising the steps of (a) incubating a mixture comprising

(aa) a CG7145, CG9547, CG5807, CG8315, or

CG3625 homologous polypeptide, particularly a polypeptide according to claim 3, or a functional fragment thereof; (ab) a binding target/agent of said CG7145,

CG9547, CG5807, CG8315, or CG3625 homologous polypeptide or functional fragment thereof; and (ac) a candidate agent under conditions whereby said polypeptide or functional fragment thereof specifically binds to said binding target/agent at a reference affinity; (b) detecting the binding affinity of said polypeptide or functional fragment thereof to said binding target to determine the affinity for the agent; and (c) determining a difference between affinity for the agent and reference affinity.

24. A method for screening for an agent, which modulates the activity of a GG7145, CG9547, CG5807, CG8315, or CG3625 homologous polypeptide, comprising the steps of

(a) incubating a mixture comprising

(aa) a CG7145, CG9547, CG5807, CG8315, or

CG3625 homologous polypeptide or a functional fragment thereof; and

(ab) a candidate agent under conditions whereby said polypeptide or functional fragment thereof exhibits a reference activity, (b) detecting the activity of said polypeptide or functional fragment thereof to determine an activity in presence of the agent; and (c) determining a difference between the activity in presence of the agent and reference activity.

25. A method of producing a composition comprising the (poly)peptide identified by the method of claim 22 or the agent identified by the method of claim 23 or 24 with a pharmaceutically acceptable carrier, diluent or/and additive.

26. The method of claim 25 wherein said composition is a pharmaceutical composition for preventing, alleviating or/and treating of metabolic diseases or dysfunctions, including metabolic syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

27. Use of a (poly)peptide as identified by the method of claim 22 or of an agent as identified by the method of claim 23 or 24 for the preparation of a pharmaceutical composition for the treatment, alleviation or/and prevention metabolic diseases or dysfunctions, including metabolic syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

28. Use of a nucleic acid molecule as defined in any one of claims 1-6 or 10 for the preparation of a medicament for the treatment, alleviation and/or prevention of metabolic diseases or dysfunctions, including metabolic syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

29. Use of a polypeptide as defined in any one of claims 1 to 6, 8 or 9 for the preparation of a medicament for the treatment, alleviation and/or prevention of metabolic diseases or dysfunctions, including metabolic syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

30. Use of a vector as defined in claim 7 for the preparation of a medicament for the treatment, alleviation and/or prevention of metabolic diseases or dysfunctions, including metabolic syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

31. Use of a host cell as defined in claim 20 or 21 for the preparation of a medicament for the treatment, alleviation and/or prevention of metabolic diseases or dysfunctions, including metabolic syndrome, obesity or/and diabetes, as well as related disorders such as eating disorder, cachexia, pancreatitis, hypertension, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, or liver fibrosis.

32. Use of a CG7145, GG9547, CG5807, CG8315, or CG3625 homologous nucleic acid molecule or/and of a fragment thereof for the production of a non-human transgenic animal which over- or under-expresses the CG7145, CG9547, CG5807, CG8315, or CG3625 homologous gene product.

33. Kit comprising at least one of (a) a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous nucleic acid molecule or/and a fragment thereof; (b) a CG7145, CG9547, CG5807, CG8315, or CG3625 homologous amino acid molecule or/and a functional fragment or/and an isoform thereof; (c) a vector comprising the nucleic acid of (a);

(d) a host cell comprising the nucleic acid of (a) or the vector of (c);

(e) a polypeptide encoded by the nucleic acid of (a), expressed by the vector of (c) or the host cell of (d);

(f) a fusion polypeptide encoded by the nucleic acid of (a); (g) an antibody, an aptamer or other modulator/effector of the nucleic acid of (a) or the polypeptide of (b) , (e) , or (f) or/and (h) an anti-sense oligonucleotide of the nucleic acid of (a).