MX2008015670A - G protein coupled receptor 39 (gpr39). - Google Patents

Abstract

The present invention relates to the functional characterization of the G protein coupled receptor GPR39 and to compounds, which modify or regulate GPR39 protein activity. In particular the present invention relates to methods of screening for agonists or antagonists of GPR39 in order to identify compounds capable of modulating carbohydrate metabolism and to the therapeutic uses of these compounds. In particular to the use of GPR39 in methods to identify compounds that are capable to enhance glucose control in a subject and which are effective for preventing and/or treating pathologies related with an impaired carbohydrate metabolism, in particular in the prevention and/or treatment of diabetes including associated complications thereof, or of the metabolic syndrome including associated complications thereof. Including Type 1 (insulin-dependent or IDDM), Type 2 (non- insulin- dependent diabetes mellitus), maturity-onset diabetes of the young (MODY) and gestational diabetes.

Description

RECEPTOR 39 COUPLED TO PROTEIN G (GPR39) FIELD OF THE INVENTION The present invention relates to the functional characterization of the GPR39 receptor coupled to G protein and to compounds, which modify or regulate the activity of the GPR39 protein. In particular, the present invention relates to methods for improving the control of glucose in a subject in need of such control, which comprises administering to the subject a composition comprising a therapeutically effective amount of a compound capable of modulating or regulating the activity of the GPR39 protein. In another embodiment, the present invention relates to the involvement of GPR39 signaling in the regulation of glucose, and thus in the glycemia observed in diabetes and in the metabolic syndrome, including obesity, diabetes, cardiovascular diseases, atherosclerosis , atherogenic dyslipidemia, hypertension, hypertriglyceridemia and adipose tissue disorders.

BACKGROUND OF THE INVENTION GTP-binding proteins (G proteins) act as intermediates between the binding of ligands such as hormones and other chemical mediators, to G-protein coupled receptors (GPCRs) and the activation of intracellular effectors. Upon binding of a ligand to a GPCR, the cytoplasmic domains of the receptor undergo conformational changes, which allow the interaction of the receptor with a G protein, which in turn allows the activation of intracellular intermediates such as adenylate cyclase, phospholipase C or channels of ions. Such a system allows the amplification of the original signal, since many second messengers can be produced in response to the binding of an individual ligand to the GPCR. Through this mechanism, the cells are able to detect and respond to alterations in their external environment. The G protein-coupled receptors form a superfamily of integral proteins of the plasma membrane, each receptor sharing the common trait of seven hydrophobic transmembrane domains, each of which is 20 to 30 amino acids in length and which are linked by sequences of hydrophilic amino acids of varying length. The amino terminus of the receptor is extracellular, with the carboxy terminus found in the cytoplasm of the cell. GPCRs are found in a wide range of tissues and cell types, and are involved in many different physiological processes. They are activated by a wide range of ligands, for example, hormones such as luteinizing hormone, follicle stimulating hormone, chorionic gonadotrophin, thyrotropin, adrenocorticotropin, glucagon and vasopressin, or neurotransmitters such as 5-HT, acetylcholine (muscarinic AchR), histamine, prostaglandins, calcitonin, leukotrienes and Ca2 +. The wide distribution and wide variety of functions of the GPCRs indicate that GPCRs can play important roles in a variety of pathological conditions. Of course, it has been found that GPCRs are implicated in diseases related to bronchoconstriction, hypertension, inflammation, hormonal alteration, diabetes, apoptosis, nociception, facilitation of neurotransmission and tremor disorders. Within the superfamily of G protein-coupled receptors, putative GPCRs for which natural ligands are unknown are termed "orphan receptors". It has been shown that G protein-coupled receptors are valuable drug targets, since they are the target of approximately 50% of the drugs sold in the market. Therefore, many orphan GPCRs are being evaluated to identify new potential targets. GPR39 was identified based on its sequence similarity to the growth hormone secretagogue receptor (GHS-R) and the neurotensin 1 and 2 receptors (NT-R1 and NT-R2) (McKee et al., 1997) . The predicted GPR39 protein of 453 amino acids contains the 7 transmembrane domains characteristic of GPCRs. By sequence comparison with other GPCRs, McKee et al. (1997) found that the protein sequence of GPR39 is 27%, 29% and 32% identical to that of GSHR, MTLR1 (motilin receptor), and neurotensin receptor 1, respectively. Northern blot analysis revealed that GPR39 has a wide distribution in tissues. An individual hybridization messenger RNA transcript of 1.8 to 2 kb was detected in most regions of the brain tested. Without However, in addition to this species, an alternate transcript, 3 kb in length, was observed in several peripheral tissues such as the stomach and small intestine. In tissues such as the pancreas, thyroid and colon, this 3 kb species was the only transcript detected (McKee et al., 1997). By means of in situ fluorescence hybridization, McKee et al. (1997) mapped the GPR39 gene for 2q21-q22. An acid residue in TM3 is conserved in GPR39. It is known that this residue is essential for the binding and activation of GHS-R by structurally different GHSs. Based on these tissue distribution studies, it has been hypothesized that GPR39 is involved in cardiovascular disease states (WO2001 / 081634 and WO2004 / 004279), cancers and in particular, brain cancers such as glioblastoma (WO2001 / 036685 and WO01042288), inflammation and neurological disease states (US 2003/232769 and WO2004 / 004279) and in gastrointestinal and liver diseases (WO2004 / 004279). Only recently has obestatin been identified, the putative ligand for GPR39 (Zhang J. V. et al, 2005). It was found that obestatin suppresses food intake and reduces gastrointestinal functions, including gastric emptying and jejunal motility. Based on these anorexigenic actions, GPR39 has been postulated as a novel antiobesity objective. However, in none of the cited references, a function for GPR39 has been provided in energy homeostasis and in particular in carbohydrate metabolism.

Diabetes is caused by the occurrence of abnormal metabolism of glucose, proteins and lipids, due to an insufficiency of the actions of insulin. Typical signs of diabetes include an abnormal increase in serum glucose levels, insulin insufficiency, and an excretion of glucose in the urine. Several clinical subclasses are recognized, including: type 1 (insulin-dependent diabetes mellitus or IDDM), type 2 (non-insulin-dependent diabetes mellitus), juvenile onset diabetes at maturity (MODY) and gestational diabetes. They differ in etiology, pathology, genetics, age of onset and treatment. Type 1 diabetes, the most severe form of diabetes, accounts for 5 to 10 percent of diabetes, and occurs more frequently in children and young adults. In this form of diabetes, the body does not produce any insulin and without regular injections of insulin, the victim falls into a coma and dies. Individuals suffering from type 1 diabetes are totally insulin-dependent. Type 2 diabetes, the most common type of diabetes, is usually characterized by gradual onset and occurs mainly in people over 40 years of age. Type 2 diabetes is a metabolic disorder that results from the body's inability to produce enough insulin, or to properly use insulin to meet the body's needs, especially when the person is overweight. It is the most common form of the disease, and accounts for 90 to 95 percent of diabetes cases.

Initially, the combination of dietary measures, weight reduction and oral medication, can keep the condition under control for a period, but most people with type 2 diabetes eventually require insulin injections. Diabetes can be controlled with insulin, and in some cases through careful diet, but there is a need for safe and effective treatment for diabetes with minimal side effects and without the invasive insulin injection procedure. The impaired metabolism of carbohydrates, and in particular diabetes or metabolic syndrome, is also associated with several complications, including (poly) neuropathy (peripheral, autonomic, proximal, focal), nephropathy, kidney disease, renal failure, bladder dysfunction, retinopathy, vascular complications of large and small vessels, cerebrovascular accident, myocardial infarction, occlusive disease of large vessels, coronary artery disease (ischemic), cerebral vascular disease, heart failure, peripheral arterial disease, hypertension, sexual dysfunction and gastroparesis. In accordance with the present invention, said complications will also benefit from a treatment of impaired carbohydrate metabolism.

BRIEF DESCRIPTION OF THE INVENTION As indicated above, the present invention relates to the identification of novel functions of the GPR39 receptor. As shown in the examples below, mutations of GPR39 in mammals affect blood glucose concentrations and designate GPR39 as a key element in the regulation of carbohydrate metabolism. This discovery provides a way for new therapeutic procedures in the treatment of diabetes, including associated complications, or metabolic syndrome, including associated complications, through the modulation of GPR39 activity. This discovery also provides new identification methods to identify compounds useful for the prevention or treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof. Therefore, a first aspect of the invention provides the use of all or part of the GPR39 protein in a method for identifying compounds that improve the control of glucose in a subject, and which are effective in preventing and / or treating pathologies. related to an impaired metabolism of carbohydrates, in particular in the prevention and / or treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof. Alternatively, the invention provides for the use of cells that express the all or part of the GPR39 protein in said method. In a specific embodiment, GPR39 is an isolated protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a splicing variant of the proteins having the SEQ ID's mentioned above, and an amino acid sequence having at least 80% and preferably at least 90%, 95%, 96%, 97% or 98% sequence identity, with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Parts of the GPR39 protein, as the phrase was used above, means that they include fragments of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, said fragments being of at least 10, for example, minus 20, 30 40, 50, 75, 100 or 150 or more amino acids in size. Such fragments can be derived from the N-terminal region of SEQ ID NO: 2 or SEQ ID NO: 4, respectively. Fragments that include the N-terminal region can be used to reconstitute the extracellular portion of the receptor to provide receptor binding sites. Preferably, the fragments will retain the ability to bind to an agent known to bind GPR39, including the natural ligand, as well as receptor agonists and / or antagonists as defined below, in particular retaining the ability to bind to the ligand. putative of GPR39, that is, to join the obestatin. The present invention also provides the use of an isolated nucleic acid sequence encoding all or part of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, in a method for identifying compounds that improve the control of glucose in a subject, and that are effective in preventing and / or treating pathologies related to a deteriorated carbohydrate metabolism, in particular in the prevention and / or treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof. The nucleic acid sequences as used in the methods of the present invention, mean that they include the isolated nucleic acid sequences consisting of SEQ ID NO: 1 or SEQ ID NO: 3, and nucleic acid sequences which have at least 80%, and preferably at least 90%, 95%, 96%, 97% or 98% sequence identity, with the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. Nucleic acids of the invention further include nucleic acids comprising a sequence having at least 80% and preferably at least 90%95%, 96%, 97% or 98% sequence identity, with the nucleic acid sequences of SEQ ID NO: 1 or SEQ ID NO: 3, or their complements. Preferably, these sequences will hybridize with the corresponding nucleic acid under controlled conditions to minimize non-specific binding. Preferably, conditions of severe to moderately severe hybridization are preferred. Suitable conditions include, for example, for the detection of sequences that are approximately 80 to 90% identical, hybridization overnight at 42 ° C in Na2HPO4 0.25 M, pH 7.2, SDS at 6.5%, dextran sulfate at 10% and a washed final at 55 ° C in 0.1 X SSC, 0.1% SDS. For detection of sequences that are more than about 90% identical, suitable conditions include overnight hybridization at 65 ° C in 0.25 M Na2HPO4, pH 7.2, SDS at 6.5%, dextran sulfate at 10% and a final wash at 60 ° C in 0.1X SSC, 0.1% SDS. It will be appreciated that such nucleic acids do not necessarily code for "full length" polypeptides, and will thus include nucleic acids representing, for example, mutant forms of the GPR39 gene in which the coding sequence has been prematurely terminated by a substitution that result in a stop codon or a structure mutation. These are also nucleic acids of the invention. The invention also provides for the use of nucleic acids which are fragments of the nucleic acids encoding a polypeptide of the invention. In one aspect, the invention provides nucleic acid primers consisting essentially of 15 to 50, for example, 15 to 35, 18 to 35, 15 to 24, 18 to 30, 18 to 21 or 21 to 24 nucleotides, of a sequence coding for a polypeptide of the invention or its complement. The nucleic acids and polypeptides of the invention can be used therapeutically to treat disease. In particular, they can be used to treat diseases whose pathology is associated with the action of GPR39 receptors, in particular that associated with the prevention and / or treatment of pathologies related to a deteriorated carbohydrate metabolism, in particular in the prevention and / or diabetes treatment, including associated complications thereof, or metabolic syndrome, including associated complications thereof. In another aspect, vectors are provided comprising the sequences of said nucleic acids, in particular, expression vectors comprising a promoter operably linked to the nucleic acid sequences of the invention. The vectors can be carried by a host cell, and expressed within said cell. After said expression, said cells can be used in the methods according to the invention. As mentioned hereinabove, it is an object of the invention to provide test methods for the identification of compounds (hereinafter also referred to as agents) that bind to, or modulate, the activity of polypeptides of the invention. In particular, it is contemplated that said compounds are an organic or inorganic assembly of atoms of any size, and include small molecules (less than about 2500 Daltons) or larger molecules, such as peptides, polypeptides, whole proteins and polynucleotides, wherein said compounds can be used in treatment methods as described above. Since the present inventors are the first to identify GPR39 as a receptor as a key element in the regulation of carbohydrate metabolism, the present invention opens up the possibility of the use of GPR39 itself and / or compounds that agonize or antagonize this receptor in therapeutic applications. Therefore, the invention further extends to a method of treating the body of a human or animal, said method comprising the use of an agonist or antagonist of GPR39. As used herein, an "agonist" refers to an agent that binds to and activates GPR39, that is, produces a pharmacological response, and includes positive allosteric modulators that bind to a site different from the binding site. agonist in the receptor, and that improves the sensitivity of the receptor towards the natural agonist of the receptor. An "antagonist," as used herein, refers to agents that attenuate the effects of an agonist for GPR39. The antagonism can be competitive and reversible (ie, it binds to a receptor region in common with the agonist, and can be replaced with a sufficiently large amount of agonist), or it can be non-competitive and / or irreversible (ie, the antagonist binds covalently to the binding site, and no amount of agonist can overcome the inhibition). Other types of antagonism are non-competitive antagonism, where the agent binds to an allosteric site of the receptor, or inverse agonism, where for a constitutive receptor, as reported for GPR39 (Holst et al., 2004), the agent is binds to the binding site to the agonist and attenuates the constitutive activity of the receptor. In particular, the present invention provides a method of treating disease conditions related to impaired carbohydrate metabolism, in particular in the prevention and / or treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof, said method comprising those conditions where a decrease in basal glucose levels is required, administering to the human or animal a therapeutically active dosage of a GPR39 receptor agonist. The use of a GPR39 agonist is of particular use in the treatment of the metabolic syndrome, including associated complications thereof, in particular comprising the use of an identifiable GPR39 agonist using a method of the present invention. Alternatively, in the treatment of diabetes, including associated complications thereof, said method comprises administering to the human or animal a therapeutically active dosage of a GPR39 receptor antagonist, in particular comprising the use of an identifiable GPR39 antagonist using a method of the present invention. These and other aspects of the invention are described herein in more detail.

BRIEF DESCRIPTION OF THE SEQUENCES SEQ ID NO: 1 is the nucleotide sequence for mouse GPR39. SEQ ID NO: 2 is the amino acid sequence for mouse GPR39.

SEQ ID NO: 3 is the nucleotide sequence for human GPR39. SEQ ID NO: 4 is the amino acid sequence for human GPR39. SEQ ID NO: 5 is obestatin of human. SEQ ID NO: 6 is monkey obestatin. SEQ ID NO: 7 is mouse obestatin. SEQ ID NO: 8 is rat obestatin. SEQ ID NO: 9 is gerbil obestatin. SEQ ID NO: 10 is pork obestatin. SEQ ID NO: 1 is obestatina de gato. SEQ ID NO: 12 is dog obestatina. SEQ ID NO: 13 is goat obestatin. SEQ ID NO: 14 is obestatin of sheep. SEQ ID NO: 15 is cattle obestatina. SEQ ID NO: 16 is the consensus sequence for obestatin.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A-1 B show the expression of GPR39-LacZ in the pancreas. In the pancreas derived from GPR39"'", expression was detected in the islets of Langerhans (enclosed in a circle), as evidenced by the blue stain (figure 1A, 40x). No staining was present in the wild-type pancreas (Figure 1 B, 40x). Figure 2 shows the comparison of the body weight of GPR39"'" and WT (wild-type) mice followed in male and female animals from 8 to 12 up to 16 to 20 weeks. The data are expressed as mean ± standard deviation. Figures 3A-3B show the comparison of the blood glucose concentration at the starting point under feeding (FIG. 3A) and fasting conditions (FIG. 3B) of GPR39"'" and WT mice, followed in male and female animals . Figure 4 shows the comparison of the glucose tolerance test of fasting GPR39"/ _ and WT mice, followed in male and female animals, the blood glucose concentration was determined at 15, 30, 45, 60, 90 and 120 minutes after the administration of glucose, and expressed as a percentage change of the values at the starting point.The data are expressed as mean ± standard deviation Figures 5A-5B show the comparison of the body composition of mice GPR39"'" and WT followed in male and female mice Fat and lean weights are expressed in percent body weight Data are expressed as mean ± standard deviation Figure 6A is an alignment of GPR39 protein sequences of human (NP 001499), mouse (AAH85285), rat (XP 22578), dog (partial) (XP 853332) and cattle (partial) (XP 589836) .The alignment shows the GPR39 protein sequence of human above, other species in order of similarity of decreasing sequence with the human. The name of each sequence reflects the access number of the sequence in the nr database of the NCBI, the gene symbol and the name of the species. The sequences of dog and cattle in the alignment differ from the original public domain sequence: the C-terminal amino acids that lack similarity with other GPR39 sequences were removed. The argument is that the delegated amino acids were from lower quality sequencing results. The human sequence is used as a reference, and the amino acids are gray. The amino acids of other sequences are in color, they are similar to the human sequence.

DETAILED DESCRIPTION OF THE INVENTION Nucleic acid Nucleic acid, as used in the methods of the present invention, includes DNA (including genomic DNA and cDNA) and RNA. Where the nucleic acid according to the invention includes RNA, reference to the sequences illustrated in the accompanying listings should be considered as reference to the equivalent RNA, with U replacing T. The nucleic acid of the invention can be single-stranded or double chain. The single-stranded nucleic acids of the invention include antisense nucleic acids. In this way, it will be understood that the reference to SEQ ID NO: 1 or sequences comprising SEQ ID NO: 1 or fragments thereof, include complementary sequences, unless the context clearly indicates otherwise. The same applies to SEQ ID NO: 3. In general, the nucleic acid according to the present invention is provided as an isolate, in isolated and / or purified form, or free or substantially free of material with which it is associated in natural form, such as free or substantially free of nucleic acid flanking the gene in the human genome, except possibly one or more regulatory sequences for expression. The nucleic acid can be completely or partially synthetic, and can include genomic DNA, cDNA or RNA. In other words, the term "isolated" indicates that a sequence of natural occurrence has been removed from its normal cellular context. In this way, the sequence can be in a cell-free solution, or it can be placed in a different nucleic acid or cell environment context. Therefore, the nucleic acids claimed herein may be present as heterologous material in whole cells or in cell lysates or in a partially, substantially or completely purified form. The invention also provides nucleic acids which are fragments of the nucleic acids encoding a polypeptide of the invention. In one aspect, the invention provides nucleic acid primers consisting essentially of 15 to 50, eg, 15 to 35, 18 to 35, 15 at 24, 18 to 30, 18 to 21 or 21 to 24 nucleotides, of a sequence encoding a polypeptide of the invention or its complement. The term A essentially consists of = refers to nucleic acids that do not include any additional 5 'or 3' nucleic acid sequence. In another aspect of the invention, the nucleic acids of the invention consisting essentially of 15 to 30 nucleotides as defined above, may be linked at the 3 'end, but preferably the 5' end, to short additional sequences ( for example, from 4 to 15, such as from 4 to 10 nucleotides), to which they are not naturally bound. Said additional sequences are preferably linkers comprising a restriction enzyme recognition site that facilitates cloning when the nucleic acid of the invention is used, for example, as a PCR primer. The primers of the invention are desirably capable of hybridizing selectively with nucleic acids encoding the polypeptides of the invention. By "Aselectives" is meant selective with respect to sequences encoding other purine receptors, and in particular with respect to different receptors of adenine receptors. The ability of the sequence to selectively hybridize can be determined by experimentation, or it can be calculated. For example, one way to calculate the Tm of an initiator is by referencing the formula to calculate the Tm of initiators for a homologous target sequence. This formula is Tm (° C) = 2 (A + T) + 4 (G + C) - 5.

This will provide the Tm under conditions of 3xSSC and 0.1% SDS (where SSC is 0.15 M NaCl, 0.015 M sodium citrate, pH 7). This formula is generally suitable for primers up to about 50 nucleotides in length. In the present invention, this formula can be used as an algorithm to calculate a nominal Tm of an initiator for a specified sequence derived from a sequence encoding a polypeptide of the invention. The Tm can be compared with a Tm calculated for GPCR sequences of humans and rats, based on the maximum number of matches with any part of these other sequences. Suitable conditions for an initiator to hybridize with an objective sequence can also be measured experimentally. Suitable experimental conditions comprise hybridizing a candidate primer with nucleic acid encoding a polypeptide of the invention, and nucleic acid encoding other G protein-coupled receptors on a solid support under low stringency hybridization conditions (eg, 6xSSC to 55 ° C), washing in reduced SSC and / or higher temperature, for example, in 0.2xSSC at 45 ° C, and increasing the hybridization temperature progressively until determining the hybridization conditions that allow the primer to hybridize with nucleic acid coding for a polypeptide of the invention, but not other nucleic acids encoding GPCR. The nucleic acids of the invention, in particular initiators, may possess a revealing label. Suitable brands include radioisotopes such as P or S, fluorescent labels, enzyme labels, or other protein brands such as biotin. Said labels can be added to poiinucleotides or initiators of the invention, and can be detected using techniques known per se. The primers of the present invention may comprise synthetic nucleic acids, such as those with modified base structures intended to improve the stability of the nucleic acid in a cell. Many different types of oligonucleotide modification are known in the art. These include methyl phosphonate and phosphorothioate base structures, and the addition of polyglycine or acridine chains at the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it will be understood that the polynucleotides described herein may be modified by any method available in the art. Such modifications can be carried out to improve the in vivo activity or the lifetime of the polynucleotides of the invention. Also included within the scope of the invention are antisense sequences based on the nucleic acid sequences described herein, preferably in the form of oligonucleotides, in particular stabilized oligonucleotides, or ribozymes. Antisense oligonucleotides can be designed to hybridize to the complementary sequence of nucleic acid, pre-messenger RNA or mature messenger RNA, interfering with the production of the encoded polypeptide by a given target DNA sequence, so that its expression is reduced or prevented altogether. The ribozymes will be designed to break the messenger RNA encoded by a nucleic acid sequence encoding the GPCR GPR39 of the invention, desirably in a specific target sequence for the GPCR GPR39, ie, one that is not common for other sequences of GPCR. The construction of antisense sequences and their use is described in Peyman and Ulman, Chemical Reviews, 90: 543-584 (1990), Crooke, Ann. Rev. Pharmacol. Toxicol., 32: 329-376 (1992), and Zamecnik and Stephenson, P. N.A. S, 75: 280-284 (1974). The construction of ribozymes and their use is described, for example, in Gibson and Shillitoe, Molecular Biotechnology 7 (2): 25-37 (997). In one embodiment, RNA of the invention can be used for the induction of RNA interference (RNAi), using double-stranded RNA (dsRNA) sequences (Fire et al., Nature 391: 806-81 1, 1998) or sequences of Short interfering RNA (siRNA) (Yu et al., Proc Nati Acad Sci USA, 99: 6047-52, 2002). "RNAi" is the process by which dsRNA induces degradation dependent on homology of complementary messenger RNA. In one embodiment, a nucleic acid molecule of the invention is hybridized by annealing of complementary bases with a "sense" ribonucleic acid of the invention, to form the double-stranded RNA. The sense and antisense nucleic acid molecules of dsRNAs corresponding to at least about 20, 25, 50, 100, 250 or 500 nucleotides or a coding strand of GPR39, or to only a portion thereof, are provided. In an alternative embodiment, the siRNA molecules have 30 nucleotides or less in length, and more preferably 21 to 23 nucleotides, with 3 'overhangs of 2 to 3 characteristic nucleotides, which are generated by the digestion of longer dsRNA molecules by ribonuclease III. See, for example, Tuschl T. (Nat Biotechnol 20: 446-48, 2002). Intracellular transcription of small RNA molecules can be achieved by cloning the siRNA templates into transcription units of RNA polymerase III (Pol III), which normally code for small nuclear RNA (snRNA) U6 or human ribonuclease P RNA H 1. Two Methods can be used to express siRNA molecules: in one embodiment, sense and antisense strands that make up the siRNA duplex are transcribed by individual promoters (Lee, et al., Nat. Biotechnol., 20, 500-505, 2002); in an alternative embodiment, the siRNA molecules are expressed as foot and loop hairpin RNA structures that give rise to siRNA molecules after intracellular processing (Brummelkamp et al., Science 296: 550-553, 2002) (incorporated citation in the present as a reference). The dsRNA / siRNA is most commonly administered by joining sense and antisense RNA strands in vitro prior to delivery to the organism. In an alternative embodiment, RNAi (RNA interference) can be carried out by administering sense and antisense nucleic acids of the invention in the same solution without binding prior to administration, and can even be performed by administering the nucleic acids in vehicles. separated within a very narrow time frame. Nucleic acid molecules encoding fragments, homologs, derivatives and analogs of a GPR39 or antisense nucleic acids complementary to a GPR39 nucleic acid sequence are also provided. Antisense sequences, siRNA molecules and ribozyme sequences of the invention can be introduced into cultured mammalian cell lines to study the function of GPCR GPR39, for example, by causing down-regulation of this gene and observing phenotypic effects, or expression or location of proteins described herein that are associated with the GPCR GPR39. In cells where aberrant expression of GPCR GPR39 occurs, said antisense, siRNA and ribozyme sequences can be used to down-regulate gene expression. The cDNA sequence of the GPCR of the invention can be cloned using standard PCR (polymerase chain reaction) cloning techniques. This involves producing a pair of primers for 5 'and 3' ends in opposite strands of SEQ ID NO: 1, by placing the primers in contact with messenger RNA or cDNA obtained from a mammalian cortical cell, performing a low polymerase chain reaction conditions that produce the amplification of the desired region, isolating the amplified fragment (e.g., purifying the reaction mixture on an agarose gel), and recovering the amplified DNA. The primers can be designed to contain enzyme recognition sites of suitable restriction, so that the amplified DNA can be cloned into a suitable cloning vector. The same applies to SEQ ID NO: 3. Polynucleotides that are not 100% homologous to the sequence of SEQ ID NO: 1 or SEQ ID NO: 3, but which code for either SEQ ID NO: 2 or SEQ ID NO: 4 or other polypeptides of the invention can be obtained in many ways. For example, site-directed mutagenesis of the sequence of SEQ ID NO: 1 or SEQ ID NO: 3 can be performed. This is useful where, for example, silent codon changes are required by sequences to optimize the codon preferences for a a particular host cell in which polynucleotide sequences are being expressed. Other sequence changes may be desired to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides. More changes may be desirable to represent particular coding changes that are required to provide, for example, conservative substitutions. The nucleic acids of the invention may comprise additional sequences at the 5 'or 3' end. For example, synthetic or natural 5 'guide sequences can be linked to the nucleic acid encoding polypeptides of the invention. Additional sequences may also include 5 'or 3' untranslated regions required for the transcription of nucleic acid of the invention in particular host cells.

In addition, GPR39 homologs of other animals, in particular mammals (e.g., humans or rabbits), more particularly primates including human, can be obtained and used in the methods of the present invention. Said sequences can be obtained by preparing or obtaining collections of cDNAs obtained from dividing tissues or cells or collections of genomic DNA from other animal species, and probing said collections with probes comprising all or part of SEQ ID NO: 1 or SEQ ID NO. : 3 under conditions of medium to high severity (eg, 0.03 M sodium chloride and 0.03 sodium citrate at a temperature of about 50 ° C to about 60 ° C). The present invention further extends to an isolated DNA sequence comprising sequences encoding a polypeptide of the invention, but in which the coding sequences are divided into up to two or more (preferably not more than five, for example, four or three) exons. Said sequences of exons can be natural, and can be obtained from genomic, or synthetic, clones. Exon sequences can be used in the construction of minisequences of genes comprising nucleic acid encoding polypeptides of the invention, whose sequences are interrupted by one or more exon sequences. Minigenes can also be constructed using heterologous exons, derived from any eukaryotic source.

Polypeptides The isolated polypeptides used in the methods of the present invention will be those as previously defined in isolated, free or substantially free form of material with which they are naturally associated, such as other polypeptides with which they are found in the cell. The polypeptides can be formulated, in fact, with diluents or adjuvants, and even for practical purposes, they can be isolated - for example, the polypeptides will usually be mixed with gelatin or other vehicles, if they are used to coat microtiter plates for use in immunoassays. . The polypeptides can be glycosylated, either naturally or by heterologous eukaryotic cell systems, or can be (e.g., if produced by expression in a prokaryotic cell) non-glycosylated. The polypeptides can be phosphorylated and / or acetylated. A polypeptide of the invention may also be in a substantially purified form, in which case it will generally comprise the polypeptide in a preparation in which more than 90%, for example, 95%, 98% or 99% of the polypeptide in the preparation, is a polypeptide of the invention. The polypeptides of the invention can be modified, for example, by the addition of histidine residues that facilitate their purification, or by the addition of a signal sequence that promotes their secretion from a cell.

As shown in the alignment in Figure 6A and Table A, the mammalian GPR39 proteins have a general sequence identity of at least 80% with the human or mouse sequence. For this reason, polypeptides having at least 80%, for example, 90%, 95%, 96%, 97% or 98% sequence identity with SEQ ID NO: 2 or SEQ ID NO: 4, are also provided by the present invention. Such polypeptides include polypeptides which are amino acid sequence variants, alleles, derivatives or mutants of SEQ ID NO: 2 or SEQ ID NO: 4, respectively. For example, said polypeptide may have an amino acid sequence that differs from that given in SEQ ID NO: 2 or SEQ ID NO: 4, by one or more of addition, substitution, deletion and insertion of one or more (such as 1). to 20, for example 2, 3, 4 or 5 to 10) amino acids. Table A provides a matrix showing identity, similarity and spaces between each sequence. There are more spaces between the sequence of dog and cattle and the other sequences, because the first sequences are partial. For overlapping fragments, the degree of identity is given by the sum of the percent identity with the percent of spaces. For example, for dogs with humans, the degree of identity for the overlapping fragment is 86%.

TABLE A Gpr39_Mus Gpr39_Rattus GPR39_Canis GPR39_Bos GPR39 Homo 80% 79% 67% 55% Amino acids 87% 88% 74% 59% identical 0% 0% 19% 37% Similar amino acids Alignment spaces Gpr39_Mus 93% 66% 53% 96% 72% 58% 0% 19% 37% Gpr39_Rattus 65% 53% 72% 57% 19% 37% GPR39 Canis 67% 73% 21% The percent identity of polypeptide sequences can be calculated using commercially available algorithms, which compare a reference sequence (eg, SEQ ID NO: 2 or SEQ ID NO: 4 of the present invention) with a query sequence. More details to evaluate the identity, are described below. Where it is determined that a query sequence has an identity with that of SEQ ID NO: 2 or SEQ ID NO: 4 of at least 80%, and preferably at least 90%, 95%, 96%, 97% or 98%, said sequence being that of a polypeptide that retains GPR39 receptor activity, said sequence forms part of the present invention. A polypeptide according to the present invention can be isolated and / or purified (for example, using an antibody), for example, after production by expression of coding nucleic acid. He Isolated and / or purified polypeptide can be used in the formulation of a composition, which can include at least one additional component, for example, a pharmaceutical composition that includes a pharmaceutically acceptable excipient, vehicle or carrier. A polypeptide according to the present invention can be used as an immunogen, or otherwise in the production of specific antibodies. The antibodies are useful in purification and other manipulation of polypeptides, diagnostic identification and therapeutic contexts. A polypeptide according to the present invention can be used in the identification of molecules that bind to it or modulate its activity or function. Said molecules may be useful in a therapeutic context (possibly, including prophylactic context). A labeled polypeptide or polypeptide of the invention or fragment thereof can also be attached to a solid phase, for example, the surface of an immunoassay cavity or gauge rod. Said labeled and / or immobilized polypeptides can be packaged in equipment in a suitable container together with suitable reagents, controls, instructions and the like. Said polypeptides and kits can be used in methods of detecting antibodies to said polypeptides present in a sample, or active portions or fragments thereof by immunoassay.

Immunoassay methods are well known in the art, and will generally comprise: (a) providing a polypeptide comprising an epitope that can be joined by an antibody against said protein; (b) incubating a biological sample with said polypeptide under conditions that allow the formation of an antigen-antibody complex; and (c) determining whether the antigen-antibody complex comprising said polypeptide is formed.

Sequence identity The percent identity of nucleic acid sequences and polypeptides can be calculated using commercially available algorithms that compare a reference sequence with a query sequence. The following programs (provided by the National Center for Biotechnology Information) can be used to determine homologies / identities: BLAST, BLAST with interstices, BLASTN and PSI-BLAST, which can be used with predetermined parameters. The GAP algorithm (Genetics Computer Group, Madison, Wl) uses the Needleman and Wunsch algorithm to align two complete sequences, which maximizes the number of matches and minimizes the number of spaces. In general, the default parameters are used, with a penalty of creation of spaces of 12 and penalty of extension of spaces of 4. Another method to determine the best overall match between a nucleic acid sequence or a portion thereof and a query sequence, is the use of the program of FASTDB computation based on the algorithm of Brutlag et al. (Comp. App. Biosci., 6; 237-245 (1990)). The program provides a global sequence alignment. The result of said global sequence alignment is in percent identity. The appropriate parameters used in a FASTDB search of a DNA sequence to calculate the percent identity are: matrix = unit, tupie (row) k = 4, penalty for mismatch = 1, penalty for union = 30, length of the randomization group = 0, limitation score = 1, space penalty = 5, penalty for space size = 0.05 and window size = 500, or length of query sequence in nucleotide bases, whichever is shorter. Parameters suitable for calculating the percent identity and similarity of an amino acid alignment are: matrix = PAM 150, tupie (row) k = 2, penalty for mismatch = 1, penalty for union = 20, length of the randomization group = 0, limitation score = 1, space penalty = 5, penalty for space size = 0.05 and window size = 500, or length of query sequence in nucleotide bases, whichever is shorter.

Vectors The isolated nucleic acid sequences of the present invention can be incorporated into vectors, in particular expression vectors. The vector can be used to replicate the nucleic acid in a compatible host cell. Thus, in another embodiment, the invention provides a method for obtaining polynucleotides of the invention by introducing an isolated polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and growing the cell under conditions that produce the vector replication. The vector can be recovered from the host cell. Suitable host cells are described below in relation to expression vectors. Preferably, an isolated polynucleotide of the invention in a vector is operably linked to a control sequence that is capable of providing expression of the coding sequence by the host cell, ie, the vector is an expression vector. The term "operably linked" refers to a juxtaposition, wherein the described components are in a relationship that allows them to function in their desired manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Suitable vectors can be selected or constructed which contain suitable regulatory sequences, including sequences of promoter, terminator fragments, polyadenution sequences, enhancer sequences, marker genes and other sequences, as appropriate. Vectors can be plasmids, viral vectors, e.g., phage, phagemid or baculovirus, cosmids, YACs, BACs or PACs, as appropriate. Vectors include gene therapy vectors, for example, vectors based on adenovirus, adeno-associated virus, retrovirus (such as HIV or MLV) or alpha virus vectors. The vectors can be provided with an origin of replication, optionally a promoter for the expression of said polynucleotide, and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes, for example, an ampicillin resistance gene in the case of a bacterial plasmid, or a neomycin resistance gene for a mammalian vector. In vitro vectors can be used, for example, for the production of RNA, or they can be used to transfect or transform a host cell. The vector can also be adapted for use in vivo, for example, in gene therapy methods. Systems for the cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast cells, and baculovirus systems. Mammalian cell lines available in the art for the expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, hamster brood kidney cells, COS cells, and many others.

Promoters and other expression regulation signals can be selected to be compatible with the host cell for which the expression vector is designed. For example, yeast promoters include promoters GAL4 and ADH of S. cerevisiae, and promoters nmtl and adh of S. pombe. Mammalian promoters include the metallothionein promoter that can be induced in response to heavy metals such as cadmium. Viral promoters such as the promoter of SV40 large T antigen or adenovirus promoters can also be used. All of these promoters are readily available in the art. The vectors may include other sequences, such as promoters or enhancers that direct expression of the inserted nucleic acid sequences, so that the polypeptide is produced as a fusion and / or nucleic acid encoding secretion signals, so that the polypeptide produced in the host cell is secreted from the cell. Vectors for the production of polypeptides of the invention for use in gene therapy, include vectors possessing a minigene sequence of the invention. Accordingly, an object of the present invention is to provide a method for treating abnormal conditions related to an underpressure of GPCR GPR39 and its activity, said method comprising the use of a polynucleotide encoding a GPCR GPR39, in particular, in a method for treat impaired carbohydrate metabolism, such as diabetes including complications associated with it. In gene therapy, a polynucleotide isolated from the invention is used to effect the endogenous production of GPCR GPR39 by the relevant cells in the subject. For example, a polynucleotide encoding a GPCR GPR39 can be designed for expression in a replication-defective retroviral vector as provided above. The retroviral expression construct can then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention, so that the packaging cell now produces infectious viral particles containing the gene of interest. These producer cells can be administered to a subject for the design of cells in vivo and expression of the polypeptide in vivo. For a review of gene therapy, see chapter 20 of Gene Therapy and other Molecular Genetic-based Therapeutic Approaches, in Human Molecular Genetics, T. Strachan and A. P. Read, BIOS Scientific Publishers Ltd. (1996). For more details see, for example, Molecular Cloning: a Laboratory Manual: 2a. edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for the manipulation of nucleic acid, for example, in the preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression and protein analysis, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley & Sons, 1992.

The vectors can be transformed into a suitable host cell as described above, to provide for the expression of a polypeptide of the invention. Thus, in another aspect, the invention provides a method for preparing polypeptides according to the invention, which comprises culturing a host cell transformed or transfected with an expression vector as described above, under conditions that provide expression by the vector of a coding sequence coding for the polypeptides, and recovering the expressed polypeptides. The polypeptides can also be systems expressed in vitro, such as reticulocyte lysate. Another embodiment of the invention provides host cells transformed or transfected with the vectors for the replication and expression of the polynucleotides of the invention. The cells will be selected to be compatible with said vector and may be, for example, bacteria, yeast, insects or mammals. The host cells can be cultured under conditions for gene expression, so that the encoded polypeptide is produced. If the polypeptide is expressed coupled to a suitable signal guide peptide, it can be secreted from the cell in the culture medium. After production by expression, a polypeptide can be isolated and / or purified from the host cell and / or culture medium, as the case may be, and then used as desired, for example, in the formulation of a composition that may include one or more components additional, such as a pharmaceutical composition that includes one or more pharmaceutically acceptable excipients, carriers or carriers. The polynucleotides according to the invention can also be inserted into the vectors described above in an antisense orientation, to provide the production of antisense RNA or ribozymes.

Tests It is an object of the present invention to provide a test for identifying compounds that improve the control of glucose in a subject and that are effective in preventing and / or treating pathologies related to a deteriorated carbohydrate metabolism, in particular in the prevention and / or the treatment of diabetes, including associated complications thereof, or of the metabolic syndrome, including associated complications thereof, which test comprises: providing all or part of a GPR39 receptor protein according to the invention, contacting said protein with a putative binding compound; and to determine if said compound is capable of interacting with said protein. As is known for GPCRs that can interact with G proteins, as well as with other accessory proteins, the tests as provided herein may comprise in addition to all or part of the GPR39 receptor protein, G proteins or other accessory proteins. GPCRs are known to interact with G proteins, as well as with other accessory proteins that include arrestins, proteins receptor activity modifiers (RAMPs), proteins that contain PDZ domain, and others. The interaction of a GPCR with said accessory protein may result in the determination of several important biological properties of a receptor, such as transport to or from the membrane, definition of its pharmacology, determination of its glycosylation status or signal transmission through of mechanisms that function independently of G protein coupling (Laburthe et al., 2002). As an example, the association of the calcitonin receptor-like receptor with RAMP1, a specific individual transmembrane protein, leads to the expression of the calcitonin receptor-like receptor capable of binding to the peptide related to the calcitonin gene, while the association with RAMP2 or RAMP3 leads to expression as an adrenomedullin binding receptor (Latchie et al., 1998). A similar interaction of RAMPs for VPAC1 receivers has been documented; glucagon, PTH1 and PHT2 (Christopoulos et al., 2001, 2003), suggesting a more generalized function for accessory proteins in the modulation of the signaling of G-protein coupled receptors. In a test modality, the receptor or subunits of the receptor they can be used in a binding test. Binding tests can be competitive or non-competitive. Such a test can accommodate the rapid identification of a large number of compounds to determine which compounds, if any, are capable of binding to the polypeptides. Then, more detailed tests can be carried out with those compounds that are found to bind, in order to further determine if said compounds act as agonists or antagonists of the polypeptides of the invention. Accordingly, it is an object of the present invention to provide a method for identifying a compound that improves the regulation of glucose in a subject, and which is effective to prevent and / or treat pathologies related to a deteriorated metabolism of carbohydrates, in particular in the prevention and / or treatment of diabetes, including associated complications thereof, or the metabolic syndrome, including associated complications thereof, which method comprises: (i) contacting host cells that express all or part of the receptor protein GPR39 according to the invention, with said compound under conditions suitable for binding, and (ii) detecting the binding of the compound to said receptor protein. In another embodiment, this invention provides a method for identifying a compound that improves the regulation of glucose in a subject, and which is effective to prevent and / or treat pathologies related to a deteriorated carbohydrate metabolism, in particular in the prevention and / or or the treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof, which method comprises: (i) contacting membrane preparations of host cells that express all or part of the receptor protein GPR39 according to the invention, with said compound under conditions suitable for binding, and (ii) detecting the binding of the compound to said receptor protein. In another embodiment, the present invention provides a method for identifying a compound that improves the regulation of glucose in a subject, and which is effective to prevent and / or treat pathologies related to a deteriorated metabolism of carbohydrates, in particular in the prevention and / or the treatment of diabetes, including associated complications thereof, or the metabolic syndrome, including associated complications, whose method involves a competitive binding test wherein: (i) host cells that express all or part of the protein receptor GPR39 according to the invention, are contacted with a compound known to bind to the GPR39 receptor protein in the presence and absence of the compound to be tested, and (ii) the effect of said compound on the binding of the compound known to bind to the GPR39 receptor protein. A decrease in the binding of the compound known to bind to the GPR39 receptor protein in the presence of the compound to be tested is an indication that said compound binds to the GPR39 receptor protein. Obestatin, the natural ligand for the GPR39 receptor protein, has recently been identified (Zhang, J.V. et al., 2004 Science Vol. 310; 996-999) as another peptide derived from the same prohormone as ghrelin. In a particular embodiment, the compound known to bind to the receptor consists of obestatin, more in particular selected from one of the obestatin sequences selected from SEQ ID NOs: 5 to 16, more in particular SEQ ID NO: 5 or SEQ ID NO: 7. Alternatively, obestatin as used herein, refers to a peptide having at least 60%, 70%, 80%, 90%, 95% , 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 5 or SEQ ID NO: 7. In an alternative embodiment, the competitive binding test mentioned above is carried out in membrane preparations of host cells that express all or part of the GPR39 receptor protein according to the invention. Methods for preparing said host cells and for obtaining membrane preparations of said cells are described below. In a specific embodiment, the GPR39 receptor protein in the binding tests mentioned above, is an isolated protein having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a splice variant of the proteins having the SEQ ID's mentioned above, and an amino acid sequence having at least 80% and preferably at least 90%, 95%, 96%, 97 %, 98% or 99% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Parts of the GPR39 protein, as the term was used above, means that it includes fragments of the SEQ polypeptide ID NO: 2 or SEQ ID NO: 4, said fragments being of at least 0, for example, at least 20, 30, 40, 50, 75, 100 or 150 or more amino acids in size. Such fragments can be derived from the N-terminal region of SEQ ID NO: 2 or SEQ ID NO: 4, respectively. Fragments that include the N-terminal region can be used to reconstitute the extracellular portion of the receptor to provide receptor binding sites. Preferably, the fragments will retain the ability to bind to the compound known to bind to the GPR39 receptor protein. More particularly, the fragments will retain the ability to bind obestatin as previously defined herein.

Membrane Preparations Cell membranes expressing the receptor protein of this invention are useful for certain types of tests including, but not limited to, ligand binding tests, GTP-and-S binding tests, and others. The details of preparation of said cell membranes can be determined in some cases by the nature of the resulting test, but typically involve harvesting whole cells and dissolving the cell, for example, by sonication in an ice-cooled pH regulator (e.g., Tris. 20 mM HCl, 1 mM EDTA, pH 7.4 at 4 ° C). The resulting crude cell lysate is subsequently cleared of cell debris by low speed centrifugation, for example, at 200 xg for 5 minutes at 4 ° C. Further purification and membrane enrichment are finally done using a high speed centrifugation step such as, for example, 40,000 xg for 20 minutes at 4 ° C, and the resulting membrane pellet is washed by suspension in pH regulator cooled in ice and repeating the step of high speed centrifugation. The final washed membrane pellet is resuspended in the test pH regulator. Protein concentrations are determined by the method of Bradford (1976) using bovine serum albumin as a standard. The membranes can be used immediately, or they can be frozen for later use.

Radiolabelled ligand binding assays Cells expressing the receptor of this invention can be used to identify ligands for said receptor. The same tests can be used to identify receptor agonists or antagonists that can be used for a variety of therapeutic purposes. Radioligand binding tests are performed by diluting membranes prepared from cells expressing the receptor in a suitable pH regulator such as, for example, 50 mM Tris pH buffer (pH = 7.4 at 0 ° C) containing bovine serum albumin. at 2.1% (Sigma), aprotinin (0.005 mg / ml, Boehringer Mannheim) and obestatin (01. mM, Sigma) as protease inhibitors. The final protein concentration in the test is typically within 12 to 40 pg / ml. The membranes are then incubated with radiolabeled ligand, either in the presence or absence of competent ligands, on ice for 60 minutes in a total volume of 96-well microtiter plates of 250 μ ?. The bound ligand is then separated from the free ligands by filtration through pre-soaked GF / B filters in polyethyleneimine (PEI) at 0.5%, using a Tomtec vacuum filtration device (Wallac). After the addition of Ready Safe (Beckman) scintillation fluid, the bound radioactivity is quantified using a Trilux scintillation counter (Wallac) (approximately 40% counting efficiency of counting counts). Alternatively, it may be preferable to collect the bound ligand, and then remove the ligand from the receptor using procedures well known in the art. The data is adjusted to non-linear curves using software to adjust the curve, such as GraphPad prism. In this manner, agonist or antagonist compounds that bind to the receptor can be identified, since they inhibit the binding of the radiolabeled ligand to the membrane protein of cells expressing said receptor. Non-specific binding is defined as the amount of radioactivity remaining after incubation of the membrane protein in the presence of 100 nM of the unlabeled peptide corresponding to the radioligand used. In equilibrium saturation binding tests, membrane preparations or intact cells transfected with the receptor are incubated in the presence of increasing concentrations of the labeled compound to determine the binding affinity of the labeled ligand. The binding affinities of the unlabeled compounds can be determined in competitive binding assays at equilibrium, using a fixed concentration of labeled compound in the presence of varying concentrations of the displacement ligands. In a particular modality, the radioligand binding test mentioned above is carried out using radiolabeled obestatin as defined hereinabove. The labeling of obestatin and receptor binding have been described in Zhang, J. V. ef al. (2004 Science Vol. 310; 996-999). In summary: iodostatin was iodinated, using the lodogen procedure (Pierce, Upland, IN). Mixtures of the peptide (20 pg) and 1 mCi [125 l] of Nal were transferred to precoated lodogen vials, and incubated for 4 minutes. The 25l-labeled peptide was applied to a Sep-Pak C18 cartridge (Waters) before elution with 60% acetonitrile / 0.1% TFA. For radioligand binding tests, rat jejunum or other tissues were washed with pH A regulator (20 mM Hepes, 5 mM EDTA, 1 mM dithiothreitol (DTT), 10 μM amidinophenyl methanesulfonyl fluoride, 5 mg / L leupeptin, 100 mM KCl, pH 7.5), were cut into small pieces, and homogenized using a motorized homogenizer. The homogenates were centrifuged at 1,000 g for 5 minutes, and the supernatant was centrifuged at 300,000 g for 1 hour at 2 ° C. The pellets (crude membrane fractions) were resuspended with pH A regulator without KCI, quickly frozen under liquid nitrogen, and stored at -80 ° C until use. Tissue homogenates were incubated in 100 μ? of saline solution regulated in its pH with phosphate containing bovine serum albumin at 0.1% for 18 hours at room temperature with variable concentrations of obestatin-125! in the presence or absence of obestatin not marked to excess of, 000 times. After incubation, the tubes were centrifuged for 10 minutes at 10,000 g, and the pellets were washed twice in PBS cooled on ice before quantification of radioactivity with a gamma spectrophotometer. The specific binding was calculated by subtracting the non-specific binding from the total binding. For displacement curves, a fixed concentration of obestatin-125! it was incubated with or without increasing concentrations of obestatin or other peptides. In addition to the binding tests mentioned above, it is also an object of the present invention to provide functional tests for identifying compounds that improve the control of glucose in a subject, characterized in that said compounds modulate the activity of the GPR39 receptor proteins in accordance with the invention. Said test comprises the steps of: (i) contacting all or parts of the GPR39 receptor protein with the compound to be tested, and (ii) measuring the activity of the GPR39 receptor protein, wherein the change of activity of GPR39 in the presence of the test compound, is an indication of the ability of the compounds to modulate carbohydrate metabolism. Given the observed involvement of GPR39 in carbohydrate metabolism, in all the described modalities that identify compounds that bind to GPR39 and / or modulate GPR39 activity, such tests can be used to identify compounds that can affect glucose homeostasis. in a subject, including humans and warm-blooded animals.

A compound that modulates the activity of a polypeptide of the invention, refers to a compound that alters the activity of the polypeptide, so that it behaves differently in the presence of the compound than in the absence of the compound. Compounds that affect modulation include agonists and antagonists. An agonist encompasses a compound that activates the function of GPCR GPR39. Alternatively, an antagonist includes a compound that interferes with the function of GPCR GPR39. Typically, the effect of an antagonist is observed as a blockade of receptor activation induced by the agonist; however, in the case of GPR39, which has recently been described as a constitutive active receptor, compounds that interfere with GPCR GPR39 function also include inverse agonists, i.e., agents that bind to the same receptor binding site as an agonist for that receptor, but that exerts the opposite pharmacological effect. Antagonists include competitive antagonists, as well as non-competitive antagonists. A competitive antagonist (or competitive blocker) interacts with or near the specific site for agonist binding. A non-competitive antagonist or blocker inactivates receptor function by interacting with a site different from the site of agonist interaction. It is in this way an object of the present invention to provide a method for identifying compounds capable of improving the control of glucose in a subject, said method comprising: (i) contacting host cells expressing the all or part of the GPR39 receptor protein with the compound to be tested, under conditions that allow the activation of said receptor protein, and (ii) detect any increase in the activity of the GPR39 receptor, thereby identifying the compound of test as a compound capable of improving the control of glucose in a subject. In another embodiment, the present invention provides a method for identifying compounds capable of decreasing the activity of GPR39, said method comprising: (i) contacting host cells that express all or part of the GPR39 receptor protein with the compound that is leaving to be tested, under conditions that allow activation of said receptor protein, and (ii) to detect any decrease in GPR39 receptor activity, thereby identifying the test compound as a compound capable of normalizing glucose tolerance in a human or animal, i.e., identifying the test compound as useful in the treatment of diabetes, including associated complications thereof. Again, as for the binding tests mentioned hereinabove, the activity tests mentioned above can also be used to identify compounds that would modulate carbohydrate metabolism. Based on the phenotype observed in the glucose tolerance test, it is expected that the compounds that increase the activity of GPR39 result in a decrease in blood glucose tolerance, and compounds that decrease the activity of GPR39 result in an increase in blood glucose tolerance. In a particular embodiment, the present invention provides a method for identifying compounds capable of decreasing blood glucose tolerance, said method comprising: (i) contacting host cells that express all or part of the GPR39 receptor protein with the compound to be tested, under conditions that allow activation of said receptor protein, and (ii) detect any increase in GPR39 receptor activity, thereby identifying the test compound as a compound capable of decreasing tolerance to blood glucose. The effect of the compounds on the activity of GPR39 may be an increase or decrease in the formation of an intracellular second messenger that is mediated by GPR39, such as intracellular calcium, cAMP or a reporter gene product. GPR39 belongs to the class of proteins known as G-protein coupled receptors (GPCRs). GPCRs transmit signals through cell membranes after ligand binding. GPCRs bound to the ligand activate intracellular signaling events mediated by heterotrimeric G proteins, such as activation of the cyclasy adenylate pathway or activation of the phospholipase C-β pathway. Tests to evaluate the activation of intracellular signaling events mentioned above are generally known in the art, and include among others, cell-based tests for signal transduction comprising chimeric transcription factors inducible by the ligand, binding tests for G protein-coupled receptors using intensity distribution analysis of fluorescence, cell signaling pathways using cyclic nucleotides coupled to luminophores or measurement of G protein-coupled receptor responses using a multiple response element or reporter test directed to cAMP response element. The description of several such tests is given below. The present invention also provides a bioassay for identifying compounds that modulate the regulatory regions of the GPCR GPR39 gene. Said test is carried out using rat or human cells capable of expressing a polypeptide of the invention (preferably of SEQ ID NO: 2 or SEQ ID NO: 4). The cells are contacted with at least one compound wherein the ability of said compound to modulate the regulatory region is known. After, the cells are monitored for the expression of the nucleic acid of the invention. Alternatively, the promoter can be linked to a reporter gene. Suitable reporter genes that can be used include, for example, the chloramphenicol acetyltransferase gene, the luciferase gene, and the like. As understood by those skilled in the art, bioassay methods for identifying compounds that modulate the activity of receptors such as the polypeptides of the invention generally require comparison with a control. One type of control is a cell or culture that is treated substantially the same as the test cell or test culture exposed to the compound, with the distinction that the cell or control culture is not exposed to the compound. Another type of control cell or culture that can be used is a cell or culture that is identical to the transfected cells, the exception being that the control cell or culture does not express functional GPR39 GPCR. Accordingly, the response of the transfected cell can be compared with that of the cell or control culture for the same compound under the same reaction conditions. Particularly preferred types of tests, include binding tests and functional tests that can be performed as follows: Binding assays Nucleic acid overexpression coding for the polypeptides of the invention can be used in cell lines (including HEK 293 mammalian cells, CHO cells and insect Sf9 cells), to produce membrane preparations possessing said polypeptides (referred to in this section as GPCR GPR39 for convenience) for ligand binding studies. These membrane preparations can be used in conventional filter-binding tests (eg, using Brandel filter test equipment), or in high-throughput scintillation proximity type binding tests (SPA technology and Cytostar-T flashplate; Amersham Pharmacia Biotech ), to detect the binding of the radiolabeled ligand and the displacement of said radioligands by competitors at the binding site. Radioactivity can be measured with a Packard Topcount, or similar instrumentation, capable of making rapid measurements of 96, 384 or 1536 microtiter cavity formats. The SPA / Cytostar-T technology is particularly subject to high performance identification, and therefore this technology is suitable for use as a sieve for compounds capable of displacing standard ligands. Another method to study the binding of ligands to GPCR protein GPR39 in an environment that approximates the native situation, makes use of a surface plasmon resonance effect exploited by the Biacore instrument (Malmqvist M., Biochem Soc Trans. 1999, 27 (2): 335-40). GPCR GPR39 in whole cell or membrane preparations could be linked to the biosensor chip of a Biacore, and ligand binding in the presence and absence of compounds could be examined to identify competitors of the binding site.

Functional tests Since GPCR GPR39 acts by means of Gi or Go (inhibitory protein G), which usually interacts with GIRK (potassium channels of inward rectification), the flow of potassium ions must result in the activation of these receptors. This ion flux can be measured in real time, using a variety of techniques that determine the agonist or antagonist effects of particular compounds. Therefore, GPCR receivers Recombinant GPR39 expressed in cell lines or, for example, Xenopus oocytes, can be characterized using the electrophysiology of individual channels and whole cells to determine the mechanism of action of compounds of interest. Electrophysiological identification can be performed for active compounds in GPCR GPR39, using conventional electrophysiological techniques, and when they become available, novel high performance methods currently under development.

Fluorescence - Calcium and sodium fluxes are measurable using various fluorescent ion-sensitive dyes, including fluo-3, fluo-4, fluo-5N, fura red, sodium green, SBFI and other similar probes from suppliers that include Molecular Probes. The influx of calcium and sodium as a result of GPCR GPR39 can be characterized in such a way in real time, using fluorometric and fluorescence imaging techniques, including fluorescence microscopy with or without confocal laser methods combined with algorithms for image analysis. Another method is a high throughput identification test for active compounds such as agonists or modulators that affect calcium transit. This test is based on an instrument called a fluorescence imaging plate reader (FLIPR®), Molecular Devices Corporation). In its most common configuration, it excites and measures the fluorescence emitted by fluorescein-based dyes.

Uses an argon ion laser to produce high-power excitation at 488 nm of a fluorophore, an optical system that scans quickly on the bottom of a 96/384-well plate, and a sensitive cooled CCD camera that captures fluorescence issued. It also contains a 96/384 cavity pipetting head that allows the instrument to provide test agent solutions in the cavities of a 96/384 cavity plate. The FLIPR test is designed to measure fluorescence signals of cell populations before, during and after the addition of compounds, in real time, of all 96/384 cavities simultaneously. The FLIPR test can be used to identify and characterize functionally active compounds in the recombinant GPR39 GPCR, for example, rat or human GPCR GPR39, expressed in cell lines. As described below, the calcium transit was measured in cells transfected with the GPCR GPR39 using the FLIPR test which measures the activation of the receptors by various substrates to determine the natural ligand of the receptor.

Cyclic AMP test (cAMP) - The stimulation or inhibition of cyclic AMP formation (cAMP) mediated by the receptor can be tested in cells expressing the receptor. An example of a protocol for a cAMP test is provided below. In this protocol, COS-7 cells are transiently transfected with the receptor gene using the DEAE-dextran method, and sown in plates of 96 cavities. 48 hours after transfection, cells are washed twice with saline buffered at pH with Dulbecco's phosphate (PBS) supplemented with 10 mM HEPES, 10 mM glucose and 5 mM theophylline, and incubated in the same pH buffer for 20 minutes at 37 ° C, in CO5 at 5%. Test compounds are added, and the cells are incubated for another 10 minutes at 37 ° C. The medium is then aspirated, and the reaction is interrupted by the addition of 100 mM HCl. The plates are stored at -20 ° C for 2 to 5 days for the measurement of cAMP, the plates are thawed, and the cAMP content in each well is measured by means of the cAMP scintillation proximity test (Amersham Pharmacia Biotech ). The radioactivity is quantified using a Trilux microbead counter (Wallac).

Microphysiometric testing - Because cellular metabolism is intricately involved in a wide range of cellular events (including the activation of multiple messenger pathways by the receptor), the use of microphysiometric measurements of cellular metabolism can provide a generic test of cellular activity that arises from the activation of any orphan receptor, despite the details of the receptor signaling pathway. General guidelines for the expression of transient receptors, cell preparation and microphysiometric recording are described elsewhere (Salon, J. A. and Cwicki, J. A., 1996). Typically, cells that express Receivers are harvested and planted at 3 x 105 cells per microphysiometer capsule, in complete medium 24 hours before an experiment. The medium is replaced with serum-free medium 16 hours before registration, to minimize non-specific metabolic stimulation, by poorly defined and distributed serum factors. On the day of the experiment, the cell capsules are transferred to the microphysiometer, and allowed to reach equilibrium in the recording medium (medium 1640 of the RPMI low pH regulator content, without bicarbonate, without serum (Molecular Devices Corporation, Sunnyvale, CA) containing BSA at 0.1 to 1% free of fatty acids), during which a basal metabolic activity measurement is established at the starting point. A standard recording protocol specifies a flow magnitude of 100 μm / min, with a total pump cycle of 2 minutes that includes a flow interruption for 30 seconds during which the rare measurement of acidification is made. The challenges of the ligand imply an exposure for 1 minute 20 seconds to the sample shortly before the first measurement of the post-challenge magnitude is made, followed by two additional pumping cycles for a total of 5 minutes 20 seconds of exposure to the sample. Typically, drugs in a primary screen are presented to the cells at a final concentration of 10 μ ?. Follow-up experiments are then performed to examine the dependence of active compounds per dose, sequentially challenging the cells with a drug concentration scale that exceed the amount needed to generate responses that vary from the threshold to maximum levels. The ligand samples are then depleted, and the reported acidification rates are expressed as a percentage of increase in the peak response over the velocity at the starting point observed shortly before the challenge. The compounds that have been found to modulate the activity of the polypeptides of the present invention have many therapeutic uses, based on the finding that GPR39 is involved in the regulation of carbohydrate metabolism. Specifically, this includes diseases related to increased blood glucose levels such as, for example, diabetes (including associated complications thereof), which includes type 1 diabetes (insulin dependent diabetes mellitus or IDDM), type 2 (non-insulin dependent diabetes mellitus) , juvenile onset diabetes at maturity (MODY) and gestational diabetes. In summary, GPCR GPR39 and related receptors can be used as a valuable tool for drug development in a variety of therapeutic areas.

Binder Agents Thus, the invention further provides novel binder agents, including modulating agents obtained by a test according to the present invention, and compositions comprising said agents. Agents that bind to the receptor and that may have agonist or antagonist activity can be used in methods to treat the diseases characterized above, whose pathology is characterized by the action by means of the GPCR GPR39 receptor, and said use forms another aspect of the invention. The agents can be administered as an effective amount of an agent of the invention. Since many of the conditions mentioned above are chronic and often incurable, it will be understood that "treatment" is intended to include the achievement of a reduction in symptoms for a period such as a few hours, days or weeks, and progression of the course of the disease. Such agents can be formulated in compositions comprising an agent together with a pharmaceutically acceptable carrier or diluent. The agent may be in the form of a physiologically functional derivative, such as an ester or a salt, such as an acid addition salt or basic metal salt, or an N- or S-oxide. The compositions can be formulated for any suitable route or means of administration. Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal, inhalable, topical (including buccal and sublingual), vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural administration). The choice of vehicle or diluent will, in fact, depend on the proposed route of administration, which may depend on the agent and its therapeutic purpose. The formulations can be conveniently presented in unit dosage form, and can be prepared by any of the methods well known in the pharmacy art. Said methods include the step of bringing the active ingredient into association with the vehicle constituting one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. For solid compositions, conventional non-toxic solid carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharine, talc, glucose, sucrose, magnesium carbonate, and similar. The active compound as defined above can be formulated as suppositories using, for example, polyalkylene glycols, acetylated triglycerides, and the like, as the carrier. Pharmaceutically administrable liquid compositions can be prepared, for example, by dissolving, dispersing, etc., an active compound as defined above, and optional pharmaceutical adjuvants in a vehicle such as, for example, water, aqueous saline dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH regulating agents, and the like, for example, sodium acetate, sorbitan monolaurate, acetate sodium triethanolamine, triethanolamine oleate, etc. Real methods to prepare said dosage forms are known, or will be apparent, to those skilled in the art; see, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania, Fifteenth Edition, 1975. The composition or formulation to be administered will, in any case, contain an amount of the active compounds in an effective amount to alleviate the symptoms of the subject being treated. Dosage forms or compositions containing the active ingredient can be prepared on the 0.25 to 95% scale, the remainder being non-toxic vehicle. For oral administration, a non-toxic pharmaceutically acceptable composition is formed by the incorporation of any of the excipients normally used such as, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, croscarmellose sodium, starch, magnesium stearate , sodium saccharin, talc, glucose, sucrose, magnesium, carbonate, and the like. Said compositions may take the form of solutions, suspensions, tablets, pills, capsules, powders, sustained release formulations, and the like. Said compositions may contain from 1% to 95% active ingredient, more preferably from 2 to 50%, most preferably from 5 to 8%. Parenteral administration is generally characterized by injection, either subcutaneously, intramuscularly or intravenously. Injectable formulations can be prepared in conventional ways, either as suspensions or liquid solutions, solid forms suitable for solution or suspension in liquid before injection, or as emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, pH regulating agents, and the like such as, for example, sodium acetate, monolaurate of sorbitan, triethanolamine oleate, triethanolamine sodium acetate, etc. The percentage of active compound contained in said parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject. However, percentages of active ingredient from 0.1% to 10% in solution are usable, and will be higher if the composition is a solid which will be subsequently diluted to the above percentages. Preferably, the composition will comprise from 0.2 to 2% of the active agent in solution. Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the treatment of impaired metabolism of carbohydrates in a subject such as, for example, diabetes (including associated complications thereof), including type 1 diabetes (insulin-dependent diabetes mellitus or IDDM), type 2 (non-insulin-dependent diabetes mellitus), juvenile onset diabetes at maturity (MODY) and gestational diabetes, said composition comprising an antagonist of the GPR39 receiver.

Diagnostic Product The present invention also provides a diagnostic product comprising an isolated nucleic acid sequence selected from the group consisting of: (i) a nucleic acid sequence encoding all or part of the polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4, (ii) a nucleic acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a nucleic acid sequence having at least 80% identity of sequence, and preferably at least 90%, 95%, 96%, 97% or 98% sequence identity, with the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3. By using the DNA, antisense DNA or siRNA of the present invention as probes, a gene abnormality of the DNA or messenger RNA encoding the GPR39 receptor or part thereof can be detected in mammals including humans, and therefore these nucleic acid sequences are diagnosis of useful genes for damage of the DNA or messenger RNA encoding the GPR39 receptor or part thereof, mutations thereof, subexpression thereof or overexpression thereof. The gene diagnosis described above can be performed, for example, by means of the Northern hybridization test publicly known or PCR-SSCP test. When underexpression of the GPR39 receptor is detected, it can be diagnosed that the subject suffers from a disease associated with GPR39 receptor dysfunction such as, for example, metabolic syndrome, including associated complications thereof. When overexpression of the GPR39 receptor is detected, it can be diagnosed that the subject suffers from a disease associated with overexpression of the GPR39 receptor such as, for example, diabetes, including associated complications thereof. The present invention also provides a diagnostic product comprising all or part of the GPR39 receptor protein. Said protein can be detected, for example, in a suitable sample, for example, a blood sample, to diagnose subexpression or overexpression of the GPR39 receptor. This invention will be better understood by reference to the following experimental details, but those skilled in the art will readily appreciate that these are only illustrative of the invention as more fully described in the claims below. In addition, several publications are cited throughout this application. The description of these publications is incorporated in this manner in this application as a reference to more fully describe the state of the art to which this invention belongs.

EXAMPLES The following examples illustrate the invention. Other embodiments will occur to those skilled in the art in light of these examples.

Materials and methods Mice with blocked expression of GPR39 Mice with blocked expression of GPR39 were obtained from Lexicon Genetics Inc. The dissolution of the open reading frame of GPR39 was achieved, replacing the coding region of the first coding exon of the GPR39 gene with a selection cassette / gene Reporter LacZ / MC1-Neo with IRES. The animals were kept in an SPF facility that met all the Belgian and European requirements for animal care. The mice were housed in groups in a climate controlled animal colony with a 12-hour light-dark cycle (light at 7:00 am Eastern Standard Time), with free access to food and water, unless It will be indicated in another way. Appropriate measures were taken to minimize pain or discomfort. All the experiments were carried out in agreement with the European Communities Council Directives (86/609 / EEC), and were approved by the local ethics committee.

Histological analysis of GPR39 expression in the pancreas The pancreas of WT (wild type) and GPR39"mice was dissected, and LacZ staining was performed on a whole slide, In brief, tissue was dissected in PBS solution on ice. and was fixed for 2 hours at 4 ° C in glutaraldehyde at 0.5% in PBS.Then the tissue was rinsed 3 times in PBS with gentle shaking.Stained overnight at 30 ° C in a pH regulator staining supplemented with 1 mg / ml of X-gal, 5 mM potassium ferricyanide and 5 mM potassium ferrocyanide After staining, the tissue was rinsed in the pH buffer of PBS wash, dehydrated in alcohol, clarified in xylol embedded in paraffin, and sectioned at 5 μ. Histological examination was performed on 10 μm tissue sections with and without counterstain with Harris haematoxylin and eosin.

Comparison of body weight in WT and GPR39 mice The animals were individually housed in cages at 22 ± 1 ° C with a cycle of 12 hours-12 hours of light-dark. Animals were fed ad libitum with a standard diet (22% protein, 4.3% fat and 4% cellulose from U.A.R., France). Body weight was followed in these animals starting at age 8 to 12 to 16 to 20 weeks.

Glucose tolerance test in WT and GPR39 mice GPR39"and" and WT mice (male and female) were fasted overnight (starting at 5:30 p.m.), and then injected subcutaneously to them with 2 g / kg of glucose (saline to a final concentration of 200 mg / ml). Blood samples were taken by venesection of the tail. First, the starting point was taken before the animals were weighed, and then glucose was administered. The glucose concentration in blood was determined at 15, 30, 45, 60, 90 and 120 minutes after glucose administration, by means of the One Touch Ultra instrument (Lifescan Inc., USA).

Comparison of body composition of WT and GPR39 mice - Body fat and body muscle mass were measured in GPR39"7" and conscious wild type mice, between 9.00 and 11.00 a.m., by quantitative nuclear magnetic resonance (NMR) (NMR analyzer Minispec mq10, Bruker, Germany). This is a method developed especially for the rapid non-invasive determination of body composition in conscious mice. The lean body, body fat and fluid were quantified by NMR. The animals were weighed before the NMR measurement. Results Histological analysis of the expression of GPR39 in the pancreas In the GPR39"'" mice at hand, the coding region of the first coding exon is replaced by the LacZ reporter gene; for the therefore, LacZ expression was examined as an indicator of the endogenous expression pattern of GPR39 in the pancreas. In the pancreas, the expression was detected by all the islets of Langerhans, the endocrine portion of the pancreas in GPR39"7" mice (figure 1A), but not in wild-type mice (figure 1B).

Comparison of body weight in WT and GPR39 mice ~ '~ Body weight was followed in these animals, starting at the age of 8 to 12 weeks up to 16 to 20 weeks. The male GPR39"'" mice gained more weight than the corresponding male wild type mice (Figure 2). No difference was observed in these female mice in this age scale.

Glucose tolerance test in WT and GPR39 mice After a 16-hour fasting period, blood glucose was not significantly changed in GPR39"'" mice (males + females) compared to wild-type mice (males + females) (from 12 to 16 weeks). Also, no difference in blood glucose between both genotypes was observed in the feeding condition (Figures 3A-3B). After glucose administration, blood glucose was significantly less increased in male GPR39"'" mice compared to male wild type mice (12 to 16 weeks) (P <0.05). In female mice, no difference was observed between both genotypes (12 to 16 weeks) (figure 4).

Comparison of body composition of mice WT and GPR39-y- Body composition was measured in mice at the age of 13 to 17 and 16 to 20 weeks. In mice from 13 to 16 weeks, no difference was observed in the composition of the body between both genotypes fed standard diet. Three weeks later, in the same animals, body fat in male GPR39"and" mice appeared to increase compared to male wild type mice (15.76 ± 5.30% vs. 1.97 ± 2.60%), while lean body mass seemed decrease in male GPR39"and" mice compared to wild-type mice (41.22 ± 3.41% vs. 43.45 ± 1.78%) (Figures 5A-5B).

Discussion Based on the first findings that the GPR39 receptor is highly expressed in pancreatic tissue, together with the results of the phenotypic analysis of GPR39"and ~ mice showing increased levels of cholesterol in both sexes of mice with blocked expression of GPR39, compared to wild-type baits (unpublished data), a possible role for the GPR39 receptor in glycemic control should be investigated, confirming the implication of GPR39 in the Control of blood glucose would validate this receptor as a good target to treat diabetic diseases that include complications of these, and all diseases related to the metabolic syndrome. For these reasons, the purpose of the present work was to first locate the GPR39 receptor in the tissue of the pancreas using the lacZ expression technique, and then to test the GPR39"'" mice against wild type mice in the tolerance test. the glucose Based on these results, the determination of insulin concentration before and after glucose administration in the glucose tolerance test will be performed, to confirm the possible function of GPR39 in the control of blood glucose. In parallel to all these studies, the composition of the body was also determined using an NMR device. Here again, depending on the preliminary results, it is possible that the metabolism of GPR39"'" mice compared to wild-type mice, in a cage structure with standard metabolism, may be investigated.

REFERENCES Bradford, M. M., A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976; 72: 248-254. Hoist, B., Holliday, N. D., Bach, A., Elling C. E., Coc, H. M. and Schwartz, T. W., Common structural basis for constitutive activity of the Ghrelin receiver family. J. Biol. Chem. 2004, 279 (51): 53806-53817. Date Y, Nakazato M, Murakami N, Kojima M, Kanga a K, Matsukura S. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun. Jan 26, 2001; 280 (3): 904-7. Ghoos Y, Maes B, Geypens B, Mys G, Hiele M, Rutgeerts P and Vantrappen G. Measurement of gastric emptying rate of solids by means of a carbon labeled octanoic acid breath test. Gastroenterol. 1993; 104: 1640-7. Inui A, Asakawa A, Bowers CY, Mantovani G, Laviano A, Meguid MM, Fujimiya M. Ghrelin, appetite, and gastric motility: the emerging role of the stomach as an endocrine organ. FASEB J. Mar, 2004; 18 (3): 439-56. Maes B, Ghoos Y, Geypens B, Mys G, Hiele M, Rutgeerts P and Vantrappen G. The combined 3C-glycine / 4C-octanoic acid breath test: a double carbon labeled breath test to monitor gastric emptying rate of liquids and solids. J. Nucí. Med. 1994; 35: 824-31. McKee KK, Tan CP, Palyha OC, Liu J, Feighner SD, Hreniuk DL, Smith RG, Howard AD and Van der Ploeg LH T. Cloning and characterization of two human G protein-coupled receptor genes (GPR38 and GPR39) related to the growth hormone secretagogue and neurotensin receptors. Genomics 46: 426-434, 1997. Trudel L, Tomasetto C, Rio M C, Bouin M, Plourde V, Eberling P and Poitras P (2002) Ghrelin / motilin-related peptide is a potent prokinetic to reverse gastric postoperative ileus in rat. Am. J. Physiol. 282, G948-G952.

Peeters TL. Central and peripheral mechanisms by which ghrelin regulates gut motility. J Physiol Pharmacol. Dec 2003; Supl. 54, 4: 95-103. Hall J. A. and Owicki J. A. Real-time measurement of receptor activity: Application of microphysiometric techniques to receptor biology. Meth. Neurosci. nineteen ninety six; 25: 201-224. Zhang J.V., Ren P-G., Avsian-Kretchmer O., Luo, C-W., Rauch R., Klein C, Hsueh A.J. W. (2004) Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science Vol. 310; 996-999. Laburthe M., Couvineau A., Marie J C. VPAC receptors for VIP and PACAP. Receptors & Channels 8: 137-153, 2002. McLatchie LM. Fraser NJ. Main MJ. Wise A. Brown J. Thompson N. Solari R. Lee MG. Foord SM. RAMPs regulate the transport and ligand specificity of the calcitonin-receptor-like receptor. Nature 393: 333-339, 1998. Christopoulos A. Christopoulos G. Morfis M. Laburthe M.

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Claims (13)

NOVELTY OF THE INVENTION CLAIMS

1. - A method for identifying compounds that improve the control of glucose in a subject and that are effective to prevent and / or treat pathologies related to a deteriorated metabolism of carbohydrates, in particular in the prevention and / or treatment of diabetes, including complications associated with it, or the metabolic syndrome, including associated complications thereof, said method comprising the use of all or part of the GPR39 receptor protein.

2. The method according to claim 1, further characterized in that the GPR39 receptor protein is being selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, a splice variant of the proteins having the SEQ ID's mentioned above, and an amino acid sequence having at least 80% and preferably at least 90%, 95%, 96%, 97% or 98% sequence identity, with the amino acid sequence of SEQ ID NO : 2 or SEQ ID NO: 4.

3. The method according to claim 1 or 2, further characterized in that a part of the GPR39 receptor protein consists of a fragment of at least 10 amino acids of the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

4. - A method for identifying a compound that improves the regulation of glucose in a subject, and that is effective to prevent and / or treat pathologies related to a deteriorated metabolism of carbohydrates, in particular in the prevention and / or treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof, which method comprises: (i) contacting host cells expressing all or part of the GPR39 receptor protein according to the invention, with said compound under conditions suitable for binding, and (ii) detecting the binding of the compound to said receptor protein.

5. - A method for identifying a compound that improves the regulation of glucose in a subject, and that is effective to prevent and / or treat pathologies related to a deteriorated metabolism of carbohydrates, in particular in the prevention and / or treatment of diabetes, including associated complications thereof, or metabolic syndrome, including associated complications thereof, which method comprises: (i) contacting membrane preparations of host cells that express all or part of the GPR39 receptor protein in accordance with the invention, with said compound under conditions suitable for binding, and (ii) detecting the binding of the compound to said receptor protein.

6. - The method according to claim 4 or 5, further characterized in that the binding of the compound to the GPR39 receptor protein is being detected by means of an associated label directly or indirectly with the compound to be tested or in a test involving competition with a marked competitor.

7. The method according to claim 6, further characterized in that the labeled competitor is labeled obestatin.

8. The method according to any of claims 4 to 7, further characterized in that in step (i), the compounds are contacted with host cells that express all or part of the GPR39 protein in accordance with the claims 2 and 3.

9. A method for identifying compounds that modulate carbohydrate metabolism, said method comprising the steps of: (i) contacting all or parts of the GPR39 receptor protein with the compound to be placed test, and (ii) measuring the activity of the GPR39 receptor protein, wherein the change in activity of GPR39 in the presence of the test compound, is an indication of the ability of the compounds to modulate carbohydrate metabolism.

10. - The method according to claim 9, characterized in that in step i), the compounds are contacted with host cells that express all or part of the GPR39 protein according to claims 2 and 3.

11. The method according to claim 9 or 10, further characterized in that the activity of the GPR39 receptor protein is being measured as the modulation of an intracellular messenger.

12. - The method according to claim 11, further characterized in that the second intracellular messenger is cAMP, calcium or a reporter gene product.

13. - The use of an isolated nucleic acid sequence selected from the group consisting of: (i) a nucleic acid sequence encoding all or part of the polypeptides of SEQ ID NO: 2 or SEQ ID NO: 4, (ii) a nucleic acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a nucleic acid sequence having at least 80% sequence identity, with the nucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3, in a method according to any of claims 1 to 12. 4. - The use of a vector comprising a nucleic acid sequence as claimed in claim 13, in a method according to any one of claims 1, 4, 5, 8, 9 or 10. 15. The use of a host cell comprising a nucleic acid sequence as claimed in claim 13, or a vector such as is claimed in claim 4, in a method according to any of the claims 1, 4, 5, 8, 9 or 10. 6. - A pharmaceutical composition for the treatment of impaired control of glucose in a human or animal, comprising an agonist or antagonist of the GPR39 receptor. 17. - The use of an agonist or antagonist of GPR39, in the manufacture of a medicament for the treatment of a condition of disease related to impaired carbohydrate metabolism, particularly diabetes (including associated complications thereof), including type 1 diabetes mellitus (insulin dependent diabetes mellitus or IDDM), type 2 (non-insulin dependent diabetes mellitus), juvenile onset diabetes at maturity (MODY) and gestational diabetes. 18. - A diagnostic product comprising an isolated nucleic acid sequence selected from the group consisting of: (i) a nucleic acid sequence encoding all or part of the polypeptides of SEQ ID NO: 2 or SEQ ID NO : 4, (ii) a nucleic acid sequence consisting of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a nucleic acid sequence having at least 80% sequence identity, with the sequence of nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 3. 19. - The diagnostic product according to claim 18, wherein in step (i), the part of the polypeptide is as defined in the claim 3. 20. - A diagnostic product comprising all or part of the GPR39 receptor protein. 21. - The diagnostic product according to claim 20, further characterized in that the GPR39 receptor protein is as defined in claim 2, and the part of the GPR39 receptor protein is as defined in claim 3.