EP1468078A1

EP1468078A1 - Method and device for identifying substances capable of modulating adipocyte differentiation

Info

Publication number: EP1468078A1
Application number: EP03712261A
Authority: EP
Inventors: Bart Staels
Original assignee: Genfit SA
Current assignee: Genfit SA
Priority date: 2002-01-18
Filing date: 2003-01-17
Publication date: 2004-10-20
Also published as: CA2472016A1; FR2834996A1; JP2005525099A; FR2834996B1; WO2003060106A1; AU2003216739A1; US7368286B2; US20050032064A1

Abstract

The invention concerns a method for identifying compounds capable of modulating adipocyte differentiation, which consists in contacting the compound to be tested with genetically modified pre-adipocyte cells overexpressing the REV-ERB-ALPHA receptor and measuring the adipocyte differentiation of said genetically modified cells with the adipocyte differentiation of same said genetically modified pre-adipocyte cells in the absence of said compound to be tested. The invention also concerns genetically modified pre-adipocyte cells overexpressing the REV-ERB-ALPHA receptor as well as the method for preparing said cells.

Description

METHOD OF IDENTIFYING SUBSTANCES CAPABLE OF MODULATING ADIPOCYTARY DIFFERENTIATION

The present invention relates to methods for screening for active molecules, in particular molecules having activity in modulating adipocyte differentiation. The invention also relates to genetic constructs, cells and compositions useful for carrying out such screening methods, for example pre-adipocyte cells genetically modified to overexpress the REV-ERB ALPHA receptor, as well as the methods of preparing said cells. The invention can be implemented for the identification of active compounds or which can be used as headers for the development of active drugs for the management of metabolic pathologies, in particular for the treatment of diabetes, obesity, insulin resistance and / or syndrome X.

The invention is based in particular on the demonstration and the characterization of the role of a particular nuclear receptor, REV-ERB ALPHA, in the adipocyte differentiation mechanisms, and in particular on the capacity of this receptor, when it is over-expressed. , to sensitize cells to the action of adipocyte differentiation factors. The invention is also based on obtaining specific vectors allowing the expression of the REV-ERB ALPHA receptor, as well as genetically modified cell lines, in particular pre-adipocytes. The results obtained show a modulation of the adipocyte differentiation of such lines when they are brought into contact with agonists or receptor antagonists intervening directly or indirectly in the process of adipocyte differentiation. White adipose tissue is the primary energy storage site for eukaryotes. Its role is to store triglycerides in abundance and to mobilize them when energy intake decreases. Deregulation of adipocyte activity results in obesity and its consequences like non-insulin dependent diabetes. The adipocytes that make up white adipose tissue are highly specialized cells that express a defined set of genes characteristic of their differentiation (Fajas et al, Curr. Opin. Cell Biol 10: 165-173 (1998); Spiegelman, Diabetes 47: 507 -514 (1998), Grégoire F, Phys. Rev 78, 783-809 (1998)).

Adipocyte differentiation is a complex process whose molecular actors are becoming better known (Fajas et al, Curr. Opin. Cell Biol 10: 165-173 (1998); Spiegelman, Diabetes 47: 507-514 (1998), Grégoire F, Phys. Rev 78, 783-809 (1998)). Adipocyte differentiation is regulated in a coordinated fashion by a network of several transcription factors. It is initiated by the exit of the cell cycle and the activation of the factors C / EBP beta, C / EBP delta and ADD1 (SREBPlc) which induce the expression of the nuclear receptor activated by the "peroxisome proliferators" of gamma type, ci after named PPAR GAMMA, the main coordinator of adipocyte differentiation.

The PPAR GAMMA receptor stimulates the exit from the cell cycle and the expression of specific adipocyte genes which allow energy storage. Finally, the transcription factor C / EBP alpha cooperates with the PPAR GAMMA receptor in the final stages of adipocyte differentiation to induce a new set of genes and to maintain the expression of said PPAR GAMMA receptor. The REV-ERB ALPHA receptor is an orphan nuclear receptor whose natural or artificial ligands are unknown. Its sequence is coded by the non-coding strand of the gene coding for the alpha-type thyroid hormone receptor (Lazar, MA et al. 1989, Mol.Cell.Biol. 9 (3), 1128-1136), (Lazar, MA et al. 1990, DNA Cell Biol. 9 (2), 77-83), (Laudet, V. et al. 1991, Nucleic Acid Res. 19 (5), 1105-1012). It acts mainly as a transcription inhibitor. Its expression seems to increase during the differentiation of pre-adipocytes into adipocytes and is correlated with the expression of markers of adipocyte differentiation (Chawla, 1993; J. Biol. Chem. 266,12, pp 16265-16269).

The REV-ERB ALPHA receptor acts as a negative regulator of transcription (Laudet, V. et al. 1995, Curr.Biol. 5 (2), 124-127). The human REV-ERB ALPHA receptor has been shown to regulate its own expression (Adelmant, G. Et al. 1996, Proc.Natl; Acad.Sci. USA 93 (8), 3553-3558). The REV-ERB ALPHA receptor mRNA is highly expressed in tissues such as adipose tissue, striated muscles, liver or brain tissue, while its expression is less abundant in other tissues.

The REV-ERB ALPHA receptor is induced during adipocyte differentiation (Chawla, A. et al. 1993, J. Biol. Chem. 268 (22), 16265-16269). However, the molecular mechanism of this regulation remains unknown. It has also been observed that the REV-ERB ALPHA receptor is involved in muscle differentiation (Downes M. et al. 1995, Mol.Endocrinol. 9 (12), 1666-1678) and in the mechanism of regulation of lipid metabolism, due to the identification of the apo AI gene (gene coding for apolipoprotein AI) in rats as a target gene for the REV-ERB receptor ALPHA in the liver (Nu-Dac, Ν. 1998, J. Biol. Chem. 273, 25713-25720). It has also been suggested that the REV-ERB ALPHA receptor acts as a modulator of thyroid hormone signals, (Lazar MA 1990, J. Biol. Chem. 265 (22), 12859-12863), (Munroe, SH et al. 1991 , J. Biol. Chem. 266 (33), 22803-22086). Indeed, the REV-ERB ALPHA receptor binds to the response element of the hormone DR4 (Spanjaard, RA et al. 1994, Mol. Endocrinol. 8 (3), 286-295) and inhibits the formation of 1 homodimer TR and heterodimers TR / RXR of TREs (Downes, M. et al. 1995, Mol.Endocrinol. 9, 1666-1678).

To date, the biological role of the REV-ERB ALPHA receptor in adipose tissue and its mechanism of action remain unknown (Chawla, A. et al. 1993, J. Biol. Chem. 268 (22), 16265-16269) .

The work carried out by the inventors has now made it possible to elucidate interactions between two receptors, REV-ERB ALPHA and PPAR GAMMA. This work has shown that the PPAR GAMMA receptor activates the transcription of the Rev-erb alpha gene via the response element DR2 of the promoter of the Rev-erb alpha gene (called "Rev-DR2"). The inventors were thus able to determine that the Rev-erb alpha gene is a target of the PPAR GAMMA receptor, that the REV-ERB ALPHA receptor is a promoter of adipocyte differentiation induced by the PPAR GAMMA receptor, and that it plays a modulating role. in the adipogenesis process.

The inventors have also shown that, surprisingly, the ^¬ receptor expression REV-ERB ALPHA in preadipocytes such as 3T3-L1 cell line, increases the differentiation of said pre-adipocytes and increases the expression of the PPAR GAMMA receptor in these cells.

The inventors have thus identified regulatory mechanisms between the REV-ERB ALPHA receptors and other receptors involved in the adipocyte differentiation program, in particular the PPAR GAMMA receptor. On the basis of this work, a new method of screening for compounds capable of interacting with either the REV-ERB ALPHA receptor or with said other receptors involved in the adipocyte differentiation program is now proposed.

Such a method is useful for identifying active compounds in the treatment of pathologies linked to metabolic abnormalities using said receptors, such as adipocyte differentiation, diabetes, obesity, insulin resistance and syndrome X.

It is known that certain compounds useful for the treatment of diseases linked to adipocyte differentiation anomalies, such as diabetes or obesity, act via their interactions with the PPAR GAMMA receptor. For example, thiazolidinediones, also known as glitazones, compounds used to treat insulin resistance, have been identified as ligands and artificial activators of the PPAR GAMMA receptor. Fatty acid derivatives have also been identified as natural ligands for the PPAR GAMMA receptor. Fibrates are also powerful regulators of lipid metabolism which act as activators of the PPAR ALPHA receptor.

The results obtained by the inventors, from in vivo and in vitro studies which are presented in the present application show that the treatment with rosiglitazone (also called BRL49653 or BRL) increases the expression of the mRNA coding for the REV-ERB ALPHA receptor. Glitazones, anti-diabetic compounds commonly used in the treatment of type 2 diabetes, therefore induce the adipocyte differentiation program via the binding and activation of the nuclear receptor PPAR GAMMA.

A first aspect of the present invention therefore relates to methods of screening for active molecules, in particular molecules having an activity in the modulation of adipocyte differentiation, based on the use of the REV-ERB ALPHA receptor as a molecular target.

Another aspect of the invention relates to genetic constructs, cells and compositions useful for the implementation of such screening methods, for example pre-adipocyte cells genetically modified to overexpress the REV-ERB ALPHA receptor, as well as the methods for preparing said cells.

A particular aspect of the invention also relates to recombinant viruses (or to viral vectors) encoding a REV-ERB ALPHA polypeptide.

Another aspect of the invention relates to the use of active compounds for the implementation of methods of therapeutic or vaccine treatment of the human or animal body. These include compounds capable of interfering with the binding of the PPAR GAMMA receptor to the Rev-DR2 site or, more generally, on the activity or expression of the REV-ERB ALPHA receptor in adipocyte differentiation. REV-ERB ALPHA Receiver

The present invention is based in particular on the identification of the role of the REV-ERB ALPHA receptor in the differentiation of adipocytes, on the characterization of the mechanisms which underlie this role, and on the exploitation of this molecule for therapeutic purposes.

Within the meaning of the present invention, the term REV-ERB ALPHA receptor designates a nuclear receptor comprising the primary amino acid sequence SEQ ID NO: 4, or a fragment or functional variant thereof.

MTTLDSNNNTGGVITYIGSSGSSPSRTSPESLYSDNSNGSFQSLTQGCPTYFPPSPT GSLTQDPARSFGSIPPSLSDDGSPSSSSSSSSSSSSFYNGSPPGSLQVAMEDSSRV SPSKSTSNITKLNGMVLLCKVCGDVASGFHYGVHACEGCKGFFRRSIQQNIQYKRCL KNENCSIVRINRNRCQQCRFKKCLSVGMSRDAVRFGRIPKREKQRMLAEMQSAMNL

ANNQLSSQCPLETSPTQHPTPGPMGPSPPPAPVPSPLVGFSQFPQQLTPPRSPSPE PTVEDVISQVARAHREIFTYAHDKLGSSPGNFNANHASGSPPATTPHRWENQGCPP APNDNNTLAAQRHNEALNGLRQAPSSYPPTWPPGPAHHSCHQSNSNGHRLCPTHV YAAPEGKAPANSPRQGNSKNVLLACPMNMYPHGRSGRTVQEIWEDFSMSFTPAVR EWEFAKHIPGFRDLSQHDQVTLLKAGTFEVLMVRFASLFNVKDQTVMFLSRTTYSL

QELGAMGMGDLLSAMFDFSEKLNSLALTEEELGLFTAWLVSADRSGMENSASVEQ LQETLLRALRALVLKNRPLETSRFTKLLLKLPDLRTLNNMHSEKLLSFRVDAQ (sequence SEQ ID NO: 4)

The term “fragment” typically designates a polypeptide comprising from 5 to 200 consecutive amino acids of SEQ ID NO: 4, preferably from 5 to 150, even more preferably from 5 to 100. Specific examples of fragments are polypeptides from 5 to 80 amino acids. Preferably, the fragments comprise a functional domain of the sequence SEQ ID NO: 4, for example a transcription inhibitor domain and / or a DNA binding domain. The term functional variant includes natural variants, in particular those resulting from polymorphism (s), splicing (s), variation (s) between species, etc. This term also includes synthetic variants, in particular polypeptides comprising a sequence derived from the sequence SEQ ID NO: 4 by one or more mutations, deletions , substitutions and / or additions of one or more residues. Preferably, a synthetic variant has 75% primary sequence homology with the sequence SEQ ID NO: 4, more preferably at least 85%. The fragments or variants can also comprise added heterologous regions or chemical, enzymatic, immunological modifications, etc. Such modifications can make it possible, for example, to facilitate the production or the purification of the receptor, to improve its stability, to increase his activity, etc.

In a preferred embodiment of the invention, the term REV-ERB ALPHA receptor designates a receptor of human origin, in particular a receptor comprising the sequence SEQ ID NO: 4 or a fragment thereof.

The term Rev-erb alpha gene generally designates any portion of the genome encoding a REV-ERB ALPHA receptor as defined above.

The term “Rev-erb alpha gene construction” or “recombinant nucleic acid encoding a REV-ERB ALPHA receptor” generally designates any nucleic acid encoding a REV-ERB ALPHA receptor as defined above. It can be DNA or RNA, for example genomic DNA, cDNA, mRNA, synthetic or semi-synthetic DNA. These can be obtained by cloning from libraries or plasmids, or by synthesis, or by any other technique known to those skilled in the art. In a particular embodiment of the invention, the Rev-erb alpha gene construct is a nucleic acid comprising the sequence SEQ ID NO: 3, a fragment thereof, or any sequence hybridizing with them in conditions of moderate stringency and coding for a REV ERB ALPHA receptor.

atg acgaccctgg actccaacaa caacacaggt 661 ggcgtcatca cctacattgg ctccagtggc tcctccccaa gccgcaccag ccctgaatcc 721 ctctatagtg acaactccaa tggcagctgcgccccccccccccccccccccccccccc

841 ccacccagcc tgagtgatga cggctcccct tcttcctcat cttcctcgtc gtcatcctcc 901 tcctccttct ataatgggag cccccctggg agtctacaag tggccatgga ggacagcagc 961 cgagtgtccc ccagcaagag caccagcaac atcaccaagc tgaatggcat ggtgttactg 1021 tgtaaagtgt gtggggacgt tgcctcgggc ttccactacg gtgtgcacgc ctgcgagggc 1081 tgcaagggct ttttccgtcg gagcatccag cagaacatcc agtacaaaag gtgtctgaag

1141 aatgagaatt gctccatcgt ccgcatcaat cgcaaccgct gccagcaatg tcgcttcaag 1201 aagtgtctct ctgtgggcat gtctcgagac gctgtgcgtt ttgggcgcat ccccaaacga 1261 gagaagcagc ggatgcttgc tgagatgcag agtgccatga acctggccaa caaccagttg 1321 agcagccagt gcccgctgga gacttcaccc acccagcacc ccaccccagg ccccatgggc 1381 ccctcgccac cccctgctcc ggtcccctca cccctggtgg gcttctccca gtttccacaa

1441 cagctgacgc ctcccagatc cccaagccct gagcccacag tggaggatgt gatatcccag 1501 gtggcccggg cccatcgaga gatcttcacc tacgcccatg acaagctggg cagctcacct 1561 ggcaacttca atgccaacca tgcatcaggt agccctccag ccaccacccc acatcgctgg 1621 gaaaatcagg gctgcccacc tgcccccaat gacaacaaca ccttggctgc ccagcgtcat 1681 aacgaggccc taaatggtct gcgccaggct ccctcctcct accctcccac ctggcctcct

1741 ggccctgcac accacagctg ccaccagtcc aacagcaacg ggcaccgtct atgccccacc 1801 cacgtgtatg cagccccaga aggcaaggca cctgccaaca gtccccggca gggcaactca 1861 aagaatgttc tgctggcatg tcctatgaac atgtacccgc atggacgcag tgggcgaacg 1921 gtgcaggaga tctgggagga tttctccatg agcttcacgc ccgctgtgcg ggaggtggta 1981 gagtttgcca aacacatccc gggcttccgt gacctttctc agcatgacca agtcaccctg

2041 cttaaggctg gcacctttga ggtgctgatg gtgcgctttg cttcgttgtt caacgtgaag 2101 gaccagacag tgatgttcct aagccgcacc acctacagcc tgcaggagct tggtgccatg 2161 ggcatgggag acctgctcag tgccatgttc gacttcagcg agaagctcaa ctccctggcg 2221 cttaccgagg aggagctggg cctcttcacc gcggtggtgc ttgtctctgc agaccgctcg 2281 ggcatggaga attccgcttc ggtggagcag ctccaggaga cgctgctgcg ggctcttcgg

2341 gctctggtgc tgaagaaccg gcccttggag acttcccgct tcaccaagct gctgctcaag 2401 ctgccggacc tgcggaccct gaacaacatg cattccgaga agctgctgtc cttccgggtg 2461 gacgcccagt ga (SEQ ID NO: 3)

Moderate stringency conditions are described for example in Maniatis et al. These are, for example, the following conditions: incubation at 42 ° C. for 12 hours in a medium comprising 50% formamide, 5 × SSPE, 5 X Denhardt's solution, 0.1% SDS.

Typically, the nucleic acid used for recombination (recombinant nucleic acid) or gene construction comprises, in addition to a region encoding the REV-ERB ALPHA receptor, one or more regions for regulation of transcription, typically a promoter and / or a terminator. transcriptional. These regulatory regions are chosen according to the cellular host considered. Preferably, these are functional regulatory regions in mammalian cells. By way of example, mention may be made of constitutive or regulated promoters, inducible or not, tissue selective or ubiquitous, strong or weak, such as for example promoters of viral origin (for example: CMV, LTR, SV40) or originating from cellular genes. In a particular embodiment, the promoter is the promoter of the Rev-erb alpha gene, comprising for example the sequence SEQ ID NO: 1 or a region thereof, for example a promoter comprising the sequence AAAAGTGTGTCACTGGGGCA (SEQ ID NO: 2).

In a particular embodiment of the invention, the gene construct Rev-erb alpha is a nucleic acid comprising the sequences SEQ ID NO: 3 and SEQ ID NO: 2, a fragment thereof or any sequence hybridizing with these under conditions of moderate stringency. In a more specific mode, the Rev-erb gene construction alpha is a nucleic acid comprising a sequence coding for a polypeptide SEQ ID NO 'A operably linked to a transcriptional promoter comprising the sequence SEQ ID NO: 1 or a fragment thereof, in particular a transcriptional promoter comprising the sequence SEQ ID NO : 2.

Genetically modified cells

A particular object of the present invention resides in a population of cells comprising a recombinant nucleic acid encoding a REV-ERB ALPHA receptor.

The cells can be any cultivable cell, preferably from a mammal, for example a human. They can be primary cells or established lines. Preferably, the host cells are pre-adipocyte cells. Such cells are generally defined as fibroblast-like cells, which are capable of differentiating into adipocytes under appropriate culture conditions. More specifically, these are cells of mesodermal origin, incapable of differentiating into chondroblasts, osteoblasts or myoblasts and which, under favorable conditions specific to the cell in question, differentiate into adipocytes and express several differentiation markers characteristics of fat cells. Examples of pre-adipocyte cells which can be used for implementing the invention are in particular the cell lines 3T3-L1 (ATCC reference: CL-173), 3T3-F442A (Green H. et al., Cell 5: 19-27 (1975)), obl7 (Negrel R. et al, Proc.Natl.Acad.Sci. USA., 75: 6054-6058 (1978)) or obl771 (Doglio A. et al. Biochem J., 238 : 123-129 (1986)). A particular object of the invention therefore resides in a genetically modified pre-adipocyte cell, characterized in that it comprises a recombinant nucleic acid encoding a REV-ERB ALPHA receptor.

Other examples of cells which can be used in the context of the invention are prokaryotic cells, yeast cells or mammalian cells, in particular embryonic cells or cells such as CHO, fibroblasts, Nero, etc.

The recombinant nucleic acid present in the cells allows these cells to express a REV-ERB ALPHA receptor, or to over-express such a receptor, when the cells already have a basal level of expression. Thus, in the case of pre-adipocyte cells, the nucleic acid generally allows the cells to overexpress a REV-ERB ALPHA receptor, that is to say to produce the receptor at a level higher than that observed in the same cells in the absence of recombinant nucleic acid construction. The term over-expression generally designates an expression increased in particular by a factor 2, more generally by a factor 3, ideally by a factor of at least 5. The cells are preferably mammalian cells, in particular human cells. It is understood that cells of other species can be used, such as, for example, mouse, rat, monkey, hamster, etc. cells.

A particular object of the present invention therefore relates to a genetically modified cell, in particular pre-adipocyte cell, overexpressing the REV-ERB ALPHA receptor. The term genetically modified indicates that the cell (or an ancestor thereof) has been modified to contain a recombinant nucleic acid encoding said receptor.

Typically, the recombinant nucleic acid or the gene construct comprises, in addition to a region coding for the REV-ERB ALPHA receptor, one or more regions for transcription regulation, typically a promoter and / or a transcriptional terminator, as defined above. . The nucleic acid can be present or incorporated into a plasmid, viral vector, etc. It can be integrated into the genome of the cells, or remain in extra-chromosomal form (replicative or not).

The subject of the invention is also a process for preparing recombinant cells expressing an REV-ERB ALPHA receptor, in particular genetically modified pre-adipocyte cells overexpressing a REV-ERB ALPHA receptor or a recombinant nucleic acid as defined above . The method of the invention generally comprises the introduction of a recombinant nucleic acid as defined above encoding a REV ERB ALPHA receptor in a host cell. The host cells can be any population of cells as described above, preferably a preadipocyte, in particular the cell lines 3T3-L1, 3T3-F442A, obl7 or obl771.

According to a first preferred embodiment of the invention, the recombinant cells are obtained by transfection of host cells using a plasmid vector comprising a gene construct Rev-erb alpha. Advantageously, the transfection is carried out in the presence of a second gene construct encoding a selection or resistance gene, and the cells are selected on the basis of the expression. of said selection or resistance gene as well as of the nucleic acid coding for REV ERB ALPHA.

Within the meaning of the invention, the term "transfection" generally designates any technique allowing the transfer of a nucleic acid into a cell. These may be chemical, physical, biological techniques, etc. As an example, mention may be made of electroporation, precipitation with calcium phosphate, the use of agents which facilitate transfection, such as, for example, lipids, polymers, peptides, etc., or even the use of physical techniques such as the "gun gene", the use of projectiles, bombardment, etc.

In a particular embodiment, the method comprises cotransfection of the cells with a plasmid vector comprising said recombinant nucleic acid and with a plasmid vector comprising a gene for resistance to an antibiotic, the cells being selected for their resistance to said antibiotic and for their expression of said recombinant nucleic acid. According to a preferred embodiment of the invention, the recombinant cells are obtained by co-transfection of host cells with a Rev-erb alpha gene construct which allows the overexpression of the REV-ERB ALPHA receptor and with a construct gene that allows the overexpression of an antibiotic resistance gene. The recombinant cells are then selected in the presence of the antibiotic and tested for their over-expression of the REV-ERB ALPHA receptor.

According to a particular embodiment of the invention, the antibiotic used is chosen from the following substances cited by way of nonlimiting example: neomycin, zeocin, hygromycin, blasticidin, etc. In a particular embodiment of the method, the nucleic acid is introduced by transfection using a plasmid vector further comprising an antibiotic resistance gene, the cells being selected for their resistance to said antibiotic and for their expression recombinant nucleic acid.

According to this variant of the invention, the Rev-erb alpha gene construct which allows overexpression of the REV-ERB ALPHA receptor also comprises a functional cassette which allows the overexpression of a gene for resistance to an antibiotic. The recombinant cells are then selected in the presence of the antibiotic and tested for their over-expression of the REV-ERB ALPHA receptor. According to a particular embodiment of the invention, the antibiotic used is chosen from the following substances cited by way of nonlimiting example: neomycin, zeocin, hygromycin, blasticidin, etc.

In another particular embodiment, the nucleic acid is introduced by transfection using a plasmid vector which also comprises a gene for resistance to an antibiotic and an origin of eukaryotic replication, the cells being selected for their resistance to said antibiotic and for their expression of the recombinant nucleic acid. According to a more particular mode, the gene construct Rev-erb alpha which allows the overexpression of the receptor REV-ERB ALPHA therefore also comprises a functional cassette which allows the ^¬ expression of a gene for resistance to an antibiotic and an origin of eukaryotic replication. The recombinant cells are then selected in the presence of antibiotic and tested for receptor expression ^¬ REV-ERB ALPHA. According to a particular embodiment of the invention, the antibiotic used is chosen from the substances following cited by way of nonlimiting example: neomycin, zeocin, hygromycin, blasticidin, etc.

According to another preferred embodiment of the method of the invention, the nucleic acid is introduced into the cells by infection by means of a viral vector comprising said nucleic acid. According to a particularly preferred embodiment of the invention, the introduction is carried out using a recombinant virus comprising the recombinant nucleic acid encoding the REV ERB ALPHA receptor and, where appropriate, the selection or resistance gene. ("Infection"). Different types of recombinant viruses can be used, such as retroviruses, adenoviruses, AAV (Adenovirus Associated Virus), herpes viruses, modified baculoviruses, etc. The preferred recombinant viruses are adenoviruses and retroviruses recombinants.

In a preferred embodiment, the recombinant cells (advantageously having an overexpression of the RNA coding for the REV-ERB ALPHA receptor) are obtained by infection of the host cells, in particular pre-adipocytes, by means of viral vectors, preferably adenoviruses or retroviruses, said vectors containing a nucleic acid encoding a REV-ERB ALPHA receptor.

In this regard, another object of the invention resides in a viral vector comprising a nucleic acid encoding a REV-ERB ALPHA receptor. Another object of the invention resides in a recombinant virus comprising, in its genome, a nucleic acid encoding a REV-ERB ALPHA receptor. Preferably, the viral vector is a vector defective for replication, that is to say incapable of autonomous replication in a cell. Typically, a viral vector is defective for one or more viral genes essential for replication. In the case of retroviruses, the main viral genes are the gag, pol and env genes. In the case of adenoviruses, the main genes are contained in the E1A, E1B, E4 and E2 regions. In AAVs, these are the Rep and Cap regions of the genome. The construction of viral vectors, defective for one or more (or all) of the viral genes and comprising a nucleic acid of interest is known to those skilled in the art. These techniques use, for example, packaging lines and / or helper vectors or viruses, as illustrated in the examples.

A particular object of the invention relates to:

- a defective recombinant adenovirus comprising, in its genome, a nucleic acid encoding a REV-ERB ALPHA receptor. V adenovirus is preferably a group C adenovirus, in particular Ad5, and / or advantageously comprises a deletion of all or part of the E1A and / or E1B and / or E4 region;

- a defective recombinant retrovirus comprising, in its genome, a nucleic acid encoding a REV-ERB ALPHA receptor. The retrovirus is preferably a retrovirus derived from MLV

(Mouse Leukemia Virus) or a lentivirus, and / or advantageously comprises a deletion of all or part of the gag and / or pol region and / or approx.

According to a particular mode of implementation of the invention, the preparation (eg, transfection, infection) of the cells, in particular of the pre-adipocytes is carried out with a Rev-erb alpha gene construction which comprises, in addition to a region encoding the receptor REV-ERB ALPHA, for example the SEQ ID NO: 3, one or more regions for transcription regulation, typically a promoter and / or a transcriptional terminator. According to a preferred embodiment of the invention, these are functional regulatory regions in mammalian cells. By way of nonlimiting examples, mention may be made of constitutive or regulated promoters, inducible or not, selective for tissue or ubiquitous, strong or weak, such as for example promoters of viral origin (for example: CMV, LTR, SV40) or from cellular genes.

According to a particular mode of implementation of the invention, the preparation (eg, transfection) of the cells (eg, pre-adipocytes) is carried out with a Rev-erb alpha gene construction which comprises, in addition to a region encoding the REV receptor -ERB ALPHA, for example SEQ ID NO: 3, the promoter of the Rev-erb alpha gene, for example comprising the sequence SEQ ID NO: 1, or comprising a region thereof, for example of sequence SEQ ID NO: 2. According to another particular mode, the preparation is carried out with a sequence chosen from or comprising the sequences SEQ ID NO: 1 and SEQ ID NO: 3.

For the preparation of the recombinant cells of the invention, the host cells can be brought into contact with the Rev-erb alpha gene or the recombinant nucleic acid or the vector or the virus under any appropriate condition, then the recombinant cells are recovered. The contacting can be carried out in any suitable support and in any culture medium suitable for the cell type (for example: DMEM, RPMI, etc.).

In a particular embodiment, after infection or transfection, the stable cell lines in culture are selected. The Preferred genetically modified cells with overexpression of the gene encoding the REV-ERB ALPHA receptor are stable lines.

Screening Methods

The present invention also relates to methods of identification, selection, characterization or optimization of compounds capable of modulating adipocyte differentiation. These methods can be carried out in cellular tests or in vitro, for example by binding tests. These methods essentially use a REV ERB ALPHA receptor (or a corresponding nucleic acid) as a molecular target.

In a first embodiment, the subject of the present invention is a method of identifying, selecting, characterizing or optimizing compounds capable of modulating adipocyte differentiation, characterized in that (i) contact a compound to be tested with cells (preferably preadipocyte) as defined above, (ii) the adipocyte differentiation of said cells is measured or determined and (iii), preferably, this differentiation is compared to the differentiation adipocyte of the same said cells in the absence of said test compound.

Preferably, the cells are pre-adipocyte cells as described above, more particularly pre-adipocyte cells (over-) expressing the REV-ERB ALPHA receptor.

According to a preferred embodiment of the method of the invention, the test compound is brought into contact with cells (preferably genetically modified cells overexpressing the REV-ERB receptor ALPHA) in the presence or absence of at least one activator of a receptor involved in the adipocyte differentiation program or of at least one activator of a gene encoding a receptor involved in the adipocyte differentiation program.

The results presented in the examples indeed show that, surprisingly, the expression of the REV-ERB ALPHA receptor in the recombinant cells of the invention sensitizes the pre-adipocytes to the action of adipocyte differentiation factors and promotes the program of differentiation. Under these conditions, the selection of compounds modulating this differentiation is greatly facilitated.

In a first variant of the invention, at least one activator of a receptor involved in the adipocyte differentiation program is used, such as, for example and without limitation, an activator of the PPAR GAMMA receptor. The activator of the PPAR GAMMA receptor is for example chosen from the group comprising in a nonlimiting manner: thiazolidinediones (rosiglitazone, troglitazone, englitazone, ciglitazone, pioglitazone, KRP-297), N- (2-benzoylphenyl) -L-tyrosines , 15-deoxy-delta 12,14-prostaglandin J2, etc.

In another variant, at least one activator of a gene for a receptor involved in the adipocyte differentiation program is used. By way of non-limiting example, the test compound is brought into contact with genetically modified cells overexpressing the REV-ERB ALPHA receptor in the presence or absence of at least one activator of the PPAR gamma gene. Preferably, 1 activator of the gamma PPAR gene is chosen from the group comprising: C / EBP beta, C / EBP delta, ADD1 (SREBPlc).

The compound and the activator can be contacted with the cells at the same time, or sequentially. Typically, the activator is added first, followed by the test compound.

The measurement of adipocyte differentiation can be carried out by staining the differentiated cells. The dye is for example chosen from the group comprising the dye Oil Red O, Sudan Black.

The measurement of adipocyte differentiation can also be carried out by determining the transport or the synthesis of fatty acids. The measurement of adipocyte differentiation can also be carried out by determining the expression of at least one specific marker for the differentiated adipocytes, preferably a marker chosen from the group comprising: aP2, adipsin and leptin.

The method of the invention is remarkable in that it allows ^" •

- to identify compounds capable of modulating the activity of the REV-ERB ALPHA receptor, such as compounds capable of modulating the expression of the Rev-erb alpha gene or of compounds constituting agonists or antagonists of the REV-ERB ALPHA receptor. Thus, the method of the invention makes it possible in particular to identify compounds capable of increasing adipocyte differentiation and constituting activators of the expression of the Rev-erb alpha gene or agonists of the REV-ERB ALPHA receptor.

- to identify indirectly, in the absence of activator of the PPAR gamma gene and activator of the PPAR GAMMA receptor, compounds capable of increasing adipocyte differentiation which act as agonists of the PPAR GAMMA receptor.

According to particular implementations, the method of the invention allows:

- to identify compounds capable of reducing adipocyte differentiation and constituting antagonists of the REV-ERB ALPHA receptor.

- to identify compounds capable of increasing adipocyte differentiation constituting agonists of the REV-ERB ALPHA receptor.

- to identify, in the presence of activator of the PPAR gamma gene and / or activator of the PPAR GAMMA receptor, compounds capable of reducing adipocyte differentiation. - to identify PPAR receptor agonist compounds

GAMMA.

The term “agonist or antagonist of a receptor” is understood to mean a compound which binds to said receptor and activates or inhibits its activity, respectively.

The test compound can be of varied origin and nature. It can be isolated compounds, biological extracts, organic or inorganic molecules, banks of molecules (synthetics, peptides, nucleic acids, etc.) or microorganisms, etc. The test compound can be brought into contact with the construction of nucleic acid or the cells on (or in) any suitable support and in particular on a plate, in a tube or a flange, a membrane, etc. Generally, the contacting is carried out in a multiwell plate. which allows to conduct, in parallel, many and varied tests. Among the typical supports one finds microtiter plates and more particularly 96 or 384 well plates (or more). Depending on the support and the nature of the test compound, variable quantities of cells can be used when implementing the methods described. Conventionally, 10 ³ to 10 ⁶ cells are brought into contact with a type of test compound, in an appropriate culture medium, and preferably between 10 ⁴ and 10 ⁵ cells. The amount (or concentration) of test compound can be adjusted by the user according to the type of compound (its toxicity, its cell penetration capacity, etc.), the number of cells, the length of the incubation period, etc. Generally, cells are exposed to amounts of test compounds which vary from 1nM to ImM. It is of course possible to test other concentrations without deviating from the present invention. Each compound can also be tested in parallel, at different concentrations. Furthermore, various adjuvants and / or vectors and / or products facilitating the penetration of the compounds into the cells can, in addition, be used if necessary. Contact can be maintained for example between a few minutes and several hours or days, particularly between 5 and 72 hours, generally between 12 and 48 hours.

According to another embodiment, the method of the invention comprises the selection of compounds capable of modulating the expression of the REV-ERB ALPHA receptor, in particular of modulating the effect of the PPAR GAMMA receptor on the promoter of the Rev-erb alpha gene . Indeed, the inventors have now demonstrated that the PPAR GAMMA receptor is responsible for a modulation of the expression of the REV-ERB ALPHA receptor, and that this modulation involves an interaction between the PPAR GAMMA receptor and the gene promoter Rev-erb alpha, especially in the Rev-DR2 region (SEQ ID NO: 2). The invention also relates to a method for identifying, selecting, optimizing or characterizing compounds capable of modulating adipocyte differentiation, characterized in that it comprises (i) bringing a test compound into contact with d a nucleic acid comprising the sequence Rev-DR2 or a functional equivalent thereof, in the presence of PPAR GAMMA receptor, (ii) revealing a binding of the PPAR GAMMA receptor on this nucleic acid and, optionally, ( iii) comparison of this binding with that observed in the absence of test compound, the test compounds modulating the binding of the PPAR GAMMA receptor being compounds modulating adipocyte differentiation.

The measurement of the possible fixation of the test compound, of the PPAR GAMMA receptor or of a complex formed of the PPAR GAMMA receptor and of said test compound on the response element can be carried out by any method known to those skilled in the art, for example by detecting a signal produced by the response element following said fixing. It can be all direct or indirect methods, such as those using a reporter gene, binding tests, etc.

Thus, a particular method of the invention comprises contacting a test compound and a nucleic acid comprising the sequence Rev-DR2 (the sequence SEQ ID NO: 1 or preferably SEQ ID NO: 2) or a functional equivalent thereof, in the presence of the PPAR GAMMA receptor, and the demonstration of a binding of the PPAR GAMMA receptor on this nucleic acid. The binding is advantageously compared to that observed in the absence of test compound. In another embodiment, the test compound and the PPAR GAMMA receptor are brought into contact with a reporter system comprising (i) a transcriptional promoter comprising one or more copies of the sequence SEQ ID NO: 1, preferably of the sequence SEQ ID NO: 2 or of a functional variant thereof and (ii) a reporter gene , and the activity of the test compound is determined by measuring its effect on the expression of the reporter gene induced by the PPAR GAMMA receptor.

A particular subject of the invention thus relates to a method of identification, selection, optimization or characterization of compounds capable of modulating adipocyte differentiation, characterized in that it comprises bringing a test compound into contact and of the PPAR GAMMA receptor with a reporter system comprising (i) a transcriptional promoter comprising one or more copies of the sequence SEQ ID NO: 1, preferably of the sequence SEQ ID NO: 2 or of a functional variant thereof and (ii) a reporter gene, and the evaluation of the activity of the test compound by measuring its effect on the expression of the reporter gene induced by the PPAR GAMMA receptor

The reporter gene can be placed under the control of any promoter (for example SEQ ID NO: 1) whose sequence comprises the sequence SEQ ID NO: 2 or a functional variant thereof. This particular sequence may be present in one or more copies in the promoter (preferably 1 to 10 and even more preferably 1 to 6), upstream or downstream or internally, in the same orientation or in the orientation opposite. In a preferred embodiment of the invention, the reporter gene is placed under the control of a promoter which comprises one or more copies of the sequence SEQ ID NO: 2. Preferably, it is a promoter whose activity differential in the absence and presence of PPAR GAMMA receptor or an equivalent functional can be detected.

In this regard, the response element to the PPAR GAMMA receptor can be associated with a minimal transcriptional promoter. The minimal promoter is a transcriptional promoter having a weak or nonexistent basal activity, and capable of being increased in the presence of a transcriptional activator (for example the PPAR GAMMA receptor). A minimal promoter can therefore be a naturally weak promoter in mammalian cells, that is to say producing a non-toxic expression and / or not sufficient to obtain a pronounced biological effect. Advantageously, a minimal promoter is a construct prepared from a native promoter, by deletion of region (s) not essential (s) to transcriptional activity. Thus, it is preferably a promoter essentially comprising a TATA box, generally of a size less than 160 nucleotides, centered around the codon for initiating transcription. A minimal promoter can thus be prepared from viral, cellular, strong or weak promoters, such as for example the promoter of the thymidine kinase (TK) gene of the herpes virus, the immediate CMV promoter, the PGK promoter. , the SV40 promoter, etc.

In a preferred embodiment, the reporter gene is placed under the control of the promoter of the Rev-erb alpha gene, for example of a promoter comprising the non-coding sequence of SEQ ID NO: 1.

Any reporter gene can be used in the screening method according to the invention. Among these, there may be mentioned, for example, the gene for chloramphenicol acetyltransferase (CAT), the gene for firefly luciferase (Luc) or Renilla (Ren), the gene for secreted alkaline phosphatase (PAS) or that of beta-galactosidase (β-Gal). The activity of proteins encoded by these genes can be easily measured by conventional methods and allows indirect knowledge of the effect of nuclear receptors on gene expression by measuring the quantity of proteins produced and / or their enzymatic activity. The reporter system is advantageously introduced into a population of cells, which can be of prokaryotic or eukaryotic origin.

Another object of the invention lies in the use of a compound identified, selected, characterized or optimized according to a process described above for the preparation of a medicament intended for the implementation of a treatment method. therapeutic or vaccine for the human or animal body, in particular for the curative or preventive treatment of metabolic pathologies, in particular diabetes, obesity, insulin resistance and syndrome X.

Another object of the invention resides in a process for preparing a medicament comprising (i) a step of selecting a compound capable of modulating adipocyte differentiation as described above and (ii) bringing into contact a selected compound, or an analog thereof, with a pharmaceutically acceptable carrier.

Another subject of the invention resides in a process for the preparation of a compound active on adipocyte differentiation, comprising (i) a step of selecting a compound capable of modulating adipocyte differentiation as described above and (ii ) synthesis of a selected compound, or an analog thereof. Other aspects and advantages of the present invention will appear on reading the examples which follow and the appended drawings, provided by way of illustration and not limitation, in which,

FIG. 1 illustrates the results of the Northern blot analyzes of the mRNAs extracted from adipose tissues of rats treated or not with rosiglitazone (BRL):

Adult male rats were treated for 14 days either with rosiglitazone (10 mg / kg / day) or with the excipient (1% carboxymethylcellulose). After sacrifice and dissection, the total RNA was extracted from the epididymal and perirenal adipose tissue. 10 μg of mRNA were subjected to the analysis by Northern blot using cDNA probes of the REV-ERB ALPHA receptor (upper panel) or β-actin (lower panel).

FIG. 2 shows the induction of expression of the REV-ERB ALPHA receptor mRNA in the 3T3-L1 pre-adipocytes by rosiglitazone (BRL).

The 3T3-L1 pre-adipocytes developed until confluence in DMEM medium containing 10% fetal calf serum. Once at confluence, the cells were changed in DMEM with 10% fetal calf serum and stimulated with a mixture containing IBMX, dexamethasone, insulin, with or without rosiglitazone (1 μM in l 'H ₂ O) for 9 days. The RNA was isolated and analyzed by Northern blot.

FIG. 3 illustrates the induction of the activity of the promoter of the Rev-erb alpha gene by rosiglitazone (BRL) and the PPAR GAMMA receptor. 3T3-L1 pre-adipocytes were transfected with a plasmid which comprises a 1.7 kb fragment of the promoter of the Rev-erb alpha gene cloned in front of the luciferase reporter gene and with the plasmid pSG5-PPAR gamma which allows the expression of the murine PPAR GAMMA receptor or the corresponding empty vector pSG5. The cells were treated with rosiglitazone (1 μM) and the luciferase activities were measured as described previously.

- Figure 4 illustrates the role of the Rev-DR2 site in the induction of the activity of the promoter of the Rev-erb alpha gene by the PPAR receptor

GAMMA.

FIG. 4A shows the effects of the PPAR GAMMA receptor on the activity of a construction of the human promoter of the gene encoding the REV-ERB ALPHA receptor containing a wild or mutated Rev-DR2 site.

FIG. 4B shows the effects of the PPAR GAMMA receptor on the activity of a construction of the promoter of the gene for the human receptor REV-ERB ALPHA containing a wild or mutated site Rev ~ DR2 cloned in two copies upstream of the promoter SV40. Cos cells were transfected with the reporter constructs indicated, and plasmids pSG5-PPAR gamma or pSG5. The cells were treated with rosiglitazone (BRL) and the luciferase activities were measured.

FIG. 5 illustrates tests for measuring the electrophoretic mobility of the PPAR GAMMA receptor which binds as a heterodimer with the nuclear receptor RXR ALPHA at the Rev-DR2 site of the promoter of the Rev-erb alpha gene. The electrophoretic mobility measurement tests were carried out using the oligonucleotides indicated, labeled at their end, in the presence of the murine PPAR GAMMA receptor, murine RXR ALPHA, human REV-ERB ALPHA produced by reticulocyte lysate, or non-lysate programmed (lysate). The binding competition experiments were carried out by adding a 0, 10 or 100-fold excess of the cold Rev-DR2 oligonucleotide.

- Figure 6 shows that the exogenous expression of the REV-ERB ALPHA receptor stimulates lipid accumulation in 3T3-L1 cells.

3T3-L1 cells were infected with a control retrovirus (MFG-Neo) or with a retrovirus which allows the over-expression of the REV-ERB ALPHA receptor (MFG-Reverb alpha). The resulting cells were induced to differentiate with or without rosiglitazone (BRL) at 1 μM for eight days. The cells were then fixed and stained with Oil red O.

- Figure 6A, C and D show microscopic views of the cells stained with Oil red O. - Figure 6B shows macroscopic views of the stained plates

1 Oil red O. - Figure 6E shows the exogenous or endogenous expression of the protein

REV-ERB ALPHA (Ecto-Rev or Endo-Rev) controlled by Western blot.

A rabbit anti-REV-ERB ALPHA polyclonal antibody directed against a synthetic peptide (constituted by amino acids 263-365 of the human sequence) was used for the immunocytochemistry and Western blotting experiments. FIG. 7 illustrates the impact of the exogenous expression of the REV-ERB ALPHA receptor on the expression of the mRNAs of the PPAR GAMMA receptor and of the aP2 gene used as a marker for adipocyte differentiation. 3T3-L1 cells were infected either with the MFG-Neo retrovirus or with the MFG-Rev retrovirus and treated for 8 days with a mixture containing IBMX and insulin (denoted Mix), with or without rosiglitazone 1 microM. The mRNAs were then extracted and analyzed by Northern blot using the indicated probes.

Other advantages and characteristics of the invention will become apparent on reading the examples which follow which reflect the work carried out by the inventors to lead to the design and the implementation of the screening method.

1- MATERIALS AND METHODS

materials

Rosiglitiazone (Ref. BRL49653) is a sample provided by A. Bril (SKB, Rennes, France), GP + E86 cells (Columbia University, New York, USA) and the plasmid pMFG (Massachusetts Institute of Technology, Cambridge, USA) .

Animals

Male Sprague-Dawley rats (10 weeks old) were treated for 14 days by gavage with rosiglitiazone (10 mg / kg / day) suspended in 1% carboxymethylcellulose. The control animals received an equivalent volume (5 ml / kg / day) of the carboxymethylcellulose solution. At the end of the experiments, the animals are sacrificed under ether anesthesia. The fatty tissue is removed immediately and frozen in liquid nitrogen.

RNA analysis.

RNA extraction and Northern blot analyzes were performed according to the protocol described above (Staels, B. et al. 1192, Arterioesclerosis and Thrombosis 12 (3), 286-294) using Rev- cDNA probes rat alpha erb, murine PPAR gamma and aP2, chicken beta-actin or human 36B4.

Transfection experiences.

Constructs which include fragments of the Rev-erb alpha gene promoter cloned into the plasmid without promoter pGL2 or the plasmid SV40pGL2 (Promega, Madison, WI, USA) have been described previously (Adelmant, G. et al. 1996, Proc. natl Acad Sci. USA, 93 (8), 3553-3558). Human hepatoma HepG2 cells were obtained from the European Collection of Animal Cell Culture (Porton Down, Salisbury, UK) and 3T3-L1 cells were obtained from the American Type Cell Culture (ATCC). The cells are cultured in DMEM medium, supplemented with 2 mM glutamine and 10% (vol / vol) of fetal calf serum (SVF), in a humidified atmosphere containing 5% CO ₂ at 37 ° C. All the transfections were carried out in triplica. Luciferase activities were determined on the total cell extracts using a luciferase test system (Promega, Madison, WI, USA).

In vitro translation and EMSAs.

Plasmids ρSG5-mPPAR gamma, pSG5 ^~ mRXR alpha and pSG5-hRev- erb alpha were transcribed in vitro with T7 polymerase and translated using the rabbit reticulocyte lysate system (Promega, Madison, WI, USA). The gel retardation experiments with the REV-ERB ALPHA, PPAR GAMMA and / or RXR ALPHA proteins were carried out as previously described (Gervois, P. et al. 1999, Molecular Endocrinology 13 (3), 400-409) and Vu -Dac, N. et al. 1994, J. Biol. Chem. 269 (49), 31012-31018). For competition experiments, increasing amounts of the indicated cold probe were added immediately before the addition of the labeled oligonucleotide. The complexes were resolved in 5% polyacrylamide gels, in 0.25 X TBE buffer (90 mM borate, 2.5 mM EDTA, pH 8.3) at room temperature. The gels were dried and exposed overnight at -70 ° C on an X-ray film (XOMAT-AR, Eastman Kodak, Rochester, NY, USA).

Viral production and infection.

The cells encapsulating the GP + E86 virus (Markowitz, D. et al. 1988, J. Virol. 62 (4), 1120-1124) are cultured in DMEM medium (4.5 g / 1 glucose) containing 10% serum heat-inactivated calf (HyClone, Logan, UT, USA), 8 μg / ml gentamicin, 50 U / ml penicillin, 50 μg / ml streptomycin, at 37 ° C in an atmosphere containing 5% CO2 and 95 % of humidified air. In order to generate cell lines which constitutively overexpress the REV-ERB ALPHA receptor, the sequence coding for the REV-ERB ALPHA receptor was inserted upstream of the entry site of the internal ribosome and of the resistance gene. neomycin pCITE, (Novagen, Madison, WI, USA) of the retroviral plasmid MFG (Dranoff, G. et al. 1993, Proc. Natl. Acac. Sci. USA, 1993, 90 (8), 3539-3543) using the NcoI-BamHI sites for generating the plasmid pMFG-Rev-erb alpha. A similar construction where the sequence encoding the REV-ERB ALPHA receptor is absent is used throughout the study as a control (pMFG-Neo). The bicistronic construct is designed to allow the simultaneous expression of the REV-ERB ALPHA receptor and the neomycin resistance gene product from infected cells. To produce the recombinant viruses, the GP + E86 cells (15,000 / cm ² ) are transfected with the constructs of the MFG plasmids (2 μg) using lipofectamine (Life Technologies-Invitrogen, Groeningen, The Netherlands) and by selecting the resistants using the geneticin analog G418 (0.8 mg / ml, Life Technologies-Invitrogen, Groeningen, The Netherlands). 3T3-L1 cells were infected with the MFG-Neo or MFG-Rev-erb alpha viruses produced by GP + E86 as described (Mattot, V. et al. 2000, Oncogene, 19 (6) 762-772) and selected for their resistance to geneticin until the establishment of stable lines (approximately 10 days).

Cell culture and differentiation.

The 3T3-L1 cells (obtained from ATCC) are cultured in a growth culture medium containing DMEM and 10% fetal calf serum. The cells are differentiated by the method of Bernlohr et al. (Bernlohr, DA et al. 1984, Proc.Natl.Acad.Sci.USA, 81 (17), 5468-5472). Post-confluent cells after two days of culture (designated day D0) are transferred to a differentiation medium (DMEM, 10% FCS, 1 μm dexamethasone, 10 μg / ml insulin and 0.5 mM 3-methyl-l- isobutylxanthine (IBMXXSigma, St Louis, MI, USA)) for two days. The cells were then cultured in a post-differentiation medium (DMEM; 10% FCS, insulin) with or without rosiglitazone. The environment is changed every day. Stable 3T3-L1 pre-adipocytes expressing the REV-ERB ALPHA receptor are cultured under the same conditions but differentiated without dexamethasone. After treatment, cells were fixed with 10% formaldehyde in PBS and stained with Oil Red 0 (Sigma, St Louis, MI, USA). Alternatively, the total RNA is extracted as described above.

RESULTS Activation of the PPAR GAMMA receptor increases the expression of the REV-ERB ALPHA receptor in the adipose tissue of the rat. To determine if activation by the PPAR GAMMA receptor affects the expression of the REV-ERB ALPHA receptor in vivo, rats were treated with rosiglitazone (noted BRL), an active and highly specific ligand for the receptor. PPAR GAMMA for 14 days. The expression of the REV-ERB ALPHA receptor was analyzed in epididymal and perineal adipose tissue by Northern blot. Compared with the control, treatment with rosiglitazone greatly increases the levels of mRNA coding for the REV-ERB ALPHA receptor in the adipose tissues studied (Figure 1). The levels of beta-actin mRNA used as a control are not affected by the treatment. These experiments demonstrate that activation of the PPAR GAMMA receptor by rosiglitazone increases the expression of the REV-ERB ALPHA receptor in adipose tissue.

The activation of the PPAR GAMMA receptor induces the REV-ERB ALPHA receptor mRNA in the 3T3-L1 pre-adipocytes.

In order to study the molecular mechanism of this induction, the inventors studied the regulation of the expression of the mRNA of the REV-ERB ALPHA receptor by rosiglitazone in the 3T3-L1 pre-adipocytes (FIG. 2). 3T3-L1 preadipocytes were cultured to confluence in medium containing 10% fetal calf serum. The confluent cells were transferred to a medium containing delipidated serum and the cells were differentiated with a mixture containing dexamethasone, IBMX, insulin, with or without rosiglitazone (1 μm).

The levels of the mRNA encoding the REV-ERB ALPHA receptor increase as the differentiation of pre-adipocytes into adipocytes progresses. However, compared with the standard differentiation treatment, the levels of mRNA coding for the REV-ERB ALPHA receptor are induced earlier when rosiglitazone is added. These levels were significantly higher after 9 days in fully differentiated 3T3-L1 adipocytes. Used as a control, the levels of beta-actin mRNA changed only slightly during adipogenesis and are not affected by treatment with rosiglitazone.

The PPAR GAMMA receptor induces the expression of the REV-ERB ALPHA receptor at the transcriptional level.

In order to elucidate whether the induction of REV-ERB ALPHA receptor mRNA occurs at the transcriptional level, the inventors tested the effects of overexpression of the PPAR GAMMA receptor and of stimulation by rosiglitazone on activity. of a construct comprising a luciferase reporter gene under the control of a 1.7 kb fragment of the promoter of the Rev-erb alpha gene. 3T3-L1 cells were transfected with the construct comprising the luciferase reporter gene under the control of a 1.7 kb fragment of the Rev-erb alpha gene promoter in the presence of the expression vector pSG5-PPAR gamma which allows expression of the murine PPAR GAMMA receptor or of the corresponding empty vector pSG5 and treated with rosiglitazone or the excipient. The activity of the promoter of the Rev-erb alpha gene is induced ^¬ expression of PPAR GAMMA receptor, an effect that is also amplified in the presence of rosiglitazone (Figure 3). On the other hand, when the construct comprising the luciferase reporter gene under the control of a 1.7 kb fragment of the promoter of the Rev-erb alpha gene is transfected alone, no effect is observed. These data indicate that transcription of the Rev-erb alpha gene is induced by rosiglitazone via activation of the PPAR gamma receptor. An element, named Rev ~ DR2, which has a strong homology, with a response element of the “DR2” type of a nuclear receptor has been identified in the promoter of the Rev-erb alpha gene. It has been shown that the said REV-ERB ALPHA receptor binds on this site and represses its own transcription via it (Adelmant, G. et al, 1996, Proc.Natl.Acad.Sci, 93 (8), 3553 -3558). This has moreover been identified as being the response element on which the PPAR alpha / RXR ALPHA heterodimer binds to confer a response to fibrates to the Rev-erb alpha gene in the liver (Gervois et al, Mol. Endocrinol ., 13, 400-409, 1999). To confirm that the Rev-DR2 site can also function as a response element of the PPAR GAMMA receptor in adipose tissue, the inventors carried out transient transfection experiments using constructs comprising the wild and truncated versions of the promoter of the Rev- gene erb alpha noted pGL2-hRev ^~ erbαδ and pGL2- hRev-erbαΔ described previously (Adelmant, G. et al, 1996, Proc.Natl.Acad.Sci, 93 (8), 3553-3558) (Figure 4). To confirm that the Rev-DR2 site can also function as a response element of the PPAR GAMMA receptor in adipose tissue, the inventors also carried out transient transfection experiments using the constructs described above (Adelmant, G. et al, 1996 , Proc.Natl.Acad.Sci, 93 (8), 3553-3558) including the versions and mutated Rev-DR2 site clones upstream of the SV40 promoter (Rev-DR2, Rev-DR2M5 'and Rev-DR2M3') (Figure 4). Cotransfection in HepG2 cells of a PPAR GAMMA receptor expression vector and of a reporter vector which comprises two copies of the wild-type Rev-DR2 site cloned upstream of the SV40 promoter and of the luciferase reporter gene leads to improved induction of a factor 2.5 of transcriptional activity compared to the level obtained with the empty expression vector pSG5. On the other hand, no effect is observed when the reporter vector used comprises two copies of the mutated Rev-DR2 site either in the 3 'position or in the 5' position. The effect of overexpression of the PPAR GAMMA receptor is amplified in the presence of rosiglitazone. These results clearly show that the activity of the promoter of the Rev-erb alpha gene is regulated by the PPAR GAMMA receptor and that this induction is carried out via the Rev-DR2 site (FIG. 4).

The PPAR GAMMA receptor binds as a heterodimer with the RXR ALPHA receptor at the Rev ~ DR2 site.

Finally, it was investigated whether the PPAR GAMMA receptor binds on the Rev ^~ DR2 site. An electromobility test (delay on gel or EMSAs) was carried out using the PPAR GAMMA and RXR ALPHA proteins synthesized in vitro. As a control, the REV-ERB ALPHA receptor produced in vitro binds to the wild Rev-DR2 site both as a monomer and as a homodimer (Figure 5). On the other hand, no binding is observed on the oligonucleotide Rev-DR2 carrying a mutation on the AGGTCA half-site located at 5 '(M5') as described previously (Adelmant, G. et al, 1996, Proc.Natl Acad. Sci, 93 (8), 3553-3558). Finally, the REV-ERB ALPHA receptor binds as a monomer to the Rev-DR2 site carrying a mutation on the AGGTCA half-site located at 3 '(M3') such as previously described (Adelmant, G. et al, 1996, Proc.Natl.Acad.Sci, 93 (8), 3553-3558). The RXR ALPHA or PPAR GAMMA receptors alone do not bind to any of the oligonucleotides indicating that PPAR GAMMA and RXR ALPHA cannot bind as monomers. Binding to the Rev-DR2 site has been observed when the PPAR GAMMA receptor is incubated with the RXR ALPHA receptor. The binding is specific since competition is established with an excess of unlabeled oligonucleotide. On the other hand, no binding of the PPAR GAMMA / RXR ALPHA complex is observed on the mutated Rev ^~ DR2 site (M5 'or M3'). These binding experiments demonstrate that PPAR GAMMA binds as a heterodimer with RXR ALPHA to the intact Rev ^~ DR2 site of the promoter of the Rev-erb alpha gene.

The REV-ERB ALPHA receptor increases the adipogenic activity of the PPAR GAMMA receptor.

In order to directly confirm the participation of the REV-ERB ALPHA receptor in adipogenesis, the entire cDNA coding for the REV-ERB ALPHA receptor was cloned into a retroviral vector. 3T3-L1 pre-adipocytes were then infected with the resulting virus. Stable lines established by selection in the presence of the antibiotic G418 (Neomycin) after infection either with the MFG-Neo virus (negative control), or with the MFG-Rev-erb alpha virus, are cultured until confluence and subsequently treated with a differentiation medium (noted Mix) containing IBMX, insulin with or without rosiglitazone noted BRL (1 μM). The endogenous expression or induced by viral infection of REV-ERB ALPHA was verified by immunocytochemical analyzes or by Western blot (Figure 6E). Cells infected with MFG-Neo express high levels of REV-ERB ALPHA receptor compared to control cells infected with MFG-Neo. In the absence of rosiglitazone, the exogenous expression of the REV-ERB ALPHA receptor induces only a weak morphological differentiation of the pre-adipocytes. In the presence of rosiglitazone (1 μM), there is an increase in the differentiation of the pre-adipocytes and of the lipid accumulation in the cells expressing the REV-ERB ALPHA receptor, compared with the control cells. Indeed, after fixation and staining with "Oil red O", a small accumulation of lipids is observed in the absence of rosiglitazone, but a strong lipid accumulation is observed in cells expressing the REV-ERB ALPHA receptor treated with rosiglitazone during 8 days (Figures 6A - 6D). To achieve the same result with rosiglitazone alone without hormonal stimulation, cells must be differentiated for 16 days (data not shown). These morphological changes occur in parallel with a similar variation in the mRNA of specific markers for adipocytes. Northern blot analyzes show a low but significant level of expression of the PPAR GAMMA receptor and of aP2 in the cells expressing the REV-ERB ALPHA receptor (FIG. 7). Surprisingly, the endogenous level of the REV-ERB ALPHA receptor is disturbed in cells overexpressing the REV-ERB ALPHA receptor. The levels of aP2 mRNA and of PPAR GAMMA receptor are high in cells expressing the REV-ERB ALPHA receptor treated with a mixture of rosiglitazone after only 4 days of differentiation (FIG. 7), or with rosiglitazone alone (FIG. 7). This phenomenon is only observed after 8-12 days in the control cells. These results show that the exogenous expression of the REV-ERB ALPHA receptor produces a weak effect without hormonal stimulation and amplifies the induction of adipogenesis by activation by the ligand PPAR GAMMA. Thus, the use of the pre-adipocyte cell line of the invention, overexpressing the REV-ERB ALPHA receptor, has made it possible to identify the Rev-erb alpha gene as a new target gene for the PPAR GAMMA receptor. in the adipogenic cascade of transcription factors. This observation of the active role of the REV-ERB ALPHA receptor in the adipocyte differentiation process has enabled the inventors to implement a new screening method to identify active compounds involved in adipocyte modulation.

Claims

1. Method for identifying compounds capable of modulating adipocyte differentiation, characterized in that (i) a test compound is brought into contact with a population of genetically modified pre-adipocyte cells, comprising a recombinant nucleic acid encoding a REV receptor -ERB ALPHA, (ii) the adipocyte differentiation of said cells is measured or determined and (iii), preferably, this differentiation is compared to the adipocyte differentiation of the same said pre-adipocyte cells in the absence of said test compound.

2. Method according to claim 1, characterized in that the compound to be tested is brought into contact with cells overexpressing the receptor

REV-ERB ALPHA in the presence of at least one activator of a receptor involved in the adipocyte differentiation process.

3. Method according to claim 2, characterized in that the activator of the receptor involved in the adipocyte differentiation process is an activator of the PPAR GAMMA receptor.

4. Method according to claim 3, characterized in that the activator of the receptor involved in the adipocyte differentiation process is chosen from the group comprising in particular: thiazolidinediones, such as rosiglitazone, troglitazone, englitazone, ciglitazone, pioglitazone, or KRP- 297, N- (2-benzoylphenyl) -L-tyrosines and 15-deoxy-delta 12,14-prostaglandin J2.

5. Method according to claim 1, characterized in that the compound to be tested is brought into contact with the genetically modified pre-adipocyte cells in the presence of at least one activator of a gene involved in the adipocyte differentiation process.

6. Method according to claim 5, characterized in that the activator of a gene involved in the adipocyte differentiation process is an activator of the PPAR gamma gene.

7. Method according to claim 6, characterized in that the activator of the PPAR gamma gene is chosen from the group comprising C / EBP beta, C / EBP delta and ADD1 (SREBPlc).

8. Method according to any one of claims 1 to 7, characterized in that the REV-ERB ALPHA receptor comprises the sequence SEQ ID

NO '-4 or a functional fragment or variant thereof.

9. Method according to any one of claims 1 to 8, characterized in that the recombinant nucleic acid comprises the sequence SEQ ID NO: 3 or a fragment thereof.

10. Method according to any one of claims 1 to 9, characterized in that the recombinant nucleic acid further comprises the sequence SEQ ID NO: 1 or a fragment thereof comprising the sequence SEQ ID NO: 2.

11. Method according to any one of claims 1 to 10, characterized in that the recombinant nucleic acid is incorporated by a plasmid vector.

12. Method according to any one of claims 1 to 10, characterized in that the recombinant nucleic acid is incorporated by a viral vector.

13. Method according to any one of claims 1 to 12, characterized in that the recombinant nucleic acid is integrated into the genome of the cell.

14. Method according to any one of claims 1 to 13, characterized in that the measurement of adipocyte differentiation is carried out (i) by coloring the differentiated cells, preferably with a dye chosen from the dye Oil Red O and Sudan Black , (ii) by determining the transport or the synthesis of fatty acid, and / or (iii) by determining the expression of at least one marker specific for the differentiated adipocytes, preferably a marker chosen from aP2, adipsin and leptin.

15. Method for identifying compounds capable of modulating adipocyte differentiation, characterized in that it comprises (i) bringing a test compound and a nucleic acid comprising the sequence SEQ ID NO: 1, preferably SEQ ID NO: 2, or a functional equivalent thereof, in the presence of PPAR GAMMA receptor, (ii) the demonstration of a binding of the PPAR GAMMA receptor on this nucleic acid and, optionally, (iii) the comparison of this binding with that observed in the absence of test compound, the test compounds modulating said binding of the PPAR GAMMA receptor being compounds modulating adipocyte differentiation.

16. Method for identifying compounds capable of modulating adipocyte differentiation, characterized in that it comprises bringing a test compound and the PPAR GAMMA receptor into contact with a reporter system comprising (i) a transcriptional promoter comprising one or more several copies of the sequence SEQ ID NO: 1, preferably of the sequence SEQ ID NO: 2, or of a functional variant thereof and (ii) a reporter gene, and the evaluation of the activity of the compound test by measuring its effect on the expression of the reporter gene induced by the PPAR GAMMA receptor.

17. Use of a compound identified by a method according to one of claims 1 to 16, or of an analog thereof, for the preparation of a medicament intended for the preventive or curative treatment of a metabolic disease.

18. Use of a compound identified by a method according to one of claims 1 to 16, or of an analog thereof, for the preparation of a medicament intended for the preventive or curative treatment of diabetes, 1 ' obesity, insulin resistance and syndrome X.

19. Genetically modified pre-adipocyte cell, characterized in that it comprises a recombinant nucleic acid encoding a REV-ERB ALPHA receptor, this recombinant nucleic acid further comprising the sequence SEQ ID NO: 1 or a fragment thereof comprising the sequence SEQ ID NO: 2.

20. Cell according to claim 19, characterized in that the REV-ERB ALPHA receptor comprises the sequence SEQ ID NO: 4 or a fragment or functional variant thereof.

21. Cell according to claim 19 or 20, characterized in that the recombinant nucleic acid comprises the sequence SEQ ID NO: 3 or a fragment thereof.

22. Cell according to one of claims 19 to 21, characterized in that the recombinant nucleic acid is incorporated by a plasmid vector.

23. Cell according to one of claims 19 to 21, characterized in that the recombinant nucleic acid is incorporated by a viral vector.

24. Cell according to one of claims 19 to 23, characterized in that the recombinant nucleic acid is integrated into the genome of the cell.

25. Genetically modified pre-adipocyte cell, characterized in that it comprises a recombinant nucleic acid encoding a REV-ERB ALPHA receptor, the recombinant nucleic acid being incorporated by a viral vector.

26. Genetically modified pre-adipocyte cell, characterized in that it comprises a recombinant nucleic acid encoding a REV-ERB ALPHA receptor, the recombinant nucleic acid being integrated into the genome of the cell.

27. Cell according to claim 25 or 26, characterized in that the REV-ERB ALPHA receptor comprises the sequence SEQ ID NO: 4 or a fragment or functional variant thereof.

28. Cell according to one of claims 25 to 27, characterized in that the recombinant nucleic acid comprises the sequence SEQ ID NO: 3 or a fragment thereof.

29. Cell according to one of claims 25 to 28, characterized in that the recombinant nucleic acid further comprises the sequence SEQ ID NO: 1 or a fragment thereof comprising the sequence SEQ ID NO: 2.

30. A method of preparing a pre-adipocyte cell according to one of claims 19 to 29, characterized in that a recombinant nucleic acid encoding a REV ERB ALPHA receptor is introduced into a pre-adipocyte cell.

31. Method according to claim 30, characterized in that the pre-adipocytes are chosen from the cell lines 3T3-L1, 3T3-F442A, obl7 and obl771.

32. Method according to one of claims 30 or 31, characterized in that the nucleic acid is introduced by transfection by means of a plasmid vector.

33. Method according to claim 32, characterized in that it comprises the cotransfection of the cells with a plasmid vector comprising said recombinant nucleic acid and a plasmid vector comprising a gene for resistance to an antibiotic, and in that the cells are selected for their resistance to said antibiotic and for their expression of said recombinant nucleic acid.

34. Method according to claim 32, characterized in that the nucleic acid is introduced by transfection by means of a plasmid vector further comprising a gene for resistance to an antibiotic, and in that the cells are selected for their resistance to said antibiotic and for their expression of recombinant nucleic acid.

35. The method according to claim 32, characterized in that the nucleic acid is introduced by transfection by means of a plasmid vector further comprising a gene for resistance to an antibiotic and an origin of eukaryotic replication, and in that the cells are selected for their resistance to said antibiotic and for their expression of the recombinant nucleic acid.

36. Method according to one of claims 30 or 31, characterized in that the nucleic acid is introduced by infection by means of a viral vector.

37. Method according to claim 36, characterized in that the infection is carried out by means of an adenovirus or of a recombinant retrovirus.

38. Method according to any one of claims 30 to 37, characterized in that the recombinant nucleic acid comprises SEQ ID No: 3 or a fragment thereof.

39. Method according to any one of claims 30 to 38, characterized in that the recombinant nucleic acid further comprises one or more regions for regulation of transcription, typically a promoter and / or a transcriptional terminator.

40. Method according to claim 39, characterized in that the recombinant nucleic acid comprises the sequence SEQ ID NO: 1 or a fragment thereof comprising the sequence SEQ ID NO -2.

41. Method according to any one of claims 30 to 40, characterized in that, after infection or transfection, the stable lines of pre-adipocytes in culture are selected.

42. Defective recombinant virus, preferably an adenovirus or a defective recombinant retrovirus, characterized in that it comprises in its genome a nucleic acid encoding a REV-ERB ALPHA receptor.