HK1161269A - Use of inhibitors of plac8 activity for the modulation of adipogenesis - Google Patents

Description

Use of inhibitors of Plac8 activity for the modulation of adipogenesis

The present invention concerns a screening test for Plac8, a novel target involved in adipogenesis modulation, and for identifying modulators of the activity of this target. Furthermore, the present invention relates to modulators of Plac8 activity and their use to modulate adipogenesis and thus treat obesity and related disorders, particularly in pharmaceutical compositions.

Obesity is a major risk factor for many disorders, including hypertension, coronary artery disease, dyslipidemia (dyslipemia), insulin resistance, and type 2 diabetes. Because of the importance of the obesity epidemic, a number of investigations have focused on adipocyte biology, including developmental pathways that produce new adipocytes. Adipogenesis is the process by which undifferentiated mesenchymal precursor cells become mature adipocytes. Throughout the past decade, considerable progress has been made in elucidating the molecular mechanisms of adipocyte differentiation that involve sequential activation of transcription factors from several families, such as CCAAT/enhancer binding proteins (C/ebpa, α, and γ) and the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPAR γ) (Rosen, e.d. et al, 2002). PPAR γ is described as a "primary regulator" of adipogenesis, as it has been shown both in vitro and in vivo to be both sufficient and essential for adipogenesis. Recently, new transcription factors have been described to be involved in lipogenesis, such as the KLF family (KLF2, 5 and KLF15) (Banerjee, S.S. et al, 2003; Gray, S.M. et al, 2002), the Ebf family (Jimenez, M.A. et al, 2007) and Krox20(Chen, Z. et al, 2005), suggesting that the transcriptional cascade occurring during lipogenesis is much more complex than previously thought. Furthermore, signaling molecules and/or receptors such as the Wnt family of secretory proteins (Kang s. et al, 2007), Shh proteins, Notch receptors have also been described to be involved in molecular events leading to adipocyte formation. Interestingly, it was noted that extracellular and intracellular events were coupled in some way to modulate adipogenesis. All of these signaling pathways converge on a tightly regulated transcriptional cascade, which requires a more complete understanding to potentially control adipocyte development and prevent obesity.

Fat stores in adipose tissue are limited, and exceeding this capacity results in the accumulation of lipids in other tissues, particularly in muscle, liver, and endocrine pancreas, and results in the secretion of various adipokines (adipokines) by adipocytes. Adipose tissue is composed of several deposits located at different anatomical sites, which may originate from distinct precursors and have different physiological functions and pathophysiological effects. In contrast to the subcutaneous fat depot, the visceral fat depot may contribute more to the deficiency associated with metabolic syndrome.

Cannabinoid 1 receptors have been identified in all organs that play a critical role in glucose metabolism and type 2 diabetes, i.e. adipose tissue, gastrointestinal tract, liver, skeletal muscle and pancreas. Rimonabant (Rimonabant), the first selective cannabinoid receptor 1(CB1R) antagonist used clinically, has been shown to reduce food intake and body weight, thus improving glucose metabolism regulation.

However, there remains a need for new therapeutic targets for the treatment of obesity.

The placental 8 protein (Plac8) is known to be a cytoplasmic signaling molecule, although it has been reported to have a putative signal peptide (Rogulski, k. et al, 2005). Recently, Plac8 knockout mice were generated and exhibited an impaired immune response to bacterial infection (Ledford, j.g. et al, 2007). The role and function of Plac8 in immune cells, and in other cell types, is unknown.

The inventors have now found that Plac8 plays a crucial role in adipocyte differentiation. Thus, Plac8 is believed to be a novel relevant target for the modulation of adipogenesis and for the treatment of obesity and related disorders. Inhibition of Plac8 can also be used to reduce adipogenesis to reduce subcutaneous and visceral fat accumulation.

Detailed Description

The present invention is drawn to methods for modulating adipogenesis and metabolic function in adipocytes.

The present invention resides in the use of an inhibitor of Plac8 activity for the modulation of adipogenesis, in particular for the treatment of obesity and related disorders. The invention also concerns pharmaceutical compositions containing such modulators of adipogenesis and related disorders and screening assays for such modulators.

The inventors have identified a role for Plac8 in adipogenesis modulation. Via the transcriptomic (transcriptomic) approach, they identified genes whose expression was associated with weight gain in the group of C57BI/6 mice fed a high fat diet. Then, they performed a second analysis to evaluate the changes in gene expression induced by treatment of high fat diet-fed mice with rimonabant. Genes remain that have not been previously described in adipocyte biology, but which may be involved in important biological processes such as signaling, modification of extracellular matrix proteins, and gene transcription. These genes may be important for adipogenesis, particularly because they may be involved in the mechanism by which rimonabant reduces fat mass in mice. In this context, Plac8 was identified as being involved in adipocyte metabolism, in particular a new signaling pathway. More generally, this gene appears to play a role in adipogenesis and in controlling adipose tissue development in obesity.

The present invention resides in the identification of modulators of Plac8 activity. Such modulators may be any compound or molecule, in particular small molecules, lipids and sirnas, capable of modulating the activity of Plac 8.

Modulators of Plac8 activity can be identified by testing the ability of an agent to modulate Plac8 activity. An inhibitor of Plac8 is any compound that is capable of reducing or inhibiting, in whole or in part, the activity of Plac 8. Inhibitors of Plac8 include, but are not limited to, agents that interfere with the interaction of Plac8 with its natural ligands in intracellular compartments, agents that decrease Plac8 expression at the transcriptional and translational levels, and agents that inhibit intracellular signaling involving Plac 8.

In one embodiment, Plac8 activity may be reduced using small molecules that inhibit Plac8 transcription, in whole or in part. Such modulators can be identified using methods well known to those skilled in the art, like reporter systems consisting in a Plac8 promoter linked in frame to a reporter gene and expressed in a suitable cell line; the activity of the reporter gene product can be measured quantitatively. Thus, compounds that inhibit the expression of a reporter gene by, for example, inhibiting an activating transcription factor, may be considered potential candidates.

Reporter genes that can be used in such reporter systems are numerous and well known in the art. For example, such reporter genes may be genes that allow for the expression of Green Fluorescent Protein (GFP), luciferase, β -galactosidase, and the like.

Accordingly, one aspect of the present invention provides a method for screening for an inhibitor of Plac8 activity, comprising the steps of:

a) transfecting a cell line with a reporter construct comprising a Plac8 promoter linked to a reporter gene;

b) culturing said cell line in conditions permitting expression of said reporter gene;

c) adding a candidate compound to the cell culture, and

d) identifying inhibitor compounds as those compounds that have the ability to decrease or inhibit the expression of the reporter gene.

The predicted Plac8 promoter to be used in the screening test described above for Plac8 transcriptional regulators corresponds to SEQ ID No. 23.

In another embodiment, Plac8 expression is regulated via RNA interference using small interfering RNA (sirna) or small hairpin RNA (shrna). Thus, in one aspect, the invention relates to double-stranded nucleic acid molecules, including small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (sirna), double-stranded RNA (dsrna), micro RNA (mirna), and short hairpin RNA (shrna) molecules capable of mediating RNA interference (RNAi) against Plac8 gene expression, including mixtures of such small nucleic acid molecules and suitable formulations of such small nucleic acid molecules.

The phenomenon of RNAi-mediated gene silencing has been described for the first time in the Caenorhabditis elegans (Caenorhabditis elegans) system, where microinjection of long double-stranded RNA molecules is reported. The mechanism of RNA-mediated gene inactivation appears to be slightly different in the various organisms that have been investigated to date. However, in all systems, RNA-mediated gene silencing is based on post-transcriptional degradation of the target mRNA induced by the endonuclease Argonaute2, which is part of the so-called RISC complex 2.The sequence specificity of degradation is determined by the nucleotide sequence of the specific antisense RNA strand loaded into the RISC complex.

Introduction of the siRNA compound into a cell produces a cell having a reduced level of the target mRNA, and thus a reduced level of the corresponding polypeptide and a concurrently reduced level of the corresponding enzyme activity.

Sirnas specific for Plac8 as described herein can be used as modulators of Plac8 activity to reduce translation of Plac8 mRNA. More specifically, sirnas specific for Plac8 can be used to reduce adipogenesis and thus treat obesity and related diseases.

In one embodiment, the invention features a double stranded nucleic acid molecule, such as an siRNA molecule, wherein one strand comprises a nucleotide sequence or portion thereof that is complementary to a predetermined Plac8 nucleotide sequence in a target Plac8 nucleic acid molecule.

Modified or unmodified RNA molecules may be used. An example of a modification is the incorporation of tricyclo (tricyclo) -DNA to allow for improved serum stability of the oligonucleotide.

In one embodiment, the determined Plac8 nucleotide sequence is a Plac8 nucleotide target sequence (SEQ ID No.1 and SEQ ID No.3) described herein.

Due to the potential for sequence variability of genomes between different organisms or different subjects, the selection of siRNA molecules for broad therapeutic applications is likely to involve conserved regions of genes. Thus, in one embodiment, the invention relates to siRNA molecules that target conserved regions of the genome or regions conserved between different targets. siRNA molecules designed to target a conserved region of various targets achieve effective inhibition of Plac8 gene expression in a diverse patient population.

In one embodiment, the invention features a double-stranded short interfering nucleic acid molecule that down-regulates expression of the target Plac8 gene or directs cleavage of a target RNA, wherein the siRNA molecule comprises about 15 to about 28 base pairs, preferably about 19 base pairs. The siRNA or RNAi inhibitor of the invention can be chemically synthesized, expressed from a vector, or enzymatically synthesized.

In a specific embodiment, the siRNA specific for Plac8 is an shRNA having the sequence SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO. 7. In a preferred embodiment, the siRNA specific for Plac8 is an shRNA having the sequence SEQ ID NO.6 or SEQ ID NO.7, while in a more preferred embodiment, the siRNA specific for Plac8 is an shRNA having the sequence SEQ ID NO. 6.

The use of the siRNA according to the invention results in a 5% to 20%, preferably 5% to 15%, more preferably 5% to 10% reduction in mRNA levels from those of the corresponding wild type cells. Wild-type cells are cells prior to introduction of a nucleic acid encoding an siRNA compound, wherein the targeted mRNA is not degraded by the siRNA compound.

Inhibitors of Plac8 activity may be administered topically or systemically by any suitable route, depending on the nature of the molecule and the desired effect. In accordance with protocols used in the art, siRNA can be administered locally directly in the targeted tissue in the case of double-stranded molecules, or via a vector in the case of shRNA.

In one embodiment, RNAi is obtained using shRNA molecules. The shRNA construct encodes a stem-loop RNA. After introduction into the cell, this stem-loop RNA is processed into a double-stranded RNA compound, the sequence of which corresponds to the stem of the original RNA molecule. Such double stranded RNA can be prepared according to any method known in the art, including in vitro and in vivo methods, such as, but not limited to, those described in Sahber et al (1987), Bhattacharyya et al (1990), or U.S. Pat. No.5,795,715.

For in vivo administration, the shRNA may be introduced into a plasmid. Plasmid-derived shrnas present the advantage of providing a combination with a reporter gene or selectable marker and the option of delivery via viral or non-viral vectors. The shRNA was introduced into the vector and then into the cells, which ensured that the shRNA was expressed continuously. The vector is typically delivered to daughter cells, allowing gene silencing to proceed.

The invention also provides vectors comprising polynucleotides for expressing shrnas of the invention. For example, these vectors are AAV vectors, retroviral vectors, particularly lentiviral vectors, adenoviral vectors, which can be administered by various suitable routes, including intravenous routes, intramuscular routes, direct injection into subcutaneous tissue, or other targeted tissues selected in accordance with common practice.

The route of administration of siRNA varies from local direct delivery to systemic intravenous administration. An advantage of local delivery is that the siRNA dose required for efficacy is substantially lower because the molecule is injected into or near the target tissue. Local administration also allows focused delivery of siRNA. For such direct delivery, naked siRNA may be used. "naked siRNA" refers to siRNA (unmodified or modified) delivered in saline or other simple excipients such as 5% dextrose. The ease of formulation and administration of such molecules makes this an attractive therapeutic approach. Naked DNA may also be formulated into lipids, particularly liposomes.

systemic application of siRNA is often less invasive and, more importantly, not restricted to tissues that are sufficiently accessible from the outside. For systemic delivery, siRNA can be formulated with cholesterol conjugates, liposomes, or polymer-based nanoparticles. Liposomes are traditionally used to provide increased pharmacokinetic profiles and/or reduced toxicity profiles. They allow for significant and repeated successful in vivo delivery. Currently, the use of lipid-based siRNA systemic delivery formulations (particularly to hepatocytes) appears to represent one of the most promising recent opportunities for the development of RNAi therapeutics. Formulation with polymers such as kinetic polyconjugates (e.g., conjugated with N-acetylglucosamine for hepatocyte targeting) and cyclodextrin-based nanoparticles allows both targeted delivery and endosomal escape mechanisms. Other polymers such as Atelocollagen and chitosan allow for therapeutic effects on subcutaneous tumor xenografts and on bone metastases.

sirnas can also be directly coupled to molecular entities designed to facilitate targeted delivery. Due to the nature of the siRNA duplex, the presence of an inactive or sense strand contributes to the ideal coupling site. Examples of conjugates are lipophilic conjugates such as cholesterol, or aptamer-based conjugates.

Cationic peptides and proteins are also used to form complexes with the negatively charged phosphate backbone of the siRNA duplex.

These different delivery methods can be used to target Plac8siRNA into relevant tissues, particularly adipose tissue. For such targeting, the siRNA may be coupled to different molecules that interact with preadipocytes and adipocytes, such as, for example, ligands that interact with lipid transporters, receptors, insulin receptors, or any molecule known in the art.

Another object of the present invention is a pharmaceutical composition comprising as an active ingredient a modulator of Plac8 according to the invention. These pharmaceutical compositions comprise an effective dose of at least one modulator according to the invention and at least one pharmaceutically acceptable excipient. The excipients are selected according to the desired pharmaceutical form and route of administration among the customary excipients known to the person skilled in the art.

The invention also resides in a method for modulating adipogenesis. The methods can be used to treat obesity or related diseases. The method may also be used to reduce fat accumulation for cosmetic purposes.

Modulators of Plac8 activity are useful in therapeutics for modulating adipogenesis, in particular for the treatment and prevention of obesity related disorders, in particular type 2 diabetes, dyslipidemia, elevated blood pressure, insulin resistance, cardiovascular disorders and more generally metabolic syndrome.

According to another aspect of the present invention, it relates to a method for treating the above pathologies, comprising the in vivo administration to a patient of an effective dose of a modulator of Plac8 according to the invention.

Suitable unit dosage forms include oral forms such as tablets, hard or soft gelatin capsules, powders, granules and oral solutions or suspensions, sublingual, buccal, intratracheal, intraocular, intranasal forms, by inhalation, topical, transdermal, subcutaneous, intramuscular or intravenous forms, rectal forms and implants. For topical application, the compounds of the present invention may be used as creams, gels, ointments or lotions.

In accordance with common practice, the dosage appropriate for each patient is determined by the physician in accordance with the route of administration, the weight of the patient and the response.

Plac8 inhibitors may also be used in cosmetic applications to reduce fat accumulation on a lost face. For cosmetic applications, Plac8 inhibitors may be incorporated in formulations suitable for topical use. The Plac8 inhibitor may be a small molecule or siRNA, as previously described.

The invention will now be described by reference to the following examples, which are illustrative only and not intended to limit the invention.

Examples

Brief Description of Drawings

FIG. 1: selection of the crucial adipose tissue regulatory gene. Venn diagram (Venn diagram) shows the selection of genes based on the following criteria. A) Similar regulation in high fat feeding in the subcutaneous (SCAT or Sq) and Visceral (VAT). 151 genes (48 for SCAT and 88 for VAT) were selected. B) Of those 151 genes, the selection of genes regulated by rimonabant treatment (14 for SCAT and 54 for VAT). This led to the selection of 34 genes regulated by high fat diet and rimonabant in both tissues. Of those genes, 16 had expression levels associated with L, M and group H weights (obesity associated), while 18 were modulated by HFD at the same level in each subgroup (obesity not associated).

FIG. 2: plac8 expression in a variety of tissues and cell types. A) Northern blot of Plac8 showing mRNA expression in various mouse tissues: spleen, muscle (gastrocnemius), heart, lung, kidney, liver, Brown Adipose Tissue (BAT), Subcutaneous (SCAT), and Visceral (VAT) adipose tissue. As a control, the membrane was stained with methylene blue. The size of Plac8mRNA is shown on the right. B to E: mRNA levels of Plac8 measured by RT-PCR. B) In SCATs and VATs of wild type and Ob/Ob mice (n-5) × p < 0.05, data are shown as mean ± sd and expressed as fold increase relative to control SCATs set at 1. C) In the Stromal Vascular Fraction (SVF) and isolated adipocytes of mice (n ═ 5 mice pooled for each extraction, the experiment was repeated 3 times, showing a representative experiment). Data are expressed as fold increase relative to SCAT SVF expression. D) In human whole tissue SCAT and VAT, isolated adipocytes, isolated preadipocytes, and in vitro differentiated adipocytes. Data are presented as levels of full tissue SCAT expression relative to an arbitrary setting of 1. E) 3T3-L1 cells on day-2 before DMI treatment and up to day 7 after DMI treatment. N-2-3 groups of cells. Data are presented as levels of expression relative to day 0.

FIG. 3: knockdown of Plac8 expression and activity by shRNA. A) shRNA was transfected into 293T cells. The pSIREN retroviral plasmid containing the shRNA sequence against Plac8 was co-transfected with the pCMVSPORT expression plasmid. As a control for shRNA constructs, we used shRNA against firefly luciferase protein (shRNA luciferase). 3 shRNAs were tested in Plac 8. B) 3T3-L1 cells were transduced with a retrovirus containing shRNA against luciferase (shLuc) or Plac8(shPlac 8). mRNA levels were measured by RT-PCR prior to differentiation. C) Photo of oil red O of differentiated 3T3-L1 on day 9. D) aP2 (differentiation marker) mRNA expression measured by RT-PCR in the same cells on day 9 as in C). Results are expressed as mean ± sd, # P < 0.05, # P < 0.01; p < 0.005. n is 3.

FIG. 4: overexpression of Plac8cDNA in the 3T3-L1 cell line. A) 3T3-L1 transduced with a retrovirus expressing the murine cDNA of Plac8 or an empty retrovirus as a control. Plac8mRNA expression measured by RT-PCR on day 0. B) Photographs of oil red O at day 4 and day 9 of differentiated 3T3-L1 dishes transduced with retroviral (control) containing constructs of Plac8cDNA or empty constructs. C) PPAR γ 2 (differentiation marker) mRNA expression measured by RT-PCR in the same cells on day 9. Results are expressed as mean ± sd, # P < 0.05, # P < 0.01. n is 3.

Materials and methods

Animal treatment

Feeding C57BL/6J mice, which are obese (Collins et al 2004), with a high-fat drinkDiet (HFD) for 6 months. After 6 months of HFD, mice exhibited discrete body weights with varying degrees of glucose intolerance (as measured by the glucose tolerance test). HFD mice were divided into 3 groups, which exhibited the same level of glucose intolerance but had low (L), medium (M) or high (H) body weight, and were treated with vehicle (vehicle) or rimonabant (10mg^-1Day of the year^-1) They were treated and fed to Normal Chow (NC) for one month to normalize (normaize) their body weight.

RNA preparation, labeling and hybridization on cDNA microarray

Using peqGOLD Trifast^TM(peqlab) and chloroform-isoamyl alcohol (24: 1) extraction RNA from 5 different mice per group was extracted from visceral and subcutaneous adipose tissues. RNA was precipitated with isopropanol and purified by flow through RNeasy columns (Qiagen). RNA quality was checked before and after amplification with Bioanalyzer 2100 (Agilent). Reverse transcription of RNA and use of MessageAmp^TMKit (Ambion) for amplification of RNA. The mouse universal reference (Clontech) was similarly amplified and both adipose tissue and reference RNA were labeled by indirect techniques with Cy5 and Cy3 according to published protocols (De fourmestaux et al, 2004). The labeled RNA was hybridized to a microarray containing 17664 cDNAs prepared at the DNA array facility at the University of Rosemory (University of Lausanne). Scanning, imaging, and quality control analyses were performed as previously published (de fourmestaux et al, j.biol.chem.2004279: 50743-53). Data are expressed in log₂The intensity ratio (Cy5/Cy3) indicates normalized (normaize) by local weighted linear regression (Lowess) method with print tip (print tip) and filtered based on spot quality and incomplete annotation. All analyses were performed with R software for statistical calculations available on a Comprehensive R Archive Network (cran.

Cell culture

3T3-L1 cells were treated in DMEM (Gibco) with 10% FBS (Gibco) in 5% CO₂And (5) culturing. After retroviral infection (see below), the cells were allowedCells were grown to confluence in 100-mm or 60-mm dishes in DMEM with 10% FBS. Once confluency is achieved, the cells are exposed to differentiation medium containing dexamethasone (1. mu.M), insulin (5. mu.g/ml), and isobutylmethylxanthine (0.5. mu.M) (DMI). After 2 days, cells were maintained in media containing insulin (5 μ g/ml) until ready for harvest on day 7.

Oil red O dyeing

After 7 to 10 days of differentiation, the cells were washed once in PBS and fixed with formaldehyde (Formalde-Fresh; Fisher) for 15 minutes. The staining solution was prepared by dissolving 0.5g of oil red O in 100ml of isopropanol; 60ml of this solution were mixed with 40ml of distilled water. After 1 hour at room temperature, the staining solution was filtered and added to the dish for 4 hours. Then, the staining solution was removed, and the cells were washed twice with distilled water.

shRNA constructs

RNAi-Ready pSIREN-RetroQ ZsGreen (Clontech) was used to construct shRNA. The target sequence of Plac8 was designed by interrogating the Whitehead siRNA algorithm (http:// jura. wi. mit. edu/bioc/siRNAext /) and the siRNA designer software from Clontech (http:// biolnfo. Clontech. com/rnadesigner /); at least two sequences presented by these two algorithms were subcloned into the pSIREN vector (Clontech) using EcoRI and BamHI restriction sites. Three of the following Plac8 target sequences were selected: SEQ ID NO.5(shPlac8-1), SEQ ID NO.6(shPlac8-2) and SEQ ID NO.7(shPlac 8-3); as a negative control, a siRNA sequence against luciferase was used, having the sequence SEQ ID No.8 (shLuc).

Transfection of shRNA constructs

The specificity of the shRNA was tested in 293T HEK cells co-transfected with an expression vector containing Plac8cDNA (SEQ ID No.21) and an RNAi-Ready pSIREN-RetroQ ZsGreen vector expressing shRNA against luciferase (control shLUC) or Plac8(shPlac8) using the calcium phosphate method described in Jordan, m., et al (2004). RT-PCR analysis was performed on cell RNA extracts 24 hours after transfection.

Generation of retroviral constructs and retroviral infection

Retroviruses were constructed either on the RNAi-Ready pSIREN-RetroQ ZsGreen (pSIREN Clontech) or pMSCV puromycin plasmid (pMSCV, Clontech). The viral construct was transfected into 293HEK packaging cells using the calcium phosphate method described in Jordan, M., et al (2004) along with constructs encoding gag-pol and VSV-G proteins. Supernatants were harvested after 48 hours in the presence of 3 μm Trichostatin (Trichostatin) a (sigma) and used immediately or snap frozen and stored at-80 ℃ for later use. Viral supernatants were added to the cells in the presence of Polybrene (4 μ g/ml) for 6 hours and diluted two-fold with fresh medium for an additional 15 hours.

Overexpression constructs

A modified pMSCV puromycin retroviral vector (from Clontech) expressing a GFP marker was used to overexpress Plac8cDNA into cells. The cDNA (SEQ ID NO.21) was inserted blunt-ended into the hpaI restriction site from the multiple cloning site of pMSCV. The resulting colonies were tested for correct orientation and selected by enzymatic cleavage. The correct clone was selected, expanded and used for retroviral infection of 3T3-L1 cells.

Isolation of adipocytes and Stromal Vascular Fraction (SVF) from adipose tissue

By CO₂Male C57BL/6J mice (n ═ 6-8) at 8 weeks of age were euthanized by inhalation, and epididymis (viscera) and subcutaneous adipose tissue were collected and placed in DMEM medium containing 10mg/ml of fatty acid-poor BSA (Sigma-Aldrich, st. The tissue was minced into small pieces and then digested in 0.12 units/mL collagenase type I (Sigma) at 37 ℃ for 1 hour in a shaking water bath (80 Hz). The samples were then filtered through sterile 250 μm nylon mesh (scr NY250HC, Milian) to remove undigested debris. The resulting suspension was centrifuged at 1100RPM for 10 minutes to separate SVF from adipocytes. Adipocytes were removed and washed with DMEM buffer. Then, the user can use the device to perform the operation,they were suspended in peqgoldtifst reagent (Axonlab) and RNA was isolated according to the manufacturer's instructions. SVF fractions were in erythrocyte lysis buffer (0.154mM NH)₄Cl，10mM KHCO₃0.1mM EDTA) for 2 minutes. Then, the cells were centrifuged at 1100RPM for 10 minutes and resuspended in 500. mu.l of peqGOLD TriFast reagent (Axolla) for RNA isolation.

RNA extraction and real-time PCR

Total RNA was isolated from cultured cells using the peqGOLD TriFast reagent according to the manufacturer's instructions (Axonlab). Random primers and Superscript II (Invitrogen) were used to synthesize first strand cDNA from 0.5. mu.g total RNA. Real-time PCR was performed using Power SYBR Green Mix (Applied biosystems). The following primers were used for the mouse genes: SEQ ID NO.9(Plac 8-Forward), SEQ ID NO.10(Plac 8-reverse), SEQ ID NO.11(PPAR γ 2-F), SEQ ID NO.12(PPPAR γ 2-R), SEQ ID NO.13(Ap2-F), SEQ ID NO.14(Ap2-R), SEQ ID NO.15 (cyclophilins A-F), SEQ ID NO.16 (cyclophilins A-R). The following primers were used for the human gene: SEQ ID NO.17 (hParac 8-F), SEQ ID NO.18 (hParac 8-R), SEQ ID NO.19(h cyclophilin A-F) and SEQ ID NO.20(h cyclophilin A-R).

Northern blotting

Total RNA from various mouse tissues was isolated using the peqGOLD TriFast reagent according to the manufacturer's instructions (Axonlab). Total RNA (8. mu.g) was split on a 1, 2% agarose/formaldehyde gel and transfected overnight to nylon membranes. To serve as a control for RNA quantity loading, membranes were stained with methylene blue, followed by subsequent hybridization. For detection of Plac8 signal, probes from full-length cDNA mouse plasmids (Open biosystems) were used. By using alpha-³²p]dCTP (Amersham) was randomly primed to label the probes. Hybridization and washing were performed using the Quickhib method according to the manufacturer's instructions (Stratagene). The blot was exposed to Hyperfilm ECL (Amersham) at-80 ℃ for 1 or several days, depending on signal intensity.

Results

Example 1: microarray results

Bioinformatic analysis of the microarray data was performed to identify genes that met the following three criteria: (i) is regulated by a high-fat diet, (ii) expression in both visceral and subcutaneous fat is similarly regulated by a high-fat diet and (iii) its expression is similarly normalized by rimonabant treatment (figure 1). Of the approximately 17,000 gene targets present on the cDNA microarray used, 34 genes meet these criteria, which are listed in table 1. Notably, 10 of these genes, Cavl, Fgfl, Fndc3b, Kif5b, Mest, Npr3, Pik3ca, Sparc, Vldlr, and Wwtrl, were previously known to be important regulators of adipose tissue development and function. Some of these genes have expression levels (shown in grey in table 1) that correlate with weight gain, suggesting a potential role in the proliferation and/or hypertrophy of adipose tissue during obesity. These results demonstrate a method for identifying potential novel targets for therapeutic treatment of obesity.

Most importantly, many of the genes cited in table 1 have not been studied in the context of adipose tissue development or biology. These genes belong to the following classes of functions: extracellular matrix/cell interactions, cytoskeleton, intracellular signaling, enzymes, and transcription factors/cofactors. They are likely to be involved in tissue reconstruction and in particular adipocyte development. One of these genes, the Plac8 gene and its role in adipocyte biology is presented herein and constitutes an aspect of the invention.

The mouse and human sequences of Plac8 as used in the present invention correspond to SEQ ID NO.1 and SEQ ID NO.3, respectively.

Table 1: list of 34 gene candidates regulated by HFD and rimonabant in SCAT and VAT. The full name and gene symbols are shown in the first column. The biological roles and indices of known genes are indicated in the second column. All of these genes were up-regulated by HFD, except Plac8 and Rp9h, which were down-regulated by HFD, and normalized by rimonabant treatment. Genes associated with weight gain are shown in grey.

Example 2: tissue and cellular expression of selected genes

To better understand the role of Plac8 in adipocyte development, its expression pattern was first characterized. mRNA levels were measured by Northern blotting and RT-PCR in various mouse tissues, in isolated preadipocytes and adipocytes, in Visceral Adipose Tissue (VAT) and subcutaneous adipose tissue (SCAT) of a mouse obesity model (Ob/Ob mouse), and in human adipose tissue.

According to Northern blotting, it was shown that Plac8 (1 kb signal indicated by arrows in fig. 2A) was expressed at the same high level in SCAT and spleen of the diet C57BL/6J mice, and at lower levels in VAT, SCAT, muscle, heart, lung and muscle (fig. 2A). Then, the expression pattern of Plac8 was observed by microarray studies. In white adipose tissue of Ob/Ob mice, Plac8 levels were reduced compared to levels in wild-type mice (fig. 2B). Values are expressed as fold increase relative to control values for SCAT arbitrarily set at 1.

Adipose tissue is a complex tissue that includes not only mature adipocytes, but also precursor cells such as preadipocytes and blood vessels, macrophages, and fibroblasts. Based on collagenase I digestion technique, Stromal Vascular Fraction (SVF), including preadipocytes, endothelium and macrophage cells, was separated from the isolated fat cell fraction. Plac8 was found to be expressed predominantly in stromal vascular fractions containing preadipocytes (fig. 2C). These results indicate that Plac8 is more expressed in preadipocytes, and thus appears to be involved in differentiation or proliferation processes.

The next step is to determine whether the Plac8 gene is conserved across species. To solve this problem, RT-PCR was performed on human adipose tissue samples. Preadipocytes and adipocytes were isolated from SCAT or VAT. Isolated preadipocytes were induced to differentiate in vitro until day 7. The results show that Plac8 is indeed expressed in human fat (fig. 2D). They indicated that these genes are present in human adipose tissue. Together, these results suggest that Plac8 is a relevant candidate gene for adipocyte development, possibly required for adipogenesis or adipose tissue augmentation in obesity.

Example 3: expression of selected genes during differentiation of 3T3-L1

Next, expression of Plac8 gene was evaluated during lipogenesis. For this purpose, mRNA levels were measured by RT-PCR during the detailed differentiation time course of 3T3-L1 (adipogenic cell line) (fig. 2E). Experiments showed that Plac8 was significantly elevated in the early steps (1 to 3 hours after DMI treatment). This pattern is interesting because known adipogenic transcription factors such as CEBP β and γ (Rosen e.d. et al, 202), Krox20(Chen, z. et al, 2005) and Ebf (Jiminez, MA et al, 2007) show similar expression, suggesting that this gene is involved in the early steps of adipogenesis.

Example 4: ShRNA knockdown of Plac8 in 3T3-L1 cells induces adipogenesis

For loss of function studies, shRNA specific for Plac8 (RNAi-Ready pSIREN-RetroQ ZsGreen or pSIREN) subcloned into a retroviral vector from Clontech was used. This plasmid contained a GFP marker, which served as a control for infection efficiency in 3T3-L1 cells. Three different shrnas against Plac8 were cloned into the pSIREN plasmid and first tested in 293T HEK cells. This experiment demonstrates the ability of shRNA specific for Plac8 to inhibit Plac8 expression. Interestingly, 75% and 40% knockdown was achieved with shPlac8-2 and shPlac8-3, respectively (FIG. 3A), so that they were both used for transduction into 3T3-L1 cells.

Then, 3T3-L1 cells were infected with a retroviral vector expressing shRNA against Plac8 (shprac 8) or luciferase (shLuc) for 6 hours. Using the GFP marker, we observed 90% infection in 3T3-L1 cells. On day 0, 50% of the Plac8 knockdown was obtained in cells infected with both shPlac8-2 and shPlac8-3 (fig. 3B), whereas no inhibition was obtained with the shLuc control. Then, the cells were allowed to reach confluence and, after one week, differentiated with DMI. After 7 to 10 days of differentiation, the cells were stained by oil red O staining to determine the amount of lipid content. Knock-down of Plac8 induced adipogenesis as shown by lipid staining and a decrease in adipogenesis markers in cells transfected with shprac 8 compared to control cells transfected with shLuc (fig. 3C and 3D).

Example 5: overexpression of Plac8 in the 3T3-L1 cell line increased adipogenesis.

For the function acquisition study, the cDNA of the murine sequence of Plac8 was subcloned into the pMSCV retrovirus plasmid from Clontech. After infection of 3T3-L1 cells, the RNA level of Plac8 was measured by RT-PCR. On day 0, we obtained 3, 5-fold induction of Plac8 in 3T3-L1 cells overexpressing Plac8 (L1 Plac8) compared to control cells infected with the empty plasmid (L1 control) before differentiation (fig. 4A). Cells were allowed to reach confluence and differentiated with DMI. On days 4 and 9, cells were stained with oil red O in terms of lipid content. As shown in fig. 4B, overexpression of Plac8 increased the adipogenic potential of 3T 3-L1. A differentiation marker (PPARg2) was also measured by RT-PCR and the results showed a 54% increase in 3T3-L1 overexpressing Plac8 at day 9 compared to control cells (fig. 4C).

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Claims (15)

1. An inhibitor of Plac8 activity for use in modulating adipogenesis.

2.The inhibitor according to claim 1, which reduces adipogenesis.

3. An inhibitor according to claim 1 or 2 for use in the treatment of obesity and related disorders.

4. The inhibitor according to claim 1 or 2 for use in reducing visceral and/or subcutaneous fat accumulation.

5. The inhibitor according to any one of the preceding claims, wherein the inhibitor is a small molecule.

6. The inhibitor according to any one of the preceding claims, wherein the inhibitor is a small interfering RNA.

7. The inhibitor according to claim 7, wherein the siRNA is shRNA having a sequence corresponding to SEQ ID No.5 or SEQ ID No.6 or SEQ ID No. 7.

Use of an inhibitor of Plac8 activity for the manufacture of a medicament for modulating adipogenesis.

9. Nucleic acid having the sequence SEQ ID NO.6 or SEQ ID NO. 7.

10. A nucleic acid which is an siRNA specific for transcription inhibition by Plac 8.

11. A method for screening for an inhibitor of Plac8 activity comprising

a) Transfecting a cell line with a reporter construct comprising a Plac8 promoter linked to a reporter gene;

b) culturing said cell line in conditions permitting expression of said reporter gene;

c) adding a candidate compound to the cell culture, and

d) identifying inhibitor compounds as those compounds that have the ability to decrease or inhibit the expression of the reporter gene.

12. A composition comprising an inhibitor of Plac8 activity and at least one pharmaceutically acceptable excipient.

13. A composition according to claim 12 for use in the treatment of obesity and related diseases.

14. A composition according to claim 12 for use in reducing visceral and/or subcutaneous fat accumulation.

15. A method of modulating adipogenesis consisting in administering to a patient in need thereof an inhibitor of Plac8 to modulate adipogenesis.