WO2011126790A1 - Methods and compositions for inducing brown adipogenesis - Google Patents

Abstract

Methods and materials for inducing brown adipose and for treating obesity using FGF6 and/or FGF9 are provided.

Description

METHODS AND COMPOSITIONS FOR INDUCING BROWN ADIPOGENESIS

CLAIM OF PRIORITY

This application claims the benefit of U.S. Patent Application Serial No. 61/318,565, filed on March 29, 2010, the entire contents of which are hereby incorporated by reference.

TECHNICAL FIELD

This document provides methods and materials related to treating obesity and related disorders. For example, this document provides methods for using fibroblast growth factors such as FGF6 and/or FGF9 for decreasing fat stores or weight in a subject, preventing weight gain in a subject, and determining if a subject is at risk for weight gain or obesity.

BACKGROUND

Obesity, and disorders associated with obesity such as diabetes, are a major global health concern. Obesity is generally associated with an abnormal accumulation of fat cells. The most commonly known fat cells are white fat cells, also known as white adipose tissue (WAT) cells, which have a thin ring of cytoplasm surrounding a lipid or fat droplet. WAT is found underneath the skin and provides heat insulation, cushioning against shock and jarring, and energy reserves. An average lean person has roughly 20 to 40 billion WAT cells. An obese person can have up to ten times more WAT than the average lean person.

The less common fat cells are the brown fat cells, also known as brown adipose tissue (BAT) cells. Energy expenditure for thermogenesis in BAT serves either to maintain body temperature in the cold or to waste food energy. It has roles in thermal balance and energy balance, and when defective, is usually associated with obesity. BAT is typically atrophied in obese animals. The importance of BAT in overall energy homeostasis is underscored by the finding that ablation of BAT in mice results in severe obesity accompanied by insulin resistance, hyperglycemia, hyperlipidemia, and hypercholesterolemia (Lowell at al., Nature 366(6457):740-2 (1993); Hamann et al, Diabetes 44(11): 1266-73 (1995); Hamann et al, Endocrinology 137(l):21-9 (1996)).

Adipose tissues contain a potential mitotic compartment, which can allow for growth and differentiation of WAT or BAT cells. Adipose tissue can be readily assayed using routine techniques. An exemplary assay for adipose cells is the Oil Red O lipophilic red dye assay. The dye is used to stain neutral lipids in cells. The amount of staining is directly proportional to the amount of lipid in the cell and can be measured spectrophotometrically. The amount of lipid accumulation is determined as a parameter of differentiation. WAT and BAT can be distinguished by routine techniques, e.g., morphologic changes specific to WAT or BAT, or evaluation of WAT-specific or BAT-specific markers. For example, BAT cells can be identified by expression of uncoupling protein (UCP), e.g., UCP-1.

Fibroblast Growth Factors (FGFs) are a large family of signaling molecules that function as important regulators of many aspects of embryogenesis and organogenesis.

Processes regulated by FGFs include, for example, cell proliferation and differentiation, tissue remodeling and regeneration, and postnatal growth. The FGF signaling cascade is initiated by the binding of FGFs to specific transmembrane receptor tyrosine kinase receptors (FGFRs) and heparan sulfate proteoglycans (HSPGs). Localization studies in human, chick, mouse, and frog tissues have demonstrated high levels of mRNA expression and protein synthesis for various FGFs and their receptors in adipose cells, epithelial and mesenchymal cells, neural tube, retina, teeth, and limb bud. Differences in spatial and temporal expression of FGFs and their receptors allow the 22 members of the FGF family to elicit different effects during embryonic development and in adult cells.

SUMMARY

This document relates to methods and materials related to adipose differentiation. The present invention is based at least in part on the discovery that FGF6 and FGF9 play an important role in adipocyte differentiation. In particular, it has been found that FGF6 and FGF9 promote brown adipocyte (BAT) differentiation. Since brown adipose tissue is specialized for energy expenditure, the methods described herein are useful for the treatment of obesity and related disorders, such as diabetes (e.g., Type 2 diabetes).

In one aspect, this document features a method of promoting brown adipocyte differentiation. The method can comprise contacting a target cell or tissue with a composition comprising a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) polypeptide or fragment thereof in an amount sufficient to promote brown adipocyte differentiation, thereby producing a differentiated brown adipose cell or tissue.

In another aspect, this document features a method of decreasing fat stores or weight in a subject or preventing weight gain. The method can comprise identifying a subject in need of decreasing fat stores or weight or at risk of obesity or an obesity-related disease; obtaining one or more target cells or tissues from the subject; contacting the target cells or tissues with a composition comprising a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) polypeptide, or fragment thereof, in an amount sufficient to promote brown adipocyte differentiation, thereby producing one or more differentiated brown adipose cells or tissues; and administering to the subject the one or more differentiated brown adipose cells or tissues.

In a further aspect, this document features a method for providing a cell culture enriched in brown adipocytes. The method can comprise providing a plurality of target cells in vitro; contacting the plurality of cells with a composition comprising a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) polypeptide, or fragment thereof, in an amount sufficient to promote brown adipocyte differentiation. The fibroblast growth factor 6 (FGF6) polypeptide can be a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO: 1. The fibroblast growth factor 9 (FGF9) polypeptide can be a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO:2. The composition can further comprise a fibroblast growth factor 2 (FGF2) polypeptide, e.g., a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO:3, or fragment thereof. The composition can further comprise a bone morphogenetic protein (BMP) polypeptide or fragment thereof, e.g., BMP6 or BMP7 or a polypeptide or fragment thereof. The target cell or tissue can comprise a brown adipocyte or brown preadipocyte. The target cell or tissue can comprise a white preadipocyte or white adipocyte. The target cell or tissue can comprise a stem cell, e.g., an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell. The target cell or tissue can be in culture. The target cell or tissue can be in or can be isolated from a living subject. The subject can be an obese human subject. The level of brown adipocyte differentiation can be further evaluated by measuring morphological changes specific to brown adipocytes.

The method can further comprise implanting the differentiated brown adipose cell or tissue in a subject. The method can further comprise evaluating brown adipocyte differentiation by measuring a level of a brown adipocyte marker selected from the group consisting of PPAR gamma 2 (PPARy2), PPAR-gamma Coactivator 1 (PGC-1), cytochrome oxidase activity, and mitochondrial DNA levels, wherein the level of the brown adipocyte marker indicates the level of brown adipocyte differentiation. The method can further comprise implanting at least one brown adipocyte from said enriched culture into a subject.

In another aspect, this document features a method of determining if a subject is at risk for weight gain or obesity. The method can comprise obtaining a test sample from the subject; evaluating the expression, protein level or activity of one or more of a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) in the test sample; and comparing the expression, protein level or activity of the FGF6 or FGF9 in the test sample to that in a control, wherein a decrease in the expression, protein level or activity of the FGF6 or FGF9 in the test sample relevant to the control indicates that the subject is at risk for weight gain or obesity. The sample can comprise one or more of a brown adipocyte, a white adipocyte, a brown preadipocyte, and a white preadipocyte.

In a further aspect, this document features a method of identifying a test compound that promotes brown adipocyte differentiation. The method can comprise contacting a target cell or tissue, e.g., a preadipocyte or adipocyte cell or tissue, in vitro with a test compound; evaluating the effect of the test compound on the target cell or tissue in vitro by measuring expression and/or activity of a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9), wherein an increase in FGF6 or FGF9 expression and/or activity indicates the test compound is a candidate agent for promoting brown adipocyte differentiation. The method can further comprise evaluating an effect of the test compound on brown adipocyte differentiation in the target cell or tissue by measuring a level of a brown adipocyte marker selected from the group consisting of PPAR gamma 2 (PPARy2), PPAR-gamma Coactivator 1 (PGC-1), cytochrome oxidase activity, and mitochondrial DNA levels, wherein the level of the brown adipocyte marker indicates the level of brown adipocyte differentiation.

In another aspect, this document features a method of identifying a candidate compound for the treatment of obesity. The method can comprise selecting a candidate agent that promotes brown adipocyte differentiation identified by the method of claim 20;

providing an animal model of obesity; administering the candidate agent that promotes BAT differentiation to the animal model; and evaluating the effect of the candidate agent on obesity in the animal model; wherein an agent that decreases obesity in the animal model is a candidate therapeutic agent for the treatment of obesity. The method can further comprise administering the candidate therapeutic agent for the treatment of obesity to a subject in a clinical trial, and evaluating the effect of the candidate therapeutic agent on obesity in the subject.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the effect on UCP-1 mRNA expression in adipocytes following treatment with various concentrations of FGF6, FGF9, and FGF2.

Figure 2 is a graph showing the effect on UCP-1 mRNA expression normalized to levels of ATP in adipocytes following treatment with various concentrations of FGF6, FGF9, and FGF2.

DETAILED DESCRIPTION

This document relates to methods and materials related to adipose differentiation. The present invention is based at least in part on the discovery that FGF6 and FGF9 are involved in adipocyte differentiation. Specifically, FGF6 and FGF9 promote brown adipocyte (BAT) differentiation. Without being bound by any particular theory, it is proposed that FGF6 and FGF9 promote adipocyte differentiation and increase expression of UCP-1, which is a key mediator of energy expenditure in brown fat. By promoting adipocyte differentiation, FGF6 and FGF9 can increase energy expenditure and promote weight loss. FGF6 and FGF9 are thus therapeutic, diagnostic, and drug discovery targets for adipose- related disorders, such as obesity and related disorders such as diabetes, insulin resistance, hyperglycemia, hyperlipidemia, and hypercholesterolemia.

As used herein, the term "preadipocyte" refers to adipocyte precursor cells which proliferate and differentiate to form mature adipocytes.

Fibroblast Growth Factors (FGFs)

Secreted FGF proteins have been used clinically for wound healing and

revascularization of tissues. See, e.g., Moya et al, Microvascular Research 78: 142-147 (2009); Muneuchi et al., Burns 31 :514-517 (2004). FGF polypeptides can be viable therapeutic agent because FGFs, most of which have amino-terminal signal peptides and are readily secreted from cells, are internalized into their target cells where they exert their activity. Although the human proteins are described herein, one of skill in the art will appreciate that when another species is the intended recipient of the treated cells, homologous proteins from that species can also be used, e.g., cow, pig, sheep, or goat. Such homologous proteins can be identified, e.g., using methods known in the art, e.g., searching available databases for homologs identified in the target species, e.g., the homologene database.

FGF6

FGF6 is a 208 amino acid protein, as shown below (SEQ ID NO: 1), and is a member of the FGF family of heparin-binding fibroblast growth factors. FGF6 induces angiogenesis, cartilage formation, cell differentiation, and tissue regeneration. FGF6 is also involved the regulation of a variety of developmental processes in the brain. See, e.g., Ozawa et al, Brain Res. Mol. Brain Res. 41 :279-288 (1996). The human FGF6 sequence is set forth in Coulier et al, Oncogene 6(8): 1437-44 (1991).

1 MALGQKLFIT MSRGAGRLQG TLWALVFLGI LVGMWPSPA GTRANNTLLD SRGWGTLLSR

61 SRAGLAGEIA GVNWESGYLV GIKRQRRLYC NVGIGFHLQV LPDGRISGTH EENPYSLLEI

121 STVERGWSL FGVRSALFVA MNSKGRLYAT PSFQEECKFR ETLLPNNYNA YESDLYQGTY

181 IALSKYGRVK RGSKVSPIMT VTHFLPRI (SEQ ID NO : 1 )

FGF9

FGF9 is a 208 amino acid protein, as shown below (SEQ ID NO:2). FGF9 induces angiogenesis, vascularization, osteoblast differentiation, and chondrocyte differentiation. FGF9 is also involved in tooth development. The sequence is set forth in Miyamoto et al, Mol. Cell Biol. 13(7):4251-9 (1993).

1 MAPLGEVGNY FGVQDAVPFG NVPVLPVDSP VLLSDHLGQS EAGGLPRGPA VTDLDHLKGI

61 LRRRQLYCRT GFHLEIFPNG TIQGTRKDHS RFGILEFISI AVGLVSIRGV DSGLYLGMNE

121 KGELYGSEKL TQECVFREQF EENWYNTYSS NLYKHVDTGR RYYVALNKDG TPREGTRTKR

181 HQKFTHFLPR PVDPDKVPEL YKDILSQS (SEQ ID NO : 2 )

Combinations

In some cases, other FGFs such as FGF2 (also known as basic fibroblast growth factor or bFGF) can be used for the methods provided herein. FGF2 belongs to the 22- member family of heparin-binding fibroblast growth factors. The sequence is set forth in Watson et al, Biochem. Biophys. Res. Commun. 187(3): 1227-31 (1992). FGF2 induces angiogenesis, fibroblast proliferation, cell differentiation, neurogenesis, and vascular remodeling. See, e.g., Garcia and Obregon, Am. J. Physiol. Cell Physiol., 282:C105-C1 12 (2002). In some cases, combinations of FGFs and other growth factors can be used for the methods provided herein. For example, the methods provided herein can be practiced using a combination of two or more growth factors. In some cases, FGF6 and/or FGF9 can be used in combination with FGF2 or another FGF (e.g., FGF6 + FGF9, FGF9 + FGF2). In other cases, FGF6 and/or FGF9 can be used in combination with members of other signaling pathways including, for example, the Bone Morphogenetic Pathway or the canonical or noncanonical Wnt pathways. For example, FGF6 and/or FGF9 can be used in combination with a member of the Bone Morphogenetic Pathway (BMP) such as BMP2, -4, -5, -7, -14 or BMP3, -6 (e.g., FGF6 + BMP7, FGF9 + BMP7, FGF6 + BMP6, FGF9 + BMP6). See, e.g., U.S. Pat. No. 7,576,052; U.S. Patent Publication No. US2005/0187154.

Pharmacokinetic Properties and Therapeutic Activity

Modifications can be made to a protein that result in pharmacokinetic properties of the polypeptide which are desirable for use in protein therapy. For example, such modifications can result in longer circulatory half-life, an increase in cellular uptake, improved distribution to targeted tissues, a decrease in clearance and/or a decrease of immunogenicity. Several art-recognized approaches useful to optimize the therapeutic activity of a protein, e.g., a therapeutic polypeptide described herein (e.g., a FGF6 and/or FGF9 polypeptide or polypeptide having at least 95% sequence identity (e.g., 95, 96, 97, 98, 99, and 100% sequence identity) to SEQ ID NO: l or SEQ ID NO:2), are summarized below.

Expression System

For recombinant proteins, the choice of expression system can influence

pharmacokinetic characteristics. Differences between expression systems in post- translational processing lead to recombinant proteins of varying molecular size and charge, which can affect circulatory half-life, rate of clearance and immunogenicity, for example. The pharmacokinetic properties of the protein may be optimized by the appropriate selection of an expression system, such as selection of a bacterial, viral, or mammalian expression system. Exemplary mammalian cell lines useful in expression systems for therapeutic proteins are Chinese hamster ovary, (CHO) cells, the monkey COS-1 cell line, and the CV-1 cell line.

Chemical Modification

A protein can be chemically altered to enhance the pharmacokinetic properties while maintaining activity. The protein can be covalently linked to a variety of moieties, altering the molecular size and charge of the protein and consequently its pharmacokinetic characteristics. The moieties are preferably non-toxic and biocompatible. In one

embodiment, poly-ethylene glycol (PEG) can be covalently attached to the protein

(PEGylation). PEG is a class of polymers comprised of repeating ethylene oxide subunits with terminal hydroxyl groups. A variety of PEG molecules are known and/or commercially available. See, e.g., Sigma-Aldrich catalog. PEG molecules are available in various lengths, molecular weights, and substitution patterns, and may be linear or branched. PEG is attached to the protein via an activated terminal hydroxyl group; preferably, the hydroxyl group is activated as an ester, carbonate, aldehyde or tresylate. The activated hydroxyl reacts with nucleophilic groups on the protein, forming a linkage between the protein and PEG. Often the nucleophilic group is the amino group of a lysine or the N-terminus of the protein. One or multiple chains of PEG may be attached to the protein. The choice of site(s) and functionality of the linkage of PEGylation and the choice of PEG molecule can be optimized to achieve the desired pharmacokinetic properties. PEGylation can increase the stability of the protein, decrease immunogenicity by steric masking of epitopes, and improve half-life by decreasing glomerular filtration. See, e.g., Poly (ethylene glycol): chemistry and biological applications, Harris and Zalipsky, eds., ACS Symposium Series, No. 680, 1997; Harris et al, Clinical Pharmacokinetics 40:7, 485-563 (2001). Examples of therapeutic proteins administered as PEG constructs include Adagen (PEG-ADA) and Oncospar (Pegylated asparaginase). In another embodiment, the protein can be similarly linked to oxidized dextrans via an amino group. See Sheffield, Current Drug Targets - Cardiovas. and Haemal Dis. 1 : 1-22 (2001).

Furthermore, the therapeutic polypeptide can be chemically linked to another polypeptide. The therapeutic polypeptide can be cross-linked carrier protein to form a larger molecular weight complex with longer circulatory half-life and improved cellular uptake. In one embodiment, the carrier protein can be a serum protein, such as albumin. The therapeutic polypeptide can be attached to one or more albumin molecules via a Afunctional cross- linking reagent. The cross-linking reagent may be homo- or heterofunctional. In another embodiment, the therapeutic protein can cross-link with itself to form a homodimer, trimer, or higher analog. Again, either heterobifunctional or homobifunctional cross-linking reagents can be used to form the dimers or trimers. See Stykowski et al, Proc. Natl. Acad. Sci. USA, 95: 1184-1 188 (1998). Increasing the molecular weight and size of the therapeutic protein through dimerization or trimerization can decrease clearance. Modification of Polypeptide Formulation

The formulation of the polypeptide may also be changed. The therapeutic polypeptide can be formulated in a carrier system.

The carrier protein can be a colloidal system. The colloidal system can be liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic protein is encapsulated in a liposome while maintaining protein integrity. As one skilled in the art would appreciate, there are a variety of methods to prepare liposomes. See Lichtenberg et al, Methods Biochem. Anal. 33:337-462 (1988); Anselem et al, LIPOSOME TECHNOLOGY, CRC Press, 1993. Liposomes can be prepared from an assortment of phospholipids varying in size and substitution, and may also contain additional components with low toxicity, such as cholesterol. The liposome can be formulated and isolated in a variety of shapes and sizes. Additionally, moieties may attached to the surface of the liposome to further enhance the pharmacokinetic properties of the carrier. The moieties may be attached to phospholipid or cholesterol molecules, and the percentage of the moiety incorporated on the surface may be adjusted for optimal liposome stability and pharmacokinetic characteristics. One

embodiment comprises a liposome with polyethylene glycol (PEG) added to the surface. Liposomal formulations can delay clearance and increase cellular uptake. See Reddy, Annals of Pharmacotherapy, 34(7/8):915-923 (2000).

The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic protein can be embedded in the polymer matrix while maintaining protein integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly(I-hydroxy) acids. Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly-lactic/glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and

nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. See Reddy, Annals of Pharmacotherapy , 34(7/8):915-923 (2000). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. See Kozarich and Rich, Chemical Biol. 2:548-552 (1998).

Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99/15154 (Tracy et al), U.S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale et al), PCT publication WO 96/40073 (Zale et al), and PCT publication WO

00/38651 (Shah et al). U.S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96/40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

Administration

An agent that modulates FGF6 and/or FGF9 signaling, e.g., an agent described herein, such as a FGF6 and/or FGF9 polypeptide, can be administered to a subject by standard methods. For example, the agent can be administered ex vivo or in vivo by any of a number of different routes including intravenous, intradermal, subcutaneous, percutaneous injection, oral (e.g., inhalation), transdermal (topical), and transmucosal. In one embodiment, the modulating agent can be administered orally. In another embodiment, the agent is administered by injection, e.g., intramuscularly, or intravenously. In some cases, administration can be ex vivo administration. An ex vivo strategy can involve transfecting or transducing cells or simply treating cells obtained from the subject to be treated (or another subject) with FGF6 and/or FGF9 polypeptides or with a polynucleotide encoding a polypeptide that modulates (e.g., inhibits or enhances) FGF6 and/or FGF9 signaling. The transfected or transduced cells are then administered to the subject. For example, a nucleic acid encoding a polypeptide such as FGF6 and/or FGF9 polypeptide can be introduced into isolated cells, and the modified cells can be administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g., U.S. Patent Nos. 4,892,538 and 5,283, 187). The cells can be any of a wide range of types including, without limitation, adipose cells, fibroblasts, epithelial cells, endothelial cells, keratinocytes, neurons, or muscle cells. The agent can be encapsulated or injected, e.g., in a viscous form, for delivery to a chosen site, e.g., a site of adipose tissue, e.g., a subcutaneous or intra-abdominal adipose pad. The agent can be provided in a matrix capable of delivering the agent to the chosen site. Matrices can provide slow release of the agent and provide proper presentation and appropriate environment for cellular infiltration. Matrices can be formed of materials presently in use for other implanted medical applications. The choice of matrix material is based on any one or more of: biocompatibility, biodegradability, mechanical properties, cosmetic appearance and interface properties. One example is a collagen matrix. The agent, e.g., a FGF6 and/or FGF9 polypeptide, nucleic acid molecule, analog, mimetic or modulators (e.g., organic compounds or antibodies (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration to a subject, e.g., a human. Such compositions typically include the polypeptide, nucleic acid molecule, modulator, or antibody and a pharmaceutically acceptable carrier. As used herein the language "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances are known. Except insofar as any conventional media or agent is incompatible with the active compound, such media can be used in the compositions of the invention. Supplementary active compounds can also be incorporated into the compositions.

A pharmaceutical composition can be formulated to be compatible with its intended route of administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an agent described herein) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRTMOGEL™ (sodium carboxymethyl starch), or corn starch; a lubricant such as magnesium stearate or

STEROTES™; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

As described herein, compositions can administered to a subject at a dosage sufficient to achieve the desired therapeutic effect. In general, therapeutically effective dosages may be determined by either in vitro or in vivo methods. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described in Freireich et al, Cancer Chemother. Rep. 50:219 (1966). Body surface area can be approximately determined from height and weight of the subject. See, e.g., Scientific Tables, Geigy Pharmaceuticals, Ardsley, N.Y., 1970, 537. Dosage values may vary according to factors such as the disease state, age, sex, and weight of the individual.

The nucleic acid molecules described herein can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al, PNAS 91 :3054-3057 (1994)). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system. The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

In some embodiments, the pharmaceutical composition is injected into a tissue, e.g., an adipose tissue.

Gene Therapy

The nucleic acids described herein, e.g., an antisense nucleic acid described herein, or a FGF6 and/or FGF9 polypeptide encoding nucleic acid, can be incorporated into a gene construct to be used as a part of a gene therapy protocol to deliver nucleic acids encoding either an agonistic or antagonistic form of an agent described herein, e.g., a FGF6 and/or FGF9. The invention features expression vectors for in vivo or ex vivo transfection and expression of a FGF6 and/or FGF9 polypeptide described herein in particular cell types. In some cases, expression vectors for in vivo or ex vivo transfection and expression of different combinations of growth factors can be used (e.g., vectors for transfecting and expressing combinations of two or more FGF polypeptides or an FGF and a BMP polypeptide).

Expression constructs of such components may be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the component gene to cells in vivo. Approaches include insertion of the subject gene in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus- 1, or recombinant bacterial or eukaryotic plasmids. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (e.g., LIPOFECTIN™) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaP0₄ precipitation carried out in vivo.

One approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid, e.g., a cDNA, encoding an alternative pathway component described herein. Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid. Additionally, molecules encoded within the viral vector, e.g., by a cDNA contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.

Retrovirus vectors and adeno-associated virus vectors can be used as a recombinant gene delivery system for the transfer of exogenous genes in vivo, particularly into humans. Retroviruses are also commonly used vector for ex vivo delivery of an exogenous gene. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. The development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are characterized for use in gene transfer for gene therapy purposes. For review, see Miller, Blood, 76:271-78 (1990). A replication defective retrovirus can be packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are known to those skilled in the art. Examples of suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include *Crip, *Cre, *2 and *Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, in vitro and/or in vivo (see for example Eglitis, et al, Science 230: 1395-1398 (1985); Danos and Mulligan, Proc. Natl. Acad. Sci. USA 85:6460-6464 (1988); Wilson et al, Proc. Natl. Acad. Sci. USA 85:3014-3018 (1988); Armentano et al, Proc. Natl. Acad. Sci. USA 87:6141-6145 (1990); Huber et al, Proc. Natl. Acad. Sci. USA 88:8039-8043 (1991); Ferry et al, Proc. Natl. Acad. Sci. USA 88:8377-8381 (1991);

Chowdhury et al, Science 254: 1802-1805 (1991); van Beusechem et al, Proc. Natl. Acad. Sci. USA 89:7640-7644 (1992); Kay et al, Human Gene Therapy 3:641-647 (1992); Dai et al, Proc. Natl. Acad. Sci. USA 89: 10892-10895 (1992); Hwu et al, J. Immunol. 150:4104- 4115 (1993); U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT Application WO 92/07573).

Another viral gene delivery system useful in the present invention utilizes adenovirus- derived vectors. The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See, for example, Berkner et al., BioTechniques 6:616 (1988); Rosenfeld et al, Science 252:431-434 (1991); and Rosenfeld et al, Cell 68: 143-155 (1992). Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are known to those skilled in the art.

Recombinant adenoviruses can be advantageous in certain circumstances in that they are not capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including epithelial cells (Rosenfeld et al. (1992), supra). Furthermore, the virus particle is relatively stable and amenable to purification and concentration, and as above, can be modified so as to affect the spectrum of infectivity. Additionally, introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situ where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA). Moreover, the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. (1998), supra; Haj-Ahmand and Graham ,J. Virol. 57:267 (1986)).

Yet another viral vector system useful for delivery of the subject gene is the adeno- associated virus (AAV). Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al, Curr. Topics in Micro, and Immunol. \5%:91 -129 (1992)). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al, Am. J. Respir. Cell. Mol. Biol. 7:349-356 (1992); Samulski et al, J. Virol. 63 :3822-3828 (1989); and McLaughlin et al., J. Virol. 62: 1963-1973 (1989)). Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector such as that described in Tratschin et al, Mol. Cell. Biol. 5:3251-3260 (1985) can be used to introduce DNA into cells. A variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al, Proc. Natl. Acad. Sci. USA 81 :6466-6470 (1984); Tratschin et al, Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al, Mol. Endocrinol. 2:32-39 (1988); Tratschin et al, J. Virol. 51 :61 1-619 (1984); and Flotte et al., J. Biol. Chem.

268:3781-3790 (1993)). In some cases, lenti-viral vectors can be used.

In addition to viral transfer methods, such as those illustrated above, non-viral methods can also be employed to cause expression of an nucleic acid agent described herein (e.g., a FGF6 and/or FGF9 polypeptide encoding nucleic acid) in the tissue of a subject. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In some embodiments, nonviral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the subject gene by the targeted cell. Exemplary gene delivery systems of this type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes. Other embodiments include plasmid injection systems such as are described in Meuli et al, J. Invest. Dermatol. 116(1): 131-135 (2001); Cohen et al, Gene Ther. 7(22): 1896-905 (2000); or Tarn et al, Gene Ther. 7(21): 1867-74 (2000).

In a representative embodiment, a gene encoding an alternative pathway component described herein can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al, No Shinkei Geka 20:547-551 (1992); PCT publication W091/06309; Japanese patent application 1047381 ; and European patent publication EP-A-43075).

In clinical settings, the gene delivery systems for the therapeutic gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art. For instance, a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the receptor gene, or a combination thereof. In other embodiments, initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized. For example, the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al, PNAS 91 : 3054-3057 (1994)). In some cases, recombinant adenovirus particles can be locally administered to the site of treatment, e.g., through the use of an injection catheter, stent or infusion pump or are directly added to cells or tissues ex vivo.

The pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery system can be produced in tact from recombinant cells, e.g. retroviral vectors, the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system..

Cell Therapy

An agent described herein for increasing FGF6 and/or FGF9 signaling, e.g., a FGF6 and/or FGF9 polypeptide or active fragment thereof, can also be increased in a subject by introducing into a cell, e.g., an adipose cell, a nucleotide sequence that encodes a FGF6 and/or FGF9 polypeptide. In some cases, a nucleotide sequence can encode a polypeptide having at least 95% amino acid sequence identity (e.g., at least 95, 96, 96, 98, or 99% sequence identity) to SEQ ID NO: 1 or SEQ ID NO:2. The nucleotide sequence can be a FGF6 and/or FGF9 encoding sequence or active fragment thereof, and any of: a promoter sequence, e.g., a promoter sequence from a FGF6 and/or FGF9 gene or from another gene; an enhancer sequence, e.g., 5' untranslated region (UTR), e.g., a 5' UTR from a FGF6 and/or FGF9 gene or from another gene, a 3' UTR, e.g., a 3 ' UTR from a FGF6 and/or FGF9 gene or from another gene; a polyadenylation site; an insulator sequence; or another sequence that modulates the expression of FGF6 and/or FGF9. The cell can then be introduced into the subject.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The determination of percent identity between two amino acid sequences is accomplished using the BLAST 2.0 program. Sequence comparison is performed using an ungapped alignment and using the default parameters (Blossom 62 matrix, gap existence cost of 11, per residue gapped cost of 1, and a lambda ratio of 0.85). The mathematical algorithm used in BLAST programs is described in Altschul et al. (Nucleic Acids Res. 25:3389-3402 (1997)).

Primary and secondary cells to be genetically engineered can be obtained from a variety of tissues and include cell types which can be maintained and propagated in culture. For example, primary and secondary cells include adipose cells, fibroblasts, keratinocytes, epithelial cells (e.g., mammary epithelial cells, intestinal epithelial cells), endothelial cells, glial cells, neural cells, formed elements of the blood (e.g., lymphocytes, bone marrow cells), muscle cells (myoblasts) and precursors of these somatic cell types. Primary cells are preferably obtained from the individual to whom the genetically engineered primary or secondary cells are administered. However, primary cells may be obtained for a donor (other than the recipient).

The term "primary cell" includes cells present in a suspension of cells isolated from a vertebrate tissue source (prior to their being plated i.e., attached to a tissue culture substrate such as a dish or flask), cells present in an explant derived from tissue, both of the previous types of cells plated for the first time, and cell suspensions derived from these plated cells. The term "secondary cell" or "cell strain" refers to cells at all subsequent steps in culturing. Secondary cells are cell strains which consist of secondary cells which have been passaged one or more times.

Primary or secondary cells of vertebrate, particularly mammalian, origin can be transfected with an exogenous nucleic acid sequence which includes a nucleic acid sequence encoding a signal peptide, and/or a heterologous nucleic acid sequence, e.g., encoding FGF6 and/or FGF9, or an agonist or antagonist thereof, and produce the encoded product stably and reproducibly in vitro and in vivo, over extended periods of time. A heterologous amino acid can also be a regulatory sequence, e.g., a promoter, which causes expression, e.g., inducible expression or upregulation, of an endogenous sequence. An exogenous nucleic acid sequence can be introduced into a primary or secondary cell by homologous recombination as described, for example, in U.S. Patent No. 5,641,670, the contents of which are incorporated herein by reference. The transfected primary or secondary cells may also include DNA encoding a selectable marker which confers a selectable phenotype upon them, facilitating their identification and isolation.

Vertebrate tissue can be obtained by standard methods such a punch biopsy or other surgical methods of obtaining a tissue source of the primary cell type of interest. For example, punch biopsy is used to obtain skin as a source of fibroblasts or keratinocytes. A mixture of primary cells is obtained from the tissue, using known methods, such as enzymatic digestion or explanting. If enzymatic digestion is used, enzymes such as collagenase, hyaluronidase, dispase, pronase, trypsin, elastase and chymotrypsin can be used.

The resulting primary cell mixture can be transfected directly or it can be cultured first, removed from the culture plate and resuspended before transfection is carried out. Primary cells or secondary cells are combined with exogenous nucleic acid sequence to, e.g., stably integrate into their genomes, and treated in order to accomplish transfection. As used herein, the term "transfection" includes a variety of techniques for introducing an exogenous nucleic acid into a cell including calcium phosphate or calcium chloride precipitation, microinjection, DEAE-dextrin-mediated transfection, lipofection, or electroporation, all of which are routine in the art.

Transfected primary or secondary cells undergo sufficient number doubling to produce either a clonal cell strain or a heterogeneous cell strain of sufficient size to provide the therapeutic protein to an individual in effective amounts. The number of required cells in a transfected clonal heterogeneous cell strain is variable and depends on a variety of factors, including but not limited to, the use of the transfected cells, the functional level of the exogenous DNA in the transfected cells, the site of implantation of the transfected cells (for example, the number of cells that can be used is limited by the anatomical site of implantation), and the age, surface area, and clinical condition of the patient.

The transfected cells, e.g., cells produced as described herein, can be introduced into an individual to whom the product is to be delivered. Various routes of administration and various sites (e.g., injection or implantation into subcutaneous, intraperitoneal (including intraomental), intramuscular, or adipose tissues) can be used. Once implanted in individual, the transfected cells produce the product encoded by the heterologous DNA or are affected by the heterologous DNA itself. For example, an individual who suffers from obesity is a candidate for administration of cells producing an agent described herein, e.g., a FGF2, FGF6, and/or FGF9 polypeptide or a fragment or analog or mimic thereof as described herein.

An immunosuppressive agent e.g., drug, or antibody, can be administered to a subject at a dosage sufficient to achieve the desired therapeutic effect (e.g., inhibition of rejection of the cells). Dosage ranges for immunosuppressive drugs are known in the art. See, e.g., Freed et al, N. Engl. J. Med. 327: 1549 (1992); Spencer et al, N. Engl. J. Med. 327: 1541 (1992); Widner et al, N. Engl. J. Med. 327: 1556 (1992)). Dosage values may vary according to factors such as the disease state, age, sex, and weight of the individual.

In some cases, cell therapy methods can also include methods of contacting pluripotent, multipotent, or progenitor cell types (e.g., stem cells or adipocyte progenitor cells) with a FGF6 and/or FGF9 polypeptide or active fragment thereof. In some embodiments, the stem cells are adult stem cells, e.g., adult stem cells derived from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood; induced pluripotent stem cells (iPS cells) obtained by in vitro manipulation of differentiated somatic cells (e.g., from adipose tissue, see Sun et al, Proc Nat Acad Sci 106: 15720-15725 (2009), or fibroblasts, see, e.g., Page et al, Cloning and Stem Cells 1 1(3):417-426 (2009); Takahashi et al, Cell.

131(5):834-5 (2007), and Yu et al, Science. 318(5858): 1917-20 (2007)); or embryonic stem cells or stem cells obtained from a placenta or umbilical cord. In some embodiments, the progenitor cells are derived from the inner ear, bone marrow, mesenchyme, skin, fat, liver, muscle, or blood. Such cell types can be obtained from the individual to whom the contacted cells are to be administered, or from a donor (i.e., an individual other than the recipient). Contacted cells can undergo a sufficient number doubling events to produce either a clonal cell strain or a heterogeneous cell strain of sufficient size to provide the individual with proteins produced following stimulation of an FGF signaling pathway in effective amounts. In some cases, the contacted cells can differentiate into brown fat cells. The contacted cells, e.g., differentiated brown fat cells or other cells contacted as described herein, can be introduced into an individual to whom the product is to be delivered. Various routes of administration and various sites can be used as known in the art or described herein.

Any appropriate method of evaluating molecular and/or morphological changes in cells contacted with a FGF6 and/or FGF9 polypeptide or active fragment thereof, or in cells following stimulation of an FGF signaling pathway, can be used. In some cases, evaluating cells can include detecting and measuring morphological changes unique to brown adipose cells. For example, Oil Red O staining can be performed. Oil Red O staining is an assay performed to stain induced adipogenic cultures to detect mature adipocytes by staining lipids. In some cases, immunohistochemical staining with antibodies directed against brown fat markers such as UCP- 1 can be performed. For example, immunohistochemical detection of UCP-1 can be used to distinguish between white and brown fat. In some cases, evaluating can include measuring the number of mitochondria and/or measuring mitochondrial activity in the contacted cells. Brown adipose cells have higher numbers of mitochondria compared to white adipose cells and other cell types. Mitochondrial activity can also be measured using methods known in the art, e.g., MITOPROFILE assays (Mitosciences), cytochrome C oxidase activity assays (e.g., from Flowgen Biosciences), or platelet A lactate assays (see, e.g., U.S. Pat No. 6261796). MitoTracker™ Green FM and MitoTracker™ Orange CM- H2TMRos (Invitrogen) are useful tools for determining respectively the mass and the oxidative activity of mitochondria, e.g., in living cells or even in vivo, see, e.g., Agnello et al., Cytotechnology 56(3): 145-149 (2008). Other methods known in the art can also be used, see, e.g., Woollacott, Journal of Biomolecular Screening, 6(6):413-420 (2001). Diagnostic Assays

The diagnostic assays described herein involve evaluating the fibroblast growth factor signaling pathway in the subject, e.g., in adipose tissue. Various art-recognized methods are available for evaluating the activity of the FGF6 and/or FGF9 signaling pathways or components thereof. For example, the method can include evaluating either the level of a FGF6 and/or FGF9 pathway component (e.g., the level of a FGF6 and/or FGF9 receptor) and/or an activity of the FGF6 and/or FGF9 pathway. Techniques for detection of FGF6 and/or FGF9 are known in the art and include, inter alia, antibody based assays such as enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.

Typically, the level in the subject is compared to the level and/or activity in a control, e.g., the level and/or activity in a tissue from a non-disease subject.

Techniques for evaluating binding activity, e.g., of FGF6 and/or FGF9 to a FGF6 and/or FGF9 binding partner, such as its receptor, include fluid phase binding assays, affinity chromatography, size exclusion or gel filtration, ELISA, immunoprecipitation (e.g., the ability of an antibody specific to a first factor, e.g., FGF6 and/or FGF9, to co- immunoprecipitate a second factor or complex, e.g., its receptor, with which the first factor can associate in nature).

Another method of evaluating the FGF6 and/or FGF9 pathway in a subject is to determine the presence or absence of a lesion in or the misexpression of a gene which encodes a component of the FGF6 and/or FGF9 pathway e.g., FGF6 and/or FGF9. The methods can include one or more of the following:

detecting, in a tissue of the subject, the presence or absence of a mutation which affects the expression of a gene encoding FGF6 and/or FGF9, or detecting the presence or absence of a mutation in a region which controls the expression of the gene, e.g., a mutation in the 5' control region;

detecting, in a tissue of the subject, the presence or absence of a mutation which alters the structure of a gene encoding FGF6 and/or FGF9;

detecting, in a tissue of the subject, the misexpression of a gene encoding

FGF6 and/or FGF9, at the mRNA level, e.g., detecting a non-wild type level of a mRNA;

detecting, in a tissue of the subject, the misexpression of the gene, at the protein level, e.g., detecting a non-wild type level of a FGF6 and/or FGF9 polypeptide. In some embodiments the methods include: ascertaining the existence of at least one of: a deletion of one or more nucleotides from a gene encoding FGF6 and/or FGF9; an insertion of one or more nucleotides into the gene, a point mutation, e.g., a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene, e.g., a translocation, inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing a probe/primer including an oligonucleotide containing a region of nucleotide sequence which hybridizes to a sense or antisense sequence from a FGF6 and/or FGF9 gene, or naturally occurring mutants thereof or 5' or 3 ' flanking sequences naturally associated with the gene; (ii) exposing the probe/primer to nucleic acid of a tissue; and detecting, by hybridization, e.g., in situ hybridization, of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion.

In some embodiments, detecting the misexpression includes ascertaining the existence of at least one of: an alteration in the level of a messenger RNA transcript of a gene encoding FGF6 and/or FGF9; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; or a non-wild type level of a gene encoding FGF6 and/or FGF9.

In some embodiments, the methods include determining the structure of a gene encoding FGF6 and/or FGF9, an abnormal structure being indicative of risk for the disorder.

In some embodiments the methods include contacting a sample from the subject with an antibody to a component of the alternative pathway protein, such as FGF6 and/or FGF9, or a nucleic acid which hybridizes specifically with the gene.

Expression Monitoring and Profiling

The presence, level, or absence of FGF6 and/or FGF9 (polypeptide or nucleic acid) in a biological sample can be evaluated by obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting the polypeptide or nucleic acid (e.g., mRNA, genomic DNA) that encodes FGF6 and/or FGF9 such that the presence of the protein or nucleic acid is detected in the biological sample. The term "biological sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject, e.g., urine. Suitable biological samples are serum or urine. The level of expression of FGF6 and/or FGF9 can be measured in a number of ways, including, but not limited to: measuring the mRNA encoded by the FGF6 and/or FGF9 gene; measuring the amount of protein encoded by FGF6 and/or FGF9; or measuring the activity of the protein encoded by the gene.

The level of mRNA corresponding to FGF6 and/or FGF9 in a cell can be determined both by in situ and by in vitro formats.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction analyses and probe arrays. One suitable diagnostic method for the detection of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA encoded by the gene being detected. The nucleic acid probe can be, for example, a full-length nucleic acid, or a portion thereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250, or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to mRNA or genomic DNA of FGF6 and/or FGF9. The probe can be disposed on an address of an array, e.g., an array described below. Other suitable probes for use in the diagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contacted with the probes, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative format, the probes are immobilized on a surface and the mRNA (or cDNA) is contacted with the probes, for example, in a two-dimensional gene chip array described below. A skilled artisan can adapt known mRNA detection methods for use in detecting the level of mRNA encoded by the gene or a component of the alternative pathway.

The level of mRNA in a sample that is encoded by a gene can be evaluated with nucleic acid amplification, e.g., by rtPCR (Mullis, U.S. Patent No. 4,683,202), ligase chain reaction (Barany, Proc. Natl. Acad. Sci. USA 88: 189-193 (1991)), self sustained sequence replication (Guatelli et al, Proc. Natl. Acad. Sci. USA 87: 1874-1878 (1990)), transcriptional amplification system (Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173-1 177 (1989)), Q-Beta Replicase (Lizardi et al, Bio/Technology 6: 1 197 (1988)), rolling circle replication (Lizardi et al, U.S. Patent No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques known in the art. As used herein, amplification primers are defined as being a pair of nucleic acid molecules that can anneal to 5' or 3 ' regions of a gene (plus and minus strands, respectively, or vice-versa) and contain a short region in between. In general, amplification primers are from about 10 to 30 nucleotides in length and flank a region from about 50 to 200 nucleotides in length. Under appropriate conditions and with appropriate reagents, such primers permit the amplification of a nucleic acid molecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared/processed and

immobilized on a support, typically a glass slide, and then contacted with a probe that can hybridize to mRNA that encodes the gene being analyzed.

In another embodiment, the methods further include contacting a control sample with a compound or agent capable of detecting mRNA, or genomic DNA of a component of the alternative pathway, and comparing the presence of the mRNA or genomic DNA in the control sample with the presence of FGF6 and/or FGF9 mRNA or genomic DNA in the test sample. In still another embodiment, serial analysis of gene expression, as described in U.S. Patent No. 5,695,937, is used to detect transcript levels of FGF6 and/or FGF9.

In some cases, the methods can further include determining the level of one or more markers of BAT in a sample. Such markers can include, without limitation, UCP-1, PPARy2, PPAR-gamma Coactivator 1 (PGC-1), cytochrome oxidase activity, and mitochondrial DNA levels. In some cases, an increase in the level of one or more BAT markers indicates an increase in BAT differentiation.

A variety of methods can be used to determine the level of FGF6 and/or FGF9 protein. In general, these methods include contacting an agent that selectively binds to the protein, such as an antibody with a sample, to evaluate the level of protein in the sample. In some embodiments, the antibody bears a detectable label. Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')₂) can be used. The term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with a detectable substance. Examples of detectable substances are provided herein.

The detection methods can be used to detect a component of the FGF6 and/or FGF9 pathway, e.g., FGF6 and/or FGF9, in a biological sample in vitro as well as in vivo. In vitro techniques for detection include enzyme linked immunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence, enzyme immunoassay (EIA),

radioimmunoassay (RIA), and Western blot analysis. In vivo techniques for detection of include introducing into a subject a labeled antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques. In another embodiment, the sample is labeled, e.g., biotinylated and then contacted to the antibody, e.g., an antibody positioned on an antibody array. The sample can be detected, e.g., with avidin coupled to a fluorescent label.

In another embodiment, the methods further include contacting the control sample with a compound or agent capable of detecting a FGF6 and/or FGF9, and comparing the presence of FGF6 and/or FGF9 protein in the control sample with the presence of the protein in the test sample.

The invention also includes kits for detecting the presence of FGF6 and/or FGF9 in a biological sample. For example, the kit can include a compound or agent capable of detecting FGF6 and/or FGF9 protein (e.g., an antibody) or mRNA (e.g., a nucleic acid probe); and a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to evaluate a subject, e.g., for risk or predisposition to diabetes-related adipose disease.

The diagnostic methods described herein can identify subjects having, or at risk of developing, adipose-related disorders, such as obesity and diabetes. The prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., FGF6 and/or FGF9 or another agent described herein) to treat an adipose-related disorder.

Mutagenesis

Generation of Variants: Production of Altered DNA and Peptide Sequences by Random Methods

A wild-type FGF6 polypeptide can have the amino acid sequence set forth in SEQ ID NO: 1. In some cases, a wild-type FGF6 polypeptide can have a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. A wild-type FGF9 polypeptide can have the amino acid sequence set forth in SEQ ID NO:2. In some cases, a wild-type FGF9 polypeptide can have a sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO:2.

Amino acid sequence variants of FGF6 and/or FGF9 polypeptides or fragments thereof can be prepared by a number of techniques, such as random mutagenesis of DNA which encodes a FGF6 and/or FGF9 or a region thereof. Useful methods also include PCR mutagenesis and saturation mutagenesis. A library of random amino acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences.

PCR Mutagenesis

In PCR mutagenesis, reduced Taq polymerase fidelity is used to introduce random mutations into a cloned fragment of DNA (Leung et al, 1989, Technique 1 : 11-15). This is a very powerful and relatively rapid method of introducing random mutations. The DNA region to be mutagenized is amplified using the polymerase chain reaction (PCR) under conditions that reduce the fidelity of DNA synthesis by Taq DNA polymerase, e.g., by using a dGTP/dATP ratio of five and adding Mn²⁺ to the PCR reaction. The pool of amplified DNA fragments are inserted into appropriate cloning vectors to provide random mutant libraries.

Saturation Mutagenesis

Saturation mutagenesis allows for the rapid introduction of a large number of single base substitutions into cloned DNA fragments (Mayers et al., Science 229:242 (1985)). This technique includes generation of mutations, e.g., by chemical treatment or irradiation of single-stranded DNA in vitro, and synthesis of a complimentary DNA strand. The mutation frequency can be modulated by modulating the severity of the treatment, and essentially all possible base substitutions can be obtained. Because this procedure does not involve a genetic selection for mutant fragments both neutral substitutions, as well as those that alter function, are obtained. The distribution of point mutations is not biased toward conserved sequence elements.

Degenerate Oligonucleotides

A library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, Tetrahedron 39:3 (1983); Itakura et al, in Recombinant DNA, Proc. 3rd Cleveland Sympos. Macromolecules, ed. AG Walton, Amsterdam:Elsevier, pp. 273-289 (1981); Itakura et al, Annu. Rev. Biochem. 53 :323 (1984); Itakura et al, Science 198: 1056 (1984); Ike et al, Nucleic Acid Res. 11 :477 (1983). Such techniques have been employed in the directed evolution of other proteins (see, for example, Scott et al., Science 249:386-390 (1990); Roberts et al, Proc. Nat. Acad. Sci. USA 89:2429-2433 (1992); Devlin et al, Science 249: 404-406 (1990); Cwirla et al., Proc. Nat. Acad. Sci. USA 87: 6378-6382 (1990); as well as U.S. Patents Nos. 5,223,409, 5, 198,346, and 5,096,815).

Generation of Variants: Production of Altered DNA and Peptide Sequences by Directed Mutagenesis

Non-random or directed mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants that include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.

Alanine Scanning Mutagenesis

Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244: 1081-85 (1989)). In alanine scanning, a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine). Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell. Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis may be conducted at the target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.

Oligonucleotide-Mediated Mutagenesis

Oligonucleotide-mediated mutagenesis is a useful method for preparing substitution, deletion, and insertion variants of DNA, see, e.g., Adelman et al, DNA 2: 183 (1983).

Briefly, the desired DNA is altered by hybridizing an oligonucleotide encoding a mutation to a DNA template, where the template is the single-stranded form of a plasmid or

bacteriophage containing the unaltered or native DNA sequence of the desired protein. After hybridization, a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA. Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single- stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al, Proc. Natl. Acad. Sci. USA, 75: 5765 (1978).

Cassette Mutagenesis

Another method for preparing variants, cassette mutagenesis, is based on the technique described by Wells et al, Gene, 34:315 (1985). The starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated. The codon(s) in the protein subunit DNA to be mutated are identified. There must be a unique restriction endonuclease site on each side of the identified mutation site(s). If no such restriction sites exist, they may be generated using the above-described oligonucleotide-mediated

mutagenesis method to introduce them at appropriate locations in the desired protein subunit DNA. After the restriction sites have been introduced into the plasmid, the plasmid is cut at these sites to linearize it. A double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then hybridized together using standard techniques. This double-stranded oligonucleotide is referred to as the cassette. This cassette is designed to have 3 ' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid. This plasmid now contains the mutated desired protein subunit DNA sequence.

Combinatorial Mutagenesis

Combinatorial mutagenesis can also be used to generate variants. For example, the amino acid sequences for a group of homo logs or other related proteins are aligned, preferably to promote the highest homology possible. All of the amino acids which appear at a given position of the aligned sequences can be selected to create a degenerate set of combinatorial sequences. The variegated library of variants is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library. For example, a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.

Primary High-Through-Put Methods for Screening Libraries of Peptide Fragments or Homologs

Various techniques are known in the art for screening peptides, e.g., synthetic peptides, e.g., small molecular weight peptides (e.g., linear or cyclic peptides) or generated mutant gene products. Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, assembly into a trimeric molecules, binding to natural ligands, e.g., a receptor or substrates, facilitates relatively easy isolation of the vector encoding the gene whose product was detected. Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.

Two Hybrid Systems

Two hybrid (interaction trap) assays can be used to identify a protein that interacts with FGF6 and/or FGF9. These may include, e.g., agonists, superagonists, and antagonists of FGF6 and/or FGF9. The subject protein and a protein it interacts with are used as the bait protein and fish proteins. These assays rely on detecting the reconstitution of a functional transcriptional activator mediated by protein-protein interactions with a bait protein. In particular, these assays make use of chimeric genes which express hybrid proteins. The first hybrid comprises a DNA-binding domain fused to the bait protein, e.g., FGF6 and/or FGF9 or active fragments thereof. The second hybrid protein contains a transcriptional activation domain fused to a "fish" protein, e.g. an expression library. If the fish and bait proteins are able to interact, they bring into close proximity the DNA-binding and transcriptional activator domains. This proximity is sufficient to cause transcription of a reporter gene which is operably linked to a transcriptional regulatory site which is recognized by the DNA binding domain, and expression of the marker gene can be detected and used to score for the interaction of the bait protein with another protein. Display Libraries

In one approach to screening assays, the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay". For example, the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al, WO

88/06630; Fuchs et al, Bio/Technology 9: 1370-1371 (1991); and Goward et al, TIBS 18: 136- 140 (1992)). This technique was used in Sahu et al, J. Immunology 157:884-891 (1996), to isolate a complement inhibitor. In a similar fashion, a detectably labeled ligand can be used to score for potentially functional peptide homologs. Fluorescently labeled ligands, e.g., receptors, can be used to detect homolog which retain ligand-binding activity. The use of fluorescently labeled ligands, allows cells to be visually inspected and separated under a fluorescence microscope, or, where the morphology of the cell permits, to be separated by a fluorescence-activated cell sorter.

A gene library can be expressed as a fusion protein on the surface of a viral particle. For instance, in the filamentous phage system, foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits. First, since these

13

phage can be applied to affinity matrices at concentrations well over 10 phage per milliliter, a large number of phage can be screened at one time. Second, since each infectious phage displays a gene product on its surface, if a particular phage is recovered from an affinity matrix in low yield, the phage can be amplified by another round of infection. The group of almost identical E. coli filamentous phages M13, fd., and fl are most often used in phage display libraries. Either of the phage gill or gVIII coat proteins can be used to generate fusion proteins without disrupting the ultimate packaging of the viral particle. Foreign epitopes can be expressed at the NH₂-terminal end of pill and phage bearing such epitopes recovered from a large excess of phage lacking this epitope (Ladner et al. PCT publication WO 90/02909; Garrard et al, PCT publication WO 92/09690; Marks et al, J. Biol. Chem. 267: 16007-16010 (1992); Griffiths et al, EMBO J. 12:725-734 (1993); Clackson et al, Nature 352:624-628 (1991); and Barbas et al, PNAS 89:4457-61 (1992)).

A common approach uses the maltose receptor of E. coli (the outer membrane protein, LamB) as a peptide fusion partner (Charbit et al, EMBO 5:3029-3037 (1986)).

Oligonucleotides have been inserted into plasmids encoding the LamB gene to produce peptides fused into one of the extracellular loops of the protein. These peptides are available for binding to ligands, e.g., to antibodies, and can elicit an immune response when the cells are administered to animals. Other cell surface proteins, e.g., OmpA (Schorr et al., Vaccines 91 :387-392 (1991)), PhoE (Agterberg, et al, Gene 88:37-45 (1990)), and PAL (Fuchs et al, Bio/Tech. 9: 1369-1372 (1991)), as well as large bacterial surface structures have served as vehicles for peptide display. Peptides can be fused to pilin, a protein which polymerizes to form the pilus-a conduit for interbacterial exchange of genetic information (Thiry et al, Appl. Environ. Microbiol. 55:984-993 (1989)). Because of its role in interacting with other cells, the pilus provides a useful support for the presentation of peptides to the extracellular environment. Another large surface structure used for peptide display is the bacterial motive organ, the flagellum. Fusion of peptides to the subunit protein flagellin offers a dense array of may peptides copies on the host cells (Kuwajima et al, Bio/Tech. 6, 1080-83 (1988)). Surface proteins of other bacterial species have also served as peptide fusion partners.

Examples include the Staphylococcus protein A and the outer membrane protease IgA of Neisseria (Hansson et al, J. Bacteriol. 174, 4239-45 (1992) and Klauser et al, EMBO J. 9, 1991-99 (1990)).

In the filamentous phage systems and the LamB system described above, the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within. An alternative scheme uses the DNA-binding protein Lacl to form a link between peptide and DNA (Cull et al, Proc. Nat. Acad. Sci. USA 89: 1865-1869 (1992)). This system uses a plasmid containing the Lacl gene with an oligonucleotide cloning site at its 3 '-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced. This fusion retains the natural ability of Lacl to bind to a short DNA sequence known as LacO operator (LacO). By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides. The associated plasmid DNA is then reintroduced into cells for amplification and DNA sequencing to determine the identity of the peptide ligands. As a demonstration of the practical utility of the method, a large random library of dodecapeptides was made and selected on a monoclonal antibody raised against the opioid peptide dynorphin B. A cohort of peptides was recovered, all related by a consensus sequence corresponding to a six-residue portion of dynorphin B (Cull et al, Proc. Natl. Acad. Sci. U.S.A. 89: 1869 (1992))

This scheme, sometimes referred to as peptides-on-plasmids, differs in two important ways from the phage display methods. First, the peptides are attached to the C-terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini. Both of the filamentous phage coat proteins, pill and pVIII, are anchored to the phage through their C-termini, and the guest peptides are placed into the outward- extending N-terminal domains. In some designs, the phage-displayed peptides are presented right at the amino terminus of the fusion protein. See, e.g., Cwirla et al, Proc. Natl. Acad. Sci. U.S.A. 87, 6378-6382 (1990). A second difference is the set of biological biases affecting the population of peptides actually present in the libraries. The Lacl fusion molecules are confined to the cytoplasm of the host cells. The phage coat fusions are exposed briefly to the cytoplasm during translation but are rapidly secreted through the inner membrane into the periplasmic compartment, remaining anchored in the membrane by their C-terminal hydrophobic domains, with the N-termini, containing the peptides, protruding into the periplasm while awaiting assembly into phage particles. The peptides in the Lacl and phage libraries may differ significantly as a result of their exposure to different proteolytic activities. The phage coat proteins require transport across the inner membrane and signal peptidase processing as a prelude to incorporation into phage. Certain peptides exert a deleterious effect on these processes and are underrepresented in the libraries (Gallop et al, J. Med. Chem. 37(9): 1233-1251 (1994)). These particular biases are not a factor in the Lacl display system.

The number of small peptides available in recombinant random libraries is enormous. Libraries of 10⁷-10⁹ independent clones are routinely prepared. Libraries as large as 10^{1 1} recombinants have been created, but this size approaches the practical limit for clone libraries. This limitation in library size occurs at the step of transforming the DNA containing randomized segments into the host bacterial cells. To circumvent this limitation, an in vitro system based on the display of nascent peptides in polysome complexes has recently been developed. This display library method has the potential of producing libraries 3-6 orders of magnitude larger than the currently available phage/phagemid or plasmid libraries. Furthermore, the construction of the libraries, expression of the peptides, and screening, is done in an entirely cell-free format.

In one application of this method (Gallop et al, J. Med. Chem. 37(9): 1233-1251 (1994)), a molecular DNA library encoding 10¹² decapeptides was constructed and the library expressed in an E. coli S30 in vitro coupled transcription/translation system. Conditions were chosen to stall the ribosomes on the mRNA, causing the accumulation of a substantial proportion of the RNA in polysomes and yielding complexes containing nascent peptides still linked to their encoding RNA. The polysomes are sufficiently robust to be affinity purified on immobilized receptors in much the same way as the more conventional recombinant peptide display libraries are screened. RNA from the bound complexes is recovered, converted to cDNA, and amplified by PCR to produce a template for the next round of synthesis and screening. The polysome display method can be coupled to the phage display system. Following several rounds of screening, cDNA from the enriched pool of polysomes was cloned into a phagemid vector. This vector serves as both a peptide expression vector, displaying peptides fused to the coat proteins, and as a DNA sequencing vector for peptide identification. By expressing the polysome-derived peptides on phage, one can either continue the affinity selection procedure in this format or assay the peptides on individual clones for binding activity in a phage ELISA, or for binding specificity in a completion phage ELISA (Barret, et al. Anal. Biochem. 204:357-364 (1992)). To identify the sequences of the active peptides one sequences the DNA produced by the phagemid host.

Secondary Screens for Inhibitors of the Alternative Pathway

The high through-put assays described above can be followed (or substituted) by secondary screens in order to identify biological activities which will, e.g., allow one skilled in the art to differentiate agonists from antagonists. The type of a secondary screen used will depend on the desired activity that needs to be tested. For example, an adipose tissue-related assay described herein can be used in which the ability to increase or mimic FGF6 and/or FGF9 activity in adipose tissue can be used to identify FGF6 and/or FGF9 agonists from a group of peptide fragments isolated though one of the primary screens described above. In some cases, an adipose tissue-related assay described herein can be used in which the ability to increase or mimic the activity of a combination of two or more growth factors (e.g., FGF6+FGF9, FGF9+FGF2, or FGF9 + BMP7) in adipose tissue can be used to identify FGF and/or BMP agonists. Peptide Mimetics

The invention also provides for production of the protein binding domains of FGF6 and/or FGF9, to generate mimetics, e.g. peptide or non-peptide agents, e.g., agonists.

Non-hydro lyzable peptide analogs of critical residues can be generated using benzodiazepine (e.g., see Freidinger et al, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)), azepine (e.g., see Huffman et al, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)), substituted gamma lactam rings (Garvey et al, in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands (1988)), keto-methylene pseudopeptides (Ewenson et al, J. Med. Chem. 29:295 (1986); and Ewenson et al., in Peptides: Structure and Function (Proceedings of the 9th American Peptide Symposium), Pierce Chemical Co., Rockland, IL (1985)), b-turn dipeptide cores (Nagai et al, Tetrahedron Lett. 26:647 (1985); and Sato et al, J. Chem. Soc. Perkin Trans. 1 : 1231 (1986)), and b- aminoalcohols (Gordon et al, Biochem. Biophys. Res. Commun. 126:419-426 (1985); and Dann et al, Biochem. Biophys. Res. Commun. 134:71-77 (1986)).

Kits

A FGF6 and/or FGF9 polypeptide, e.g., a FGF6 and/or FGF9 polypeptide described herein, can be provided in a kit. The kit includes (a) FGF6 and/or FGF9, e.g., a composition that includes FGF6 and/or FGF9, and (b) informational material. The informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and/or the use of FGF6 and/or FGF9 for the methods described herein. For example, the informational material relates to adipose tissue, obesity, or diabetes.

In one embodiment, the informational material can include instructions to administer FGF6 and/or FGF9 in a suitable manner to perform the methods described herein, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein). Suitable doses, dosage forms, or modes of administration are percutaneous, i.v., and oral and implantation into an adipose tissue. In another embodiment, the informational material can include instructions to administer FGF6 and/or FGF9 to a suitable subject, e.g., a human, e.g., a human having, or at risk for, obesity.

The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is contact information, e.g., a physical address, email address, website, or telephone number, where a user of the kit can obtain substantive information about FGF6 and/or FGF9 and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.

In addition to FGF6 and/or FGF9, the composition of the kit can include other ingredients, such as a solvent or buffer, a stabilizer, a preservative, a fragrance or other cosmetic ingredient, and/or a second agent for treating a condition or disorder described herein, e.g., insulin or an obesity drug. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than FGF6 and/or FGF9. In such embodiments, the kit can include instructions for admixing FGF6 and/or FGF9 and the other ingredients, or for using FGF6 and/or FGF9 together with the other ingredients.

FGF6 and/or FGF9 can be provided in any form, e.g., liquid, dried or lyophilized form. It is preferred that FGF6 and/or FGF9 be substantially pure and/or sterile. When FGF6 and/or FGF9 is provided in a liquid solution, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being preferred. When FGF6 and/or FGF9 is provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition containing FGF6 and/or FGF9. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of FGF6 and/or FGF9. For example, the kit can include a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of FGF6 and/or FGF9. The containers of the kits can be air tight and/or waterproof. The kit optionally includes a device suitable for administration of the composition, e.g., a syringe, inhalant, pipette, forceps, measured spoon, dropper (e.g., eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In one embodiment, the device is a syringe.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1 - Clonal Expansion and Differentiation of Brown Fat Precursor Cells To screen for secreted proteins associated with brown fat differentiation,

immortalized brown preadipocytes (YHR cells) were obtained (see, e.g., Klein et al, J. Biol. Chem. 274:34795 (1999); Fasshauer et al, Mol. Cell Biol. 21 :319 (2001); and Tseng et al, Mol. Cell Biol. 24: 1918 (2004)). The cells were removed from liquid nitrogen storage and diluted with 50 mL Growth Media (GM) containing DMEM with 10% fetal bovine calf serum and 1% penicillin/streptomycin). Cells were transferred to a T150 culture flask and incubated at 37°C in 5% C0₂.

On incubation Day 1 , the flask was removed from the incubator and GM was removed by aspiration. To the cells, 3 mL IX MediaTech Trypsin EDTA (TE) was added to cover the cells and then quickly removed by aspiration. Another 3 mL of TE was added and the cells were placed in the incubator for 2 minutes. Immediately thereafter, 17 mL of GM was added to the flask and the incubation surface was flushed several times prior to transfer to a 50 mL centrifuge tube for centrifugation at 1200 rpm for 4 minutes. Subsequently, the GM was aspirated and the precipitated cells were flushed and mixed with 20 mL GM. Two 10 mL portions were transferred in 40 mL GM each to two T150 culture flasks. Cells were incubated at 37°C in 5% C0₂.

On incubation Day 2, the above protocol was repeated for both flasks, with the combined cells brought to a volume of 20 mL in GM. The number of viable cells per mL was determined by adding a 1 mL aliquot of the cell culture to a Vi-cell™ XR Cell Viability Analyzer (Beckman-Coulter, Brea, Calif). For the Vi-cell™ analyzer configuration, the "Fibroblast" setting was used. Based on the count of cells per mL, approximately 6 x 10⁶ cells were added to five T 150 flasks with a final volume of 50 mL GM. Cells were incubated at 37°C in 5% C0₂. On incubation Day 5, the YHR cells were induced to form adipocytes using Induction Media (IM) dexamethasone, IBMX, and Indomethacin. The compounds were added to each flask for a final concentration of 5 μΜ dexamethasone, 0.25 mM IBMX, and 0.125 mM Indomethacin. The cells were incubated for 48 hours at 37°C.

After the 48-hour incubation, the IM was aspirated from the adipocytes and replaced with 50 mL GM. The cells were returned to the incubator for approximately 24 hours.

Following this incubation, the GM was aspirated from the cells and the cells were harvested by treating each flask with 3 mL TE as described for incubation Day 1. The viable cell count was measured as described for incubation Day 2. To the cell surface of each flask, 17 mL GM was added and flushed several times prior to centrifugation and washing of the cells with additional GM. The viable cell count was taken after washing and diluting the adipocytes to a final cell count of approximately 800,000 cells/mL. 100 aliquots were added to each well of a 96-well plate and incubated for 6 hours.

Following the incubation, cell media was removed and replaced with Test Media (TM) containing FGFs in DMEM with 10% fetal bovine calf serum. TM was prepared for each of FG2, FGF6, and FGF9. The final concentration of each FGF stock solution was 400 ng/mL. Serial dilutions of each FGF solution were made in GM to yield FGF solutions of 200, 100, 50, 10, 2, 0.4, 0.08, and 0.016 ng/mL.

Example 2 - Assaying UCP-1 Expression

UCP-1 mRNA induction was assayed using a QuantiGene® Plex 2.0 reagent system (Panomics, Fremont, Calif). Cells obtained in Example 1 were lysed to extract total R A using 200 μϊ_^ Procarta™ lysis buffer (Procarta™ Transcription Factor Whole Cell Lysis Kit (Panomics)) per well of a 96-well plate. Each cell lysis mixture was pipetted repeatedly (about 10 to 20 times) to reduce viscosity.

A working probe set was prepared by combining the reagents in the order listed in Table 1. To each well of a capture (reaction) plate, 20 of the working probe set and 80 μΐ_^ of the cell lysis mixture were added. The plate was sealed with an adhesive plate seal. To ensure that the contents of each well were at the bottom of the well, the capture plate was centrifuged at 240 x g for 20 seconds. The capture plate was immediately placed in a 55°C incubator to begin an overnight (approximately 16-20 hours) hybridization. Table 1. Probe Set Reagents

The 2.0 Pre-Amplifier Working Reagent was prepared according to the

manufacturer's instructions. Briefly, 1 1 μϊ_^ of the Pre-Amplifier reagent was added to 11 mL of Amplifier/Label Probe Diluent and mixed. The capture plate was washed 3 times with 200 μL of the manufacturer's wash buffer. To each well of the capture plate, 100 μϊ_^ of Pre- Amplifier reagent was added. The capture plate was re-sealed and incubated at 55°C for 60 minutes.

The Amplifier Working Reagent was prepared according to the manufacturer's instructions. Briefly, 1 1 μϊ_^ of the Amplifier reagent was added to 1 1 mL of Amplifier/Label Probe Diluent and mixed. The capture plate was washed 3 times with 200 μϊ_^ of the manufacturer's wash buffer. To each well of the capture plate, 100 μϊ_^ of Amplifier reagent was added. The capture plate was re-sealed and incubated at 55°C for 60 minutes.

To hybridize the Label Probe, the Label Probe Working Reagent was prepared according to the manufacturer's instructions. Briefly, 1 1 μϊ_^ of the Label Probe reagent was added to 11 mL of Amplifier/Label Probe Diluent and mixed. The capture plate was washed 3 times with 200 μϊ_^ of the manufacturer's wash buffer. To each well of the capture plate, 100 μί_^ of Label Probe reagent was added. The capture plate was re-sealed and incubated at 55°C for 60 minutes. The capture plate was washed another 3 times with 200 μΐ_^ of the wash buffer. 100 μί_^ of 2.0 Substrate was added to each well of the capture plate. The plate was sealed, incubated at room temperature for 5 minutes, and read in a luminometer within 15 minutes.

Assays were performed in duplicate. The effect of each FGF solution on UCP-1 expression is presented in Table 2 and Figure 1. FGF9 increased UCP-1 mRNA expression 15-fold from the basal state. FGF2 increased UCP-1 about 10-fold from the basal state. Increased UCP-1 is a unique characteristic of brown fat, and the uncoupling activity of UCP- 1 is integral to heat production in brown fat cells. The magnitude of the UCP-1 expression observed in the current study strongly suggested that the FGF9 and FGF2 can induce differentiations of brown fat precursors. It was also noted that FGF6 induced UCP-1 expression to a lesser extent. Thus, it may be possible to use FGF9 and FGF6, as well as FGF2, therapeutically to induce differentiation from of brown adipocyte precursors.

Normalization of the results to account for mitogenic activity of FGF was performed as follows. A set of duplicate plates were treated with FGFs and assayed for cell number. In addition, ATP levels per well were used as surrogates for cell number. ATP abundance was measured by CellTiter-Glo® Luminescent Cell Viability Assays (Promega, Madison, Wise.) performed according to the manufacturer's instructions. ATP levels are presented in Table 3. Normalization coefficients were calculated using the ratio of ATP level per well to untreated samples. Normalization coefficients are presented in Table 4. Using the calculated normalization coefficients, normalized UCP- 1 levels per well were calculated. The results are presented in Table 5 and Figure 2. In summary, these data demonstrate that FGF2, FGF6, and FGF9 induce UCP-1 mRNA expression in adipocytes and promote brown fat cell differentiation in a dose-dependent manner.

Table 2. FGF Induction of UCP-1

Cone (ng/mL) FGF-9 FGF -2 FGF-6

400 89.26 89.305 54.1 45 70.718 49.1 12 32.74

200 83.246 91.526 72.5 57 82.54 34.363 35.453

100 78.055 81.013 59.1 33 64.197 22.432 25.45

50 56.146 59.501 31 .688 42.686 21 .71 1 23.381

10 34.282 39.197 16.754 25.083 21 .656 14.739

2 8.843 1 1.145 9.931 5.887 14.58 13.571

0.4 1 1 .102 4.613 5.463 8.61 1 13.073 7.519

0.08 4.089 4.917 9.289 7.592 9.257 6.9

0.016 8.134 6.063 6.438 4.778 7.182 7.484

0 5.721 5.733 5.1 78 9.317 4.333 5.048

Table 3. ATP Levels Following FGF Treatment

Cone (ng mL) FGF 6 FGF 6 FGF 9 FGF 9 FGF 2 FGF 2

400 182 179 229 205 186 190

200 180 176 212 200 184 189

100 176 176 219 192 189 193

50 168 174 213 179 192 182

10 157 166 196 168 166 175

2 152 152 170 152 151 157

0.4 139 151 154 138 139 146

0.08 144 144 139 132 132 142

0.016 137 146 142 129 138 138

0 138 152 135 126 133 136

Table 4. Normalization Coefficients

Cone (ng mL) FGF 6 FGF 6 FGF 9 FGF 9 FGF 2 FGF 2

400 1.32 1.18 1.69 1.63 1.39 1.40

200 1.30 1.16 1.57 1.59 1.38 1.39

100 1.27 1.16 1.62 1.53 1.41 1.42

50 1.22 1.15 1.58 1.42 1.44 1.34

10 1.14 1.10 1.45 1.34 1.25 1.28

2 1.09 1.00 1.26 1.21 1.13 1.15

0.4 1.00 0.99 1.14 1.09 1.05 1.08

0.08 1.04 0.95 1.03 1.05 0.99 1.04

0.016 0.99 0.96 1.05 1.02 1.04 1.01

0 1.00 1.00 1.00 1.00 1.00 1.00

Table 5. Normalized UPC-1 Expression

Cone (ng/mL) FGF 9 FGF 9 FGF 2 FGF 2 FGF 6 FGF 6

400 52.67 54.81 38.89 50.55 36.73 28.34

200 53.17 57.61 52.62 59.49 26.04 31.36

100 48.21 53.08 41.80 45.33 17.39 22.39

50 35.56 41.80 22.03 31.90 17.58 20.91

10 23.65 29.34 13.42 19.51 18.77 13.77

2 7.04 9.24 8.76 5.11 13.11 13.87

0.4 9.77 4.22 5.22 8.00 12.82 7.74

0.08 3.96 4.69 9.38 7.29 8.76 7.44

0.016 7.75 5.93 6.20 4.71 7.17 7.97

0 5.72 5.73 5.18 9.31 4.27 5.17

OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. For example, FGF2 can be used in addition to or in place of FGF6 or FGF9. FGF2 (also known as basic fibroblast growth factor or bFGF) is a 288 amino acid protein, as shown below (SEQ ID NO:3), and belongs to the 22-member family of heparin-binding fibroblast growth factors. FGF2 induces fibroblast proliferation, cell differentiation, angiogenesis, neurogenesis, and vascular remodeling. The sequence is set forth in Watson et al, Biochem. Biophys. Res. Commun. 187(3): 1227-31 (1992).

1 MVGVGGGDVE DVT PRPGGCQ I SGRGARGCN G I PGAAAWEA ALPRRRPRRH PSVNPRSRAA

61 GS PRTRGRRT EERPSGSRLG DRGRGRALPG GRLGGRGRGR APERVGGRGR GRGTAAPRAA

12 1 PAARGSRPGP AGTMAAGS I T TLPALPEDGG SGAFPPGHFK DPKRLYCKNG GFFLRIHPDG 18 1 RVDGVREKSD PHI KLQLQAE ERGWS I KGV CANRYLAMKE DGRLLASKCV TDECFFFERL

24 1 ESNNYNTYRS RKYTSWYVAL KRTGQYKLGS KTGPGQKAI L FLPMSAKS ( SEQ I D NO : 3 )

Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of promoting brown adipocyte differentiation, the method comprising:

contacting a target cell or tissue with a composition comprising a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) polypeptide or fragment thereof in an amount sufficient to promote brown adipocyte differentiation, thereby producing a differentiated brown adipose cell or tissue.

2. A method of decreasing fat stores or weight in a subject or preventing weight gain, the method comprising:

identifying a subject in need of decreasing fat stores or weight or at risk of obesity or an obesity -related disease;

obtaining one or more target cells or tissues from the subject;

contacting the target cells or tissues with a composition comprising a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) polypeptide, or fragment thereof, in an amount sufficient to promote brown adipocyte differentiation, thereby producing one or more differentiated brown adipose cells or tissues; and

administering to the subject the one or more differentiated brown adipose cells or tissues.

3. A method for providing a cell culture enriched in brown adipocytes, the method comprising:

providing a plurality of target cells in vitro;

contacting the plurality of cells with a composition comprising a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) polypeptide, or fragment thereof, in an amount sufficient to promote brown adipocyte differentiation.

4. The method of claim 1, 2, or 3, wherein the fibroblast growth factor 6 (FGF6) polypeptide is a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO: 1.

5. The method of claim 1, 2, or 3, wherein the fibroblast growth factor 9 (FGF9) polypeptide is a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO:2.

6. The method of claim 1, 2, or 3, wherein the composition further comprises a fibroblast growth factor 2 (FGF2) polypeptide, e.g., a polypeptide having at least 95% amino acid sequence identity to SEQ ID NO:3, or fragment thereof.

7. The method of claim 1, 2, or 3, wherein the composition further comprises a bone morphogenetic protein (BMP) polypeptide or fragment thereof, e.g., BMP6 or BMP7 or a polypeptide or fragment thereof.

8. The method of claim 1, 2, or 3, wherein the target cell or tissue comprises a brown adipocyte or brown preadipocyte.

9. The method of claim 1, 2, or 3, wherein the target cell or tissue comprises a white preadipocyte or white adipocyte.

10. The method of claim 1, wherein the target cell or tissue comprises a stem cell, e.g., an adult stem cell, an embryonic stem cell, or an induced pluripotent stem cell.

11. The method of claim 1, wherein the target cell or tissue is in culture.

12. The method of claim 1, wherein the target cell or tissue is in or is isolated from a living subject.

13. The method of claim 2 or 12, wherein the subject is an obese human subject.

14. The method of claim 1 or 2, wherein the level of brown adipocyte differentiation is further evaluated by measuring morphological changes specific to brown adipocytes.

15. The method of claim 1, further comprising implanting the differentiated brown adipose cell or tissue in a subject.

16. The method of claim 1, 2, or 3, further comprising evaluating brown adipocyte differentiation by measuring a level of a brown adipocyte marker selected from the group consisting of PPAR gamma 2 (PPARy2), PPAR-gamma Coactivator 1 (PGC-1), cytochrome oxidase activity, and mitochondrial DNA levels, wherein the level of the brown adipocyte marker indicates the level of brown adipocyte differentiation.

17. The method of claim 3, further comprising implanting at least one brown adipocyte from said enriched culture into a subject.

18. A method of determining if a subject is at risk for weight gain or obesity, the method comprising:

obtaining a test sample from the subject;

evaluating the expression, protein level or activity of one or more of a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9) in the test sample; and

comparing the expression, protein level or activity of the FGF6 or FGF9 in the test sample to that in a control,

wherein a decrease in the expression, protein level or activity of the FGF6 or FGF9 in the test sample relevant to the control indicates that the subject is at risk for weight gain or obesity.

19. The method of claim 18, wherein the sample comprises one or more of a brown adipocyte, a white adipocyte, a brown preadipocyte, and a white preadipocyte.

20. A method of identifying a test compound that promotes brown adipocyte differentiation, the method comprising:

contacting a target cell or tissue, e.g., a preadipocyte or adipocyte cell or tissue, in vitro with a test compound;

evaluating the effect of the test compound on the target cell or tissue in vitro by measuring expression and/or activity of a fibroblast growth factor 6 (FGF6) or fibroblast growth factor 9 (FGF9), wherein an increase in FGF6 or FGF9 expression and/or activity indicates the test compound is a candidate agent for promoting brown adipocyte

differentiation.

21. The method of claim 20, further comprising evaluating an effect of the test compound on brown adipocyte differentiation in the target cell or tissue by measuring a level of a brown adipocyte marker selected from the group consisting of PPAR gamma 2 (PPARy2), PPAR- gamma Coactivator 1 (PGC-1), cytochrome oxidase activity, and mitochondrial DNA levels, wherein the level of the brown adipocyte marker indicates the level of brown adipocyte differentiation.

22. A method of identifying a candidate compound for the treatment of obesity, the method comprising:

selecting a candidate agent that promotes brown adipocyte differentiation identified by the method of claim 20;

providing an animal model of obesity;

administering the candidate agent that promotes BAT differentiation to the animal model; and

evaluating the effect of the candidate agent on obesity in the animal model;

wherein an agent that decreases obesity in the animal model is a candidate therapeutic agent for the treatment of obesity.

23. The method of claim 22, further comprising administering the candidate therapeutic agent for the treatment of obesity to a subject in a clinical trial, and evaluating the effect of the candidate therapeutic agent on obesity in the subject.