GB2381866A

GB2381866A - Assays for liver X receptor (LXR) modulators

Info

Publication number: GB2381866A
Application number: GB0127132A
Authority: GB
Inventors: Jan-Ke Gustafsson; Gertrud Schuster; Hilde Irene Nebb
Original assignee: Karo Bio AB
Current assignee: Karo Pharma AB
Priority date: 2001-11-12
Filing date: 2001-11-12
Publication date: 2003-05-14
Also published as: GB0127132D0

Abstract

Assays for identifying agents for the treatment of diabetes or disorders of fatty acid or cholesterol metabolism, by identifying modulators (ligands, agonists or antagonists) that bind to or modulate the biological activity of liver X receptor alpha or beta (LXR a or LXR b ). Such modulators may alter the amount of at least one of SREBP-1, cholesterol 7a-hydroxylase (Cyp7A), fatty acid synthetase, human cholesterol ester transfer protein (CETP), LXR a or LXR b protein or mRNA levels. The assay may be performed in hepatocyte, dipocyte or preadipocyte cells. The assay may be performed in combination with a retinoid X receptor (RXR). Use of agents identified by such assays in the preparation of medicaments and methods of treating diabetes are also claimed.

Description

Assay The present invention relates to an assay for identifying agents for use in treating diabetes or disorders of fatty acid or cholesterol metabolism. The present invention also relates to novel agents identified by the assay, the use of agents identified by the assay in the treatment of diabetes or disorders of fatty acid or cholesterol metabolism.

Insulin plays a major role in the regulation of carbohydrate and lipid metabolism in the liver, adipose tissue and muscle. Hepatic fatty acid oxidation, lipogenesis, and glycerolipid synthesis are subject to regulation by insulin (for review, see O'Brien et al., Physiol. Rev., 1109-1161,1996). Control of lipid synthesis is especially important in the liver, which synthesizes lipids from glucose precursors, for its own uses but also for export into plasma as lipoproteins. Hepatic fatty acid synthesis is elevated when plasma insulin rises, as in states of obesity and noninsulin-dependent diabetes mellitus (type 2 diabetes). The fatty acids are then exported from the liver in lipoproteins, and reach extra-hepatic organs in which they are either utilized or stored.

The factors mediating the insulin regulation in lipid metabolism have for a long time been unknown, but recently several reports have identified sterol regulatory-element-binding protein-l c (SREBP-l c) as a necessary transcription factor activating fatty acid synthesis in response to insulin (Osboume et al., J. Biol. Chern.,

275, 32379-32382, 2000). SREBP-lc activates transcription of the major genes of fatty acid synthesis including acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), stearoyl-CoA desaturase-l (SCD-1) (Azzout-Mamiche et al., Biochem. J., 350, t-93, 2000 ; Foretz et al., Proc. Natl. Acad. Sci. U. S. A, 96, 12737-12742, 1999 ; and Kun et al., J Clin. Invest., 101, 1-9,1998).

The liver X receptors (LXRs) belong to a subclass of nuclear hormone receptors that form obligate heterodimers with retinoid X receptors (RXRs) and are bound and activated by oxysterols, and have in the last few years been proposed to act as sterol sensors that function to ameliorate the effects of high free cholesterol levels (Willy et

al., Genes Dev.,, 289-298, 1997, Repa et al., Annu. Rev. Cell Dev. Bol, 16, 459-481, 2000 ; and Repa et al., Curr. Opin. Biotechnol., 10, 557-563, 1999). LXRa has a relatively restricted expression pattern (liver, kidney, intestine, adipose tissue and adrenals) (NR ! H3 from the Nuclear Receptors Nomenclature Committee, Cell, 97, 161-163, 1999 and Willy et al., Genes Dev., 9, 1033-1045, 1995), whereas LXRss is 1 ~, 1033-1045, 1995), whereas LXRP is ubiquitously expressed (NR1H2). LXRa and LXRP share a high degree of amino acid similarity (78 %) (Alberti et al., Gene, 243, 93-103,2000) and have thus been proposed to be paralogues.

The first responsive element for LXRa was found in the promoter of cholesterol 7a-hydroxylase (Cyp7A), an essential enzyme in the initial and rate-limiting step in the conversion of cholesterol to bile acids (Lehmann et al., J. Biol. Chern., 272, 3137-3140, 1997). Consistent with these in vitro observations, LXRa knock-out mice lose the capacity to regulate catabolism of excess dietary cholesterol in liver resulting in a rapid accumulation of hepatic cholesteryl esters that eventually leads to liver failure, an effect the related isoform, LXRss, cannot compensate for (Peet et al., Cell, 93, 693-704,1998 and Alberti et al., J. Clin. Invest., 107, 565-573,2001). Additional LXRa target genes involved in lipid metabolism include the human cholesteryl ester transfer protein (CETP), which translocates cholesteryl ester between lipoprotein fractions (Luo et al., J. Clin. Invest., 105, 513-520,2000), and the ATP-binding cassette transporters, ABCl and ABC8, which are implicated in the flux of cellular free cholesterol (Repa et al., Science, 289, 1524-1529,2000 ; Venkateswaran et al., J. Biol. Chem., 275,

14700-14707, 2000 ; and Chawla et al., Mol. Cell., L 161-171, 2001). In addition, , 161-171, 2001). In addition, SREBP-lc is regulated by LXRs and has LXR regulatory elements in its promoter (Repa et al., Genes Dev., 14, 2819-2830, 2000 and Yoshikawa et al., Mol. Cell. Biol., 1L 2991-3000,2001).

In view of the important cross regulation between hormonal signaling pathways and lipid homeostasis, the inventors determined whether LXRa expression was under control by insulin. The inventors show that LXRa is regulated by the insulin hormonal signaling pathway in primary cultures of hepatocytes, and in vivo in rodents injected with insulin. We further show that LXRs are necessary for the insulin dependent

upregulation of several lipogenic genes. The insulin mediated upregulation of LXRa and its widespread involvement in cholesterol metabolism is indicative of a key role for LXRa in mediating insulin effects in lipid metabolism.

The present invention provides an assay for indentifying an agent for use in the treatment of diabetes or a disorder of fatty acid or cholesterol metabolism, comprising determining if a candidate agent modulates the biological activity of a liver X receptor alpha (LXRa) and/or a liver X receptor beta (LXRss).

Preferably the assay comprises determining if a candidate agent modulates the biological activity of LXRa. However, it is known that the functions of LXRa and LXRss overlap and each receptor can compensate, at least to some degree, for the loss of the other receptor.

As indicated above, it has been found that insulin exerts some of its effects through the liver X receptor alpha (LXRa). h pabular, LXRa mediate at least some of insulin's effects on lipid and cholesterol metabolism. Accordingly, by inhibiting or activating the biological activity of LXRa the metabolism of lipids and cholesterol can be altered.

The assay of the present invention allows one skilled in the art to identify an agent that modulates the biological activity of LXRa.

An agent that modulates the biological activity of LXRa may inhibit or activate the biological activity of LXRa. The modifying agent may therefore be an agonist or antagonist of LXRa.

An agent that activates the biological activity of LXRa (i. e. an agonist) is an agent that binds LXRa and leads to an increase in the amount of at least one of LXRa mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester transfer protein (CETP) in a cell comprising LXRa.

An agent that inhibits the biological activity of LXRa (i. e. an antagonist) is an agent that binds LXRa and prevents or reduces an agonist induced biological activity. For example, the agent prevents or reduces an increase in the amount of at least one of LXRa mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester transfer protein (CETP), caused by contacting a cell comprising LXRa with an agonist (e. g. insulin).

An agent that modulates the biological activity of LXR ? may inhibit or activate the biological activity of LXRP. The modifying agent may therefore be an agonist or antagonist of LXRP.

An agent that activates the biological activity ofLXRR (i. e. an agonist) is an agent that binds LXRss and leads to an increase in the amount of at least one of LXRP mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester

transfer protein (CETP) in a cell comprising LXRss.

An agent that inhibits the biological activity of LXRP (i. e. an antagonist) is an agent that binds LXRP and prevents or reduces an agonist induced biological activity. For example, the agent prevents or reduces an increase in the amount of at least one of LXRss mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester transfer protein (CETP), caused by contacting a cell comprising LXRP with an agonist (e. g. insulin).

Preferably, the assay of the present invention comprises contacting a cell containing LXRa and/or LXRss with a candidate agent and determining whether there is a change in the amount of at least one ofSREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRss mRNA or protein.

In a particularly preferred embodiment of the present invention there is provided an assay for identifying an agent that activates the biological activity ofLXRa and/or LXRp comprising contacting a cell containing LXRa and/or LXRss with a candidate agent and determining whether there is an increase in the amount of at least one of SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRss mRNA or protein. An increase can be determined by comparing the amount with that of a control cell which has not been contacted with a candidate agent. Preferably the assay is for indentifying an agent that activates the biological activity ofLXRa.

In a further particularly preferred embodiment of the present invention there is provided an assay for identifying an agent that inhibits the biological activity ofLXRa and/or LXR. ss comprising contacting a cell containing LXRa and/or LXRP with a candidate agent and a known agonist (e. g. insulin) and determining whether the agent inhibits the agonist induced biological activity of LA. Inhibition of agonist induced biological activity can be determined by comparing the amount of at least one of SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, protein fatty acid synthetase (FAS) mRNA or protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRP mRNA or protein, in a cell contacted with the candidate agent and the agonist with the amount in a cell only contacted with the agonist. Preferably the assay is for indentifying an agent that inhibits the biological activity of LXRa.

In order to identify candidate agents for use in the assay of the present invention LXRa and/or LXRss can be used to screen libraries of compounds using any one of the variety of drug screening techniques. Candidate agents may be isolated from, for example, cells, cell-free preparations, chemical libraries, or natural product mixtures. These candidate compounds may be natural or modified substrates, ligands, enzymes or

structural or functional mimetics. For a suitable review of such screening techniques, see Coligan et ai., Current Protocols in Immunology 1 (2): Chapter 5 (1991).

The present invention also provides a method of identifying a candidate agent comprising identifying agents that bind to LXRa and/or LXRss. Suitable candidate agents preferably bind to LXRa and/or LXRss with an IL50 of at least 500nM, more preferably at least 100nM.

Numerous techniques for performing such binding assays are well known to those skilled in the art. A particularly preferred technique comprises contacting LXRa or LXRP with a candidate agent in the presence of a labelled known LXR ligand, e. g. 25 (OH) cholesterol or 24,25 epoxy cholesterol, and measuring the degree of competition in a proximity scintillation assay, FP, or FRET.

In the assays of the present invention the LXRa and/or LXRss may be comprised in a

cell or on a solid support. Preferably the LXRa and/or LXRP is comprised in a cell.

Any cell can be used that comprises LXRa. Preferably the cell used is a hepatocyte or adipocyte or preadipocyte.

The assay of the present invention can be performed in vitro using cell cultures, such as cultures of hepatocytes, adipocytes, or preadipocytes, or may be performed in vivo.

Preferably such zn VIVO tests are performed in rodents such as rats and mice.

The agent may be a protein molecule (i. e. an enzyme, an antibody molecule, or receptor ligand), a polypeptide, a peptide, a carbohydrate, or an organic or inorganic chemical compound. Preferred agents are those that bind to LXRa and/or LXRss at the site of its interaction with another agonist.

A protein agent may be an antibody molecule or other molecule that exhibits affinity for LXRa and/or LXRss. The term"antibody molecule"refers to polyclonal antibodies, monoclonal antibodies or antigen binding fragments thereof, such as Fv, Fab, F (ab') 2 fragments and single chain Fv fragments. The antibody molecule may be a recombinant antibody molecule, such'as a chimeric antibody molecule preferably having human constant regions and mouse variable regions, a humanised CDR-grafted antibody molecule or a fragment thereof. Methods for producing such antibodies are well known to those skilled in the art and are described in published European patent applications EP-A-0120694 and EP-A-0125023.

A carbohydrate agent can be any carbohydrate that binds LXRa and/or LXRS.

Organic and inorganic chemical compounds may be naturally-occurring or chemically synthesised compounds that bind LXRa and/or LXRp. There are numerous commercially available libraries of chemical compounds that can be assayed for their activity. Preferably the organic and inorganic chemical compounds are small molecules having a molecular weight of less than 1000. Libraries of small molecules are commercially available and can be analysed using the assay of the present invention.

The term"diabetes"as used herein refers to type 1 or type 2 diabetes. Preferably, the term"diabetes"as used herein refers to type 2 diabetes characterised by high blood glucose levels that cannot sufficiently be reduced by insulin.

Disorders of fatty acid or cholesterol metabolism include any disorders which can be prevented or treated by increasing or reducing the biological activity of LXRa and/or LXRss. Particular disorders of fatty acid metabolism include hypolipidemia and hyperlipidemia. Particular disorders of cholesterol metabolism include hypocholesterolernia and hypercholesterolemia.

Preferably, the agent preferentially binds to LXRa and/or LXRss. The term "preferentially binds"means that the agent has greater binding affinity for LXRa and/or LXRss than for any other molecule.

The LXRa can be any LXRa, which when contacted with an agonist, leads to an increase in the amount of LXRa mRNA, SREBP-I mRNA, cholesterol 7a-hydroxylase (Cyp7A), protein, fatty acid synthetase (FAS) mRNA or human cholesteryl ester transfer protein (CETP). The LXRa may be rat LXRa having the amino acid sequence given in accession no. AAA 53633 (GenBank). Preferably, the LXRa is human LXRa havmg the amino acid sequence given in accession no. AAA 85856 (GenBank) or a functional homolog thereof. A functional homolog is an LXRa that when contacted with an agonist leads to an increase in the amount of LXRa mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester transfer protein (CETP). Preferably the functional homolog has at least 80%, more preferably at least 90% and most preferably at least 95% amino acid sequence homology with the wild type human sequence of LXRa given in accession no. AAA 85856. Preferably the sequence homology is measured using the BLAST program. Preferably, such homologs differ by between only 1 to 20 amino acids from the human LXRa. These differences are preferably conservative amino acid substitutions. Conservative changes are those that replace one ammo acid with one from the family of amino acids that are related in their side chains. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on the biological activity of the protein.

Mutations which increase the number of amino acids that are capable of formmg disulphide bonds with other amino acids in the protein may be desirable in order to increase the stability of the protein.

The LXRss used in the assay can be a full length LXRα or a functional fragment of an LXRa. A functional fragment of an LXRa is one that when contacted with insulin, or other LXRa agonist, leads to an increase in the amount of LXRa mRNA or protein,

SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester transfer protein (CETP).

The LXRa used in the assay of the present invention is preferably in combination with a retinoid X receptor (RXR). By expressing LXRa in a cell that also expresses RXR a functional complex will formed.

The LXRp can be any LXRss. The LXRss may be rat LXRO having the amino acid sequence given in accession no. AAA69522 (GenBank). Preferably, the LXRss is human LXRss having the amino acid sequence given in accession no. AAA61783 (GenBank) or a functional homolog or fragment thereof. A functional homolog is an LXRss that when contacted with an agonist leads to an increase in the amount of LXRss mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein or human cholesteryl ester transfer protein (CETP). A functional fragment of an LXRss is one that when contacted with insulin, or other LXRss agonist, leads to an increase in the amount of LXRss mRNA or protein, SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, or human cholesteryl ester transfer protein (CETP). Preferably the functional homolog has at least 80%, more preferably at least 90% and most preferably at least 95% amino acid sequence homology with the wild type human sequence of LXRP given in accession no.

AAA85856. Preferably the sequence homology is measured using the BLAST program. Preferably, such homologs differ by between only 1 to 20 amino acids from the human LXRss. These differences are preferably conservative amino acid substitutions. Conservative changes are those that replace one amino acid with one from the family of amino acids that are related in their side chains. For example, It is

reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a significant effect on the biological activity of the protein. Mutations which increase the number of amino acids that are capable of forming disulphide bonds with other amino acids in the protein may be desirable in order to increase the stability of the protein.

The present invention also provides a cell expressing a heterologous LXRa and/or a heterologous LXRss. The term"heterologous"refers to LXRa and/or LXRss which is not endogenously expressed in the cell.

The present invention also provides a novel agent that modulates the biological activity of LXRa and/or LXRss. The agent can be a protein (i. e. an enzyme or antibody molecule), a polypeptide, a peptide, a carbohydrate or an organic or inorganic chemical compound as described above.

The present invention also provides the novel agent of the present invention for use in therapy. Preferably the therapy is the treatment of diabetes or a disorder of fatty acid or cholesterol metabolism.

The present invention also provides the use of an agent that modulates the biological activity of LXRa in the manufacture of a medicament for the treatment of diabetes or a disorder of fatty acid or cholesterol metabolism.

The present invention also provides a method of treating diabetes or a disorder of fatty acid or cholesterol metabolism comprising administering to a patient in need of such treatment an effective dose of an agent that modulates the biological activity of LXRa and/or LXRss.

The invention also provides a pharmaceutical composition comprising the novel agent of the invention in combination with a pharmaceutically acceptable carrier. The pharmaceutical composition preferably comprises a therapeutically effective amount of the novel agent of the invention. The term"therapeutically effective amount"as used herein refers to an amount of a therapeutic agent needed to treat, ameliorate, or prevent diabetes or a disorder of fatty acid or cholesterol metabolism.

For any agent, the therapeutically effective dose can be estimated initially either in cell culture assays, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.

The precise effective amount for a human subject will depend upon the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination (s), reaction sensitivities, and tolerance/response to therapy. This amount can be determined by routine experimentation and is within the judgement of the clinician. Generally, an effective dose will be from 0.01 mg/kg to 50 mg/kg, preferably 0.05 mg/kg to 10 mg/kg.

Pharmaceutical compositions of this invention comprise the novel agent of the present invention with any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

The pharmaceutical compositions of this invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. We prefer oral administration or administration by injection.

The pharmaceutical compositions of this invention may contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants or vehicles. The term parenteral as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intra-articular, intrasynovial, intrastemal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are mannitol, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant such as Ph.

Helv or a similar alcohol.

The pharmaceutical compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and com starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending

u agents. If desired, certain sweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing a novel agent of this invention with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

Topical administration of the pharmaceutical compositions of this invention is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For application topically to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active components suspended or dissolved in a carrier. Carriers for topical administration of the novel agents of this invention include, but are not limited to, mineral oil, liquid petroleum, white petroleum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable lotion or cream containing the active agent suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of this invention may also be topically applied to the lower intestinal tract by rectal suppository formulation or in a suitable enema formulation. Topically-transdermal patches are also included in this invention.

The pharmaceutical compositions of this invention may be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption pro- moters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs or hormones.

The present invention also provides an assay kit for identifying agents that modify the biological activity ofLXRa and/or LXRss. Preferably, the kit is for identifying agents that modify the biological activity ofLXRa and comprises a cell comprising LXRa, and reagents for detecting an increase in the amount of LXRa. mRNA, SREBP-1 mRNA, cholesterol 7a-hydroxylase (Cyp7a) protein, fatty acid synthase (FAS) mRNA or human cholesterol ester transfer protein (CETP).

The kit may also comprise a known LXRa agonist, particularly when the kit is for identifying an agent that inhibits the biological activity of LXRa.

The present invention is now described by way of example only, with reference to the accompanying figures in which: Figure 1A shows the effects of insulin on LXRa mRNA expression cultured rat hepatocytes. The steady-state mRNA level of LXRa was measured using Northern analysis (see Materials and Methods) of total RNA (20 ug) from control cells and after treatment with 400 nM insulin for 24 hours. The average of five independent experiments is given relative to control.

Figure 1B shows the effects of insulin on LXRa mRNA expression in cultured rat hepatocytes. Dose-response curve showing results from treatment of hepatocytes with 0, 10, 100 and 400 nM insulin for 24 hrs. The average of three independent experiments is given relative to control.

Figure 1 (C) shows the effects of insulin on LXRa mRNA expression m cultured rat hepatocytes. The time-response curve was obtained for insulin after treatment (400 nM) for up to 24 h, presented as a average of three separate experiments. The values are presented relative to control (control = 1) and given as the mean SEM.

Figure 1 (D) effects of insulin on LXRa protein in cultured rat hepatocytes. Inununoblotting was performed using protein lysates from primary hepatocytes treated with 400 nM insulin for 24 h.

Figure 2 shows the effects of insulin on LXRa mRNA stability. Cultured rat hepatocytes were incubated with insulin (400 nM) compared to controls for 24 h before addition of actinomycin D (ActD, 2.5 mg/ml). The cells were harvested at different timepoints up to 12 h. The LXRa mRNA levels was measured by Northern analysis (see Materials and Methods).

Figure 3 shows the effects of insulin on LXRa mRNA in Sprague-Dawley rats.

Expression of LXRa mRNA in Sprague-Dawley rats. Sprague-Dawley rats were given 2 units Actrapid insulin (i. p. ) and 2 units Insulatard insulin (s. c. ) (Novo Nordisk,

Denmark). Control animals were given vehicle (PBS). After 90 min of insulin stimulation, livers were rapidly taken out and frozen in liquid nitrogen until RNA extraction was performed and assayed by Northern blot analysis. Quantative measurements of the Northern Blot showing the relative increases in the mRNAs are also given in the Figure.

Figure 4 shows the effects of insulin on lipogenic enzyme genes in LXRoJP-/-mice LXRo/P-/-mice and corresponcing wildtype mice were given 0.2 units Actrapid insulin (i. p. ) (Novo Nordisk, Denmark)., and control animals were given vehicle (PBS).

After 90 min of insulin stimulation, livers were rapidly taken out and frozen in liquid nitrogen until RNA extraction was performed and assayed by Northern blot analysis.

EXAMPLES MATERIALS AND METHODS Materials Ham's F-10 medium, horse serum, calf serum, anti-pleuropneumonia-like organisms (PPLO), fungizone, penicillin, streptomycin, and Trizol Reagent for total RNA

extraction were from Gibco (Grand Island, NY, USA). Dulbecco's Modified Eagle's Medium was from Bio Whittaker (Boehringer Ingelheim, Belgium). Guanidium isothiocyanate was obtained from Merck (Hoenbrunn, Munchen, Germany). Multiprime DNA labeling system, the ECL Western Blotting Kit and Hybond C-extra nitrocellulose membrane were purchased from Amersham Pharmacia (Buckinghamshire, UK). Bio Trans nylon filter was from ICN (Irvine, USA). Polyclonal antibodies against LXRa were purchased from Santa Cruz Biotechnology (# SC-1206, CA, USA).

Animals All animal use was approved by the Norwegian Animal Research Authority (NARA) and registered by the authority. Male Wistar or Sprague-Dawley rats (B & K Universal Ltd, Norway) of approximately 200-250 g were kept in cages at a constant temperature (22 C) with a fixed 12 h light-dark cycle with free access to water and a standard low-fat diet. Rats were given 2 units Actrapid insulin (s. c. ) and 2 units Insulatard insulin (i. p.) (Insulin from Novo Nordisk, Denmark), and killed 90 mm later. Control animals received vehicle (Phosphate buffered saline, PBS).

Wild-type or LXRo/p-null mice on a mixed genetic background based on C57BL/6 and SV129 strains were used (see Alberti et ai., 1. Clin. Invest., 106, 565-573,2001). Mice were given 2 units Actrapid insulin (i. p. ), and killed 90 minutes later.

Rats were anesthetized using Hypnorm Donnicum and blood was collected from the abdominal aorta. Livers were rapidly frozen in liquid nitrogen and stored at-70 C until isolation of RNA. The blood was collected m vials containing EDTA and serum was collected by centnfugation. Plasma insulin was analyzed using a rat insulin RIA kit (RI-13K Linco, St. Louis, MO) and triglycendes (TG) and glucose were analyzed spectrophotometrically on the Cobas Mira plus (Hoffman la Roche, Basel, CH) using Calibrator Human (07 3718 6, Roche, Basel, CH) as calibrant. For TG the enzymatic kit "Triglycerides/Glycerol Blanking" (450032, Boehringer Mannheim. Indianapolis, IN) was used, and for glucose"Glucose HK" (07 3672 4, Roche, Basel, CH).

Cell culture Hepatocytes were isolated by the method of Berry and Friend (Berry et al., J. Cell. Biol.

1l, 506-520,1969) with modifications according to Seglen (Seglen et al., Expt. Cell. Res., 82, 391-398,1973). The culture conditions were as previously described (Sorensen et al., Eur. J. Biochem., 208. 705-711,1992). Insulin was added as described in legends to figures, and 2.5 mg/ml actinomycin D was used.

RNA extraction and Northern blot analysis Total RNA from cultured hepatocytes was extracted by the guanidium thiocyanate method Chrigwin et al., Biochemistry, 18, 5294-5299,1979), whereas total RNA from liver-tissue was extracted by Trizol Reagent for total RNA extraction (Gibco, Grand Island, NY, USA). Northern blot analysis of RNA was essentially performed as described earlier (Steineger et al., Eur. J. Biochem., 225, 967-974,1994).

The cDNA for rat LXR. a, human ribosomal protein L27 (ATCC # 107385) were used as probes in the hybridizations. Hybndization with the ribosomal protein L27 or ethidium bromide staining of the agarose gel was used as a control to show that the treatments did not cause a general alteration in gene expression. The [a-32P]dCTP-Iabeled cDNA-probes were prepared using a standard multiprime DNA-labeling kit (Amersham, RPN 1601Y). Specific activities from 2 to 6 x 10g cpm/mg DNA were obtained. The sizes of the mRNA transcnpts were calculated on the basis of the migration of the 18S and 28S rRNA, which were visualized by ethidium bromide. Semi-quantitative results were obtained from scanning of autoradiograms using XRS 3sc scanner and the Bio Image System from Millipore (USA), showing linear increments within the working range used (5-30 mg RNA). After autoradiography the RNA filters were washed using 50 % formamide and 10 mM sodium phosphate, pH 6. 5 for I h at 65 C and reprobed.

Immunoblotting Cultured primary hepatocytes were lysed in 0.1 % Triton-X100 in PBS including

protease inhibitors (Complete, Boehringer Mannheim, Germanv). Li Lye

lysats were prepared by homogenisation of 100 mg tissue in phosphate buffered saline . 7 9 (PBS) containing 1% NP-40,0. 5 % sodium deoxycholate, 0.1 % SDS and the same protease inhibitor as above. Total protein lysates were obtained after centrifugation, and protein concentration was determined with the BioRad colorimetric assay system.

Proteins (150 mg) was separated on a 10 % SDS-polyacrylamide gel, and transferred to nitrocellulose filters (Hybond-C Extra, Amersham Pharmacia, Buckinghamshire, UK).

The LXRa protein was immunochemically detected using commercially available antibodies (SC-1206, Santa Cruz, California, USA), and signal detection was achieved using ECL chemiluminescence (Amersham Pharmacia, Buckinghamshire, UK) according to the manufacturer's instructions.

Densitometry and statistics Senuquantitative results were obtained by scanning of autoradiograms using an XRS 3sc scanner and the Bio Image System from Millipore (USA). For statistical analysis of the mRNA results, the mean control values were set to unity and variation within the

group calculated accordingly. Corresponding relative values (expressed as mean SEM) were calculated for the experimental groups.

Example 1 Effects of insulin on LXRa mRNA expression in hepatocytes in culture A dose dependent increase in LXRa mRNA expression was seen after 24 h insulin stimulation of primary rat hepatocytes in culture. The maximum (10-fold) was obtained with 100 nM insulin (Fig. la and b). The induction of LXRa mRNA levels became evident as early as 6 h after addition of insulin, with maximal levels obtained after 12 h (Fig Ic). Insulin also induced LXRa protein levels; first evident 24 h after addition of hormone (Fig. lad).

We have previously shown that maximal effects on gene expression were obtained with insulin concentrations in excess of 250 nM (Steineger et al., Eur. J. Biochem., 225, 967-974,1994 and Sorensen et al., Biochim. Biophys. Acta, 1171. 263-271,1993).

Since 400 nM insulin did not result in reduced cell viability, as judged by the

JL ? Trypan-Blue-exclusion test (0. 4% Trypan Blue), this concentration was used in further experiments (Sorensen et al., Eur. J. Biochem., 208, 705-711, 1992 ; Steineger et al., Eur. J. Biochem., 225, 967-974, 1994 ; Sorensen et al., Biochim. Biophys. Acta, 1171, 263-271 1993 ; and Steineger et al., J. Lipid. Res., 39, 744-754, 1998).

5 Example 2 Effect of the transcription inhibitor actinomycin D on LXRa steady-state mRNA expression To further define the mechanism underlying the elevated LXRa steady state mRNA level observed after insulin administration, we tested whether insulin treatment affected the stability of LXRa mRNAs by using the RNA polymerase n inhibitor, actinomycin D. Primary hepatocytes were treated with 400 nM insulin for 24 h before addition of actinomycin D (2. 5 mglml), and cells were harvested at different time points within a period of 12 h. Actinomycin D increased the LXRa mRNA level by itself, as also seen in previous studies on oxidative enzymes (Sorensen et al., Biochim. Biophys. Acta, 1171, 263-271 1993). Insulin added together with actinomycin D induced LXRa mRNA in a additive or synergistic manner (Fig. 2). This indicates that the increased LXRa mRNA level following addition of insulin is not dependent on continous transcription, hence, at least partly, the insulin effect could be caused by stabilization of the transcripts. The absolute values of the mRNA transcripts were plotted in a time-curve, and the half-lives of the transcripts were estimated by extrapolation in the linear part of the mRNA time curve (data not shown). The LXRa mRNA half-life increased from 6 to 10 h after treatment by insulin (Fig. 2), indicating that the insulin induction of LXRa mRNA could be caused by stabilization of the transcripts.

Example 3 Effect of insulin injections in Sprague-Dawley rats Next, we investigated whether the LXRa gene is regulated by insulin in a physiological setting by giving rats injections of insulin. Sprague-Dawley rats were given 2 units Actrapid insulin (i. p.) and 2 units Insulatard insulin (s. c.) (Insulin from Novo Nordisk,

Denmark). Control animals were given vehicle (PBS). Table 1 shows plasma-values of triglyceride (Tg), glucose and insulin after 90 min of insulin administration. There were no variations in triglyceride levels in the two groups, but the plasma concentration of insulin was 20 times higher than the control group, and accordingly the glucose level decreased to about 30 % of the control group. After 90 mm of insulin stimulation, liver LXRa rnRNA was increased by 1.7 fold. In comparison, another known insulin responsive gene, SREBP1-c, was increased by 1.9 fold (Fig. 3). The cDNA probe used recognizes both the SREBP-lc and SREBP-la isoforms. In rodent liver, however, the expression of SREBP-1c predominates over that of SREBP-1 a by a 9 : 1 ratio (Shimomura et al., J. Clin. Invest., 99, 838-845,1997). We have thus considered that the signal obtained in Northern blots was representative of SREBP-1c expression.

Table 1. Blood serum values from Sprague-Dawley rats given injections of insulin.

The values represent 4 animals in both groups and are given as the mean SEM.

Triglyceride (mM) Glucose (mM) Insulin (nM) Controls T70 # 0.21 # 12.2 # 1.7 1.1 # 0.5 Insulin 0.6 # 0.05 3.6 # 0. 2 21. 3 : f : 11. 9 treatment Example 4 Effect of insulin injections in LXRaJ -/- D ce A mouse line where both the LXRa and the LXR genes have been impaired (LXRα/ -/-, (Alberit et al., Gene., 243, 93-103,2000)) was used to investigate whether LXRs were involved in upregulation of insulin responding gene transcription. LXRa/ and corresponding wildtype mice were given one injection of insulin (0.2 units Actrapid insulin, Novo Nordisk, Denmark), 90 min later blood was collected and livers excised for mRNA analysis. Table 2 shows plasma-values of triglycerides (Tg), glucose and insulin after 90 mm of insulin administration. Interestingly, the basal level for both triglycendes, glucose and insulin was lower in LXRa/ -1- mice than in wildtype. However, following insulin administration, tnglyceride levels in plasma were not different in control and insulin treated animals, as also seen in rats (table 1). As

expected, both wildtype and LXRa/p-/-mice had a higher level of plasma insulin after insulin treatment (2. 7-fold and 12-fold respectively), and corresponding to this, the concentration of plasma glucose values was lower after insulin treatment (table 2).

After 90 min of insulin administration the LXRa mRNA level in liver was increased only by 1. 8-fold in wild type mice compared to mice given vehicle only (Fig. 4). In comparison, SREBP-1 mRNA was strongly induced (4. 5-fold) following insulin administration. Interestingly, the inductions following insulin administration did not occur in LXRoYp-/-mice. This strongly indicates LXRs as candidates in the insulin signaling pathway (Fig. 4).

In light of the several reports on the importance of SREBP-1c on transcriptional regulation of hepatic lipogenic enzymes, and the fact that SREBP-1c is a LXR target gene, we wanted to study if the insulin induction of lipogenic enzymes was affected in LXR (x/p-/-niice. Figure 4 show northern blots for enzymes involved in fatty acid synthesis (ACC, FAS), cholesterol synthesis (HMG-CoA synthase, HMG-CoA reductase, squalene synthase, FPP synthase) and other branches of the lipogenic program (glucokinase (GK), malic enzyme (ME), S14). As seen in figure 4, several known insulin regulated genes are also induced by the hormone in this experiment (FAS, GK, squalene synthase, ME, ACC, S14), but as also seen for SREBP-1c this insulin induction was not observed in LXRa/ -1- mice. A couple of the genes (HMG-CoA synthase and FPP synthase) were not induced by insulin. Interestingly, the basal mRNA level of these genes was increased in livers from LXRo/p-/-mice. This indicates a suppressive role for LXRa in the basal transcription level for some genes in lipid metabolism.

Table 2. Blood serum values from LXRa/b-/-mice and corresponding wildtype mice given injections of insulin. The values represent 4 animals in both groups and are given as the mean SEM.

Treatment Triglyceride (mM) Glucose (mM) Insulin (nM) Wildtyp, PBS 1.0 0. 4 9. 9 3. 2'0. 9 0. 5 Wildtype Insulin 1. 2 0. 44. 1 1. 4 2.4 # 1. 0 LXRa/b-/-, PBS 0.5 # 0.1 6. 8 : 1 : 2.3 I 0. 3 : 1 : 0. 2 LXRa/b -1- I Insulin 0.6 0. 21. 5 0. 83. 6 1. 6 DISCUSSION The current results clearly demonstrates that insulin induces LXRa gene transcription in liver. The increase in LXRa mRNA is followed by an increase in LXRa protein level, and this in turn leads to an increase in the transcription of LXRa target genes, where one of them is SREBP-lc, and downstream target genes, many of which are lipogenic enzyme genes. Furthermore, deletion of the LXR genes markedly suppressed insulin mediated induction of an entire class of lipogenic enzymes. This work strongly suggests LXRs as a insulin mediating factor important in lipogenesis.

SREBP-1c is stimulated by insulin in liver and adipose tissue (Kim et al., J. Clin. Invest., 101, 1-19, 1998 and Foretz et al., Mol Cell Bol, 19, 3760-3768, 1999) but the mechanism by which this occurs has been unknown. We show in this study that LXRa is induced by insulin in a similar manner, and the fact that SREBP-lc is an LXR-target gene adds another link to the insulin signalling pathway mediated by SREBP-1c (Repa et al., Genes. Dev.,, 2819-2830, 2000 and Yoshikawa et ai., Mol. Cell. Biol., 2l, 2991-3000,2001). SREBP-lc has been focused on as a major factor for insulin induction of many genes in lipid metabolism. However, there are reports showing a rapid insulin induction of the glucokinase gene (GK) which could not be explained by the amount of nuclear SREBp-1c, indicating a need for another factor mediating the insulin effect together with SREBP-lc (Azzout-Mamiche et al., Biochem. J., 350, t-93,

2000 and Km1 et al., Mol Cell Bol, 15, 2852-2599, 1995). In addition, in SREBP-1-/mice the refeeding response in some hepatic lipogenic enzymes were completely abolished, whereas others were only partially suppressed, indicating other factors to be involved in the refeeding response (Shimamo et al., Mol. Cell. Biol.,, 2582-2599, 1995). LXR appears to be such a factor.

The LXRa induction by insulin is dose and time dependent, and could, at least partly, be due to stabilization of the transcripts. Further studies of the LXRa promoter will be required to understand the mechanisms of this regulation and the corresponding effects on cholesterol and lipid metabolism. However, we were not able to obtain induction of a reporter gene containing the LXRa-promoter when primary hepatocytes were transected with this construct and stimulated with insulin (data not shown).

Similarly, changes in the level of Peroxisome Proliferator Activated Receptora (PPARa) and RXRa in addition to PPARa target genes in rat liver cells by insulin and glucocorticoids are due to a major effect on steady-state mRNA levels giving rise to corresponding alterations in protein levels explained by mRNA stability changes and/or

translation effects (Sorensen et al., Eur. J. Biochem., 208, 705-711, 1992 ; Steineger et al., Eur. J. Biochem., 225, 967-974, 1994, Sorensen et al., Biochim. Biophys. Acta., 1171, 263-271, 1993 ; Lemberger et al., J. Biol. Chem., 269, 24527-24530, 1994 ; and Wan et al., Lab. Invest., 70, 547-552,1994).

We have earlier shown a PPARa dependent fatty acid upregulation of LXRa (Tobin

al., Mol. Endocrinol., 14, 741-752, 2000), and PPARa have previously been shown to , 741 be phosphorylated in response to insulin, resulting in stimulation of basal as well as ligand-dependent transcriptional activity (Shalev et al., Endocrinology., 137, 4499-4502,1996 and Juge-Aubry et al., J. Biol. Chem., 274, 10505-10510,1999).

PPARa could therefore be an upstream factor mediating the insulin effect on LXRa.

The insulin effect on LXRa mRNA level in the in vivo experiment in mice was quite modest, and does not correlate to the increase by insulin of the target genes. Therefore one might speculate if other mechanisms are involved, such as phosphorylation of LXRa itself, as a known insulin effect. The cooperation between LXRa and SREBP-lc is another possibility. Other factors which has been under investigation for mediating insulin response are the ubiquitouous bHLHLZ proteins USF1 and USF2. FAS mRNA was expressed at reduced levels during refeeding in animals where either USF 1 or USF2 genes were inactivated by homologous recombination. However, USFs are not

subjected to regulation by hormonal and nutritional manipulations as are SREBPs, suggesting USFs to function as essential factors that are required to maintain lipid metabolism irrespective of nutritional state.

The present information is clinically relevant to understanding the link between glucose and fatty acid metabolism, because LXR-a seems to have a major role in hepatic lipogenesis as seen in this study as well as differentiation of adipocytes.

All documents referred to above are incorporated herein by reference.

Claims

1. An assay for indentifying an agent for use in the treatment of diabetes or a disorder of fatty acid or cholesterol metabolism, comprising determining if a candidate agent modulates the biological activity of a liver X receptor alpha (LXRa) and/or liver X receptor beta (LXRss).

2. The assay of claim 1, comprising determining if a candidate agent modulates the biological activity of a LXRa.

3. The assay of claim 1, comprising contacting a cell containing LXRs and/or LXRO with a candidate agent and determining whether there is a change in the amount of at least one ofSREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, human cholesteryl

ester transfer protein (CETP), LXRa mRNA or protein and/or LXRO mRNA or protein.

4. The assay of claim 1, wherein the agent activates the biological activity of LXRa.

5. The assay of claim 1, wherein the agent inhibits the biological activity of LXRa.

6. The assay of claim 4, wherein the agent binds LXRa and/or LXRP and leads to an increase in the amount of at least one of SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or

protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRss mRNA or protein, in a cell comprising LXRa and/or LXRP.

7. The assay of claim 5, wherein the agent binds LXRa and/or LXRp and prevents or reduces the agonist induced biological activity of LXRa and/or LXRP.

8. The assay of claim 7, wherein the agent prevents or reduces an increase in the amount of at least one of SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRss mRNA or protem caused by contacting a cell comprising LXRa and/or LXRss with an agonist.

9. An assay for identifying an agent that activates the biological activity of LXRa and/or LXRss comprising contacting a cell containing LXRa and/or LXRss with a candidate agent and determining whether there is an increase in the amount of at least one of SREBP-1 mRNAor protein, cholesterol 7a-hydroxylase (Cyp7A) mRNA or protein, fatty acid synthetase (FAS) mRNA or protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRss mRNA or protein.

10. The assay of claim 9, wherein an increase in the amount of at least one of SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7A) MARNA or protein, fatty acid synthetase (FAS) mRNA or protein, human cholesteryl ester transfer protein (CETP), LXRa mRNA or protein and/or LXRp mRNA or protein, is determined by comparing the amount with that of a control cell which has not been contacted with a candidate agent.

11. An assay for identifying an agent that mhibits the biological activity of LXRa and/or LXRP comprising contacting a cell containing LXRa and/or LXRss with a candidate agent and a known agonist and determining whether the agent inhibits the agonist induced biological activity of LXRa and/or LXRss.

12. The assay of claim 11 wherein the inhibition of the agomst induced biological activity of LXRa and/or LXRss is determined by comparing the amount of at least one of SREBP-1 mRNA or protein, cholesterol 7a-hydroxylase (Cyp7a) mRNA or protem, fatty acid synthetase (FAS) mRNA or protein, human cholesterol ester transfer protein

(CETP), LXRa mRNA or protein and/or LXR ? mRNA or protein, in a cell contacted with the candidate agent and a known agonist with the amount in a cell contacted with the known agonist only.

13. The assay of any. one of the preceding claims, wherein the LXRa and/or LXRP is comprised in a cell.

14. The assay according to claim 13, wherein the cell is a hepatocyte, a dipocyte or preadipocyte.

10

15. The assay according to any one of the preceding claims which is performed in vitro using a cell culture.

16. The assay according to any one of claims 1 to 14, which is performed in vivo.

17. The assay according to any one of the preceding claims, wherem the candidate agent is a protein molecule, a polypeptide, a peptide, a carbohydrate, an organic or inorganic chemical compound.

18. The assay according to any one of the preceding claims, wherein the LXRa is human LXRa having the amino acid sequence shown in accession no. AAA 85856 (GenBank) or a functional homolog or fragment thereof

19. An assay according to claim 1, wherein the LXRP is human LXRss having the amino acid sequence shown in accession no. AAA61783 (GenBank) or a functional homolog or fragment thereof

20. The assay according to claim 18, wherein the LXRa used m the assay of the present invention is m combination with a retmoid X receptor (RXR).

21. A method for identifying a candidate agent comprising identifying an agent that binds to LXRa and/or LXR, B.

22. A novel agent that modulates the biological activity of LXRa and/or LXRss.

23. The novel agent according to claim 21, which activates the biological activity of

LXRa and/or LXRss.

24. The novel agent according to claim 21, which inhibits the biological activity of LXRa and/or LXRss.

25. The novel agent according to any one of claims 22 to 24, which is a protein, a polypeptide, a peptide, a carbohydrate or an organic or inorganic chemical compound.

26. The novel agent according to any one of claims 22 to 25 for use in therapy.

27. The novel agent of claim 26, wherein the therapy is the treatment of diabetes or a disorder of fatty acid or cholesterol metabolism.

28. Use of an agent that modulates the biological activity of LXRa and/or LXRss in the manufacture of a medicament for the treatment of diabetes or a disorder of fatty acid or cholesterol metabolism.

29. A method of treating diabetes or a disorder of fatty acid or cholesterol metabolism comprising administering to a patient in need of such treatment an effective dose of an agent that modulates the biological activity of LXRa and/or LXR.

30. A pharmaceutical composition comprising the novel agent according to any one of claims 22 to 25 in combination with a pharmaceutically acceptable carrier.