WO1999055904A1 - Screening assay for identifying therapeutics useful for modulating body weight - Google Patents

Abstract

The invention relates to methods for screening pharmaceutical agents to find useful therapeutics for modulating body weight. In particular, changes in selenoprotein activity result in metabolic changes associated with obesity and other weight disorders. Hence, drugs which modulate selenoprotein activity are useful for treating obesity and other weight related disorders.

Description

SCREENING ASSAY FOR IDENTIFYING THERAPEUTICS USEFUL FOR MODULATING BODY WEIGHT

This application is a continuation-in-part of Ser. No. 60/083,512, filed April 29, 1998.

FIELD OF THE INVENTION The invention relates to methods for screening pharmaceutical agents to find useful therapeutics for modulating body weight. In particular, changes in selenoprotein activity result in metabolic changes associated with obesity and other weight disorders. Hence, drugs which modulate selenoprotein activity are useful for treating obesity and other weight related disorders.

BACKGROUND OF THE INVENTION Obesity is the result of an imbalance between energy intake and energy expenditure. Most attempts to treat obesity focus on reducing food intake. Although appetite suppressants can be effective in controlling weight gain, the average weight loss is modest and typically ranges from 2 to 10 kg, even in excessively obese people. (Oeser, 1977). Since 2 to 10 kg is usually a small fraction of excessive weight, abnormal appetite cannot be the sole cause of obesity. The imbalance also results from reduced energy expenditure. There are numerous indications that metabolic defects are involved. In particular, obese people who have lost weight (the "post- obese") have a low rate of fat oxidation (Larson et al., 1995), defective diet-induced thermogenesis (Jequier and Schutz, 1985), enhanced metabolic efficiency (Astrup, 1996), increased lipoprotein lipase activity (Kern et al., 1990), and a lower overall mean metabolic rate (Shah et al., 1988). Post-obese individuals consume less energy than their lean counterparts to maintain a similar body weight (Jequier and Schutz, 1985).

A study comparing 5 inbred strains of laboratory mice found that dietary consumption was not correlated with obesity (Ismail et al., 1986). The authors

1 concluded that differences in energy deposition result primarily from variations in the level of energy expenditure rather than variations in energy intake. Similarly, a comparison of 9 inbred strains of mice found that obesity induced by a high fat diet was generally not attributable to increased consumption of energy (West et al., 1992). Relatively small differences in energy expenditure can have a large impact on weight gain. One computer model predicts that a 20% reduction of the resting metabolic rate will turn a lean person obese with no change in diet (Wiensier et al., 1993). In fact, "most available studies of energy intake have shown that obese subjects do not eat more than their thin counterparts" (Ravussin et al., 1988). Such studies indicate that weight accumulation is highly sensitive to defects in energy expenditure.

It has been discovered that changes in the activity of selenoproteins result in changes in body weight. For example, obese people or animals have less selenoprotein activity at comparable body weights and increasing selenoprotein activity leads to weight loss.

Selenoproteins contain selenocysteine which is incorporated during protein biosynthesis and has been recognized as a 21st amino acid (reviewed in Stadtman, 1996). The tRNA which transports and inserts selenocysteine into selenoproteins is first charged with serine which is then converted through at least two steps to selenocysteine (Burk, 1991). The selenium donor in selenocysteine synthesis is selenophosphate. Directed by a hairpin loop within the 3' untranslated region of selenoprotein encoding mRNAs, selenocysteine is incorporated during translation by a mechanism utilizing a unique tRNA and translation factor. Incorporation of selenocysteine into a selenoprotein is specified by the codon UGA, which in most genes is read as a stop codon (Low and Berry, 1996). ⁷⁵Se-labeling studies in rats indicate the existence of a total of more than 30 mammalian selenium-containing proteins (Behne et al., 1993).

Type I iodothyronine 5'-deiodinase (5'DI) is a selenoprotein found primarily in the liver and kidney. This enzyme converts the inactive thyroid prohormone thyroxine (T4) to the metabolically-active hormone 3,3',5-triiodothyronine (T3) (Daniels, 1996). Underexpression of 5'DI results in reduced synthesis of T3. 5'DI contains selenocysteine and histidine residues at its active site (Kohrle, 1994). Using site-directed mutagenesis, Berry et al. (1991) demonstrated that selenium is responsible for the biochemical properties which characterize type I iodothyronine monodeiodination. Other selenoproteins include glutathione peroxidase (GPx), selenoprotein P (SelP) and thioredoxin reductase (TRR). GPx and SelP contribute to the antioxidant capacity of numerous tissues and plasma.

Glutathione peroxidase catalyzes the reduction of hydrogen peroxide and has a highly specific cofactor, glutathione (GSH), as shown in the equation below. Selenocysteine is the catalytic moiety of GPx (Spallholz and Boylan, 1991).

GPx + 2 GSH 2 H₂O₂ > 2 H₂O + O₂

There are three different forms of GPx. One is located in the cytosol and is referred to as "cytosolic" or "classical" glutathione peroxidase. The second form is bound to the cell membrane and is referred to as "phospholipid" glutathione peroxidase. The third form is found in the plasma and is referred to as "plasma" or "extracellular" glutathione peroxidase (Daniels, 1996). While each type differs in structure, all three require selenocysteine for activity and facilitate a similar antioxidant reaction.

Selenoprotein P is the major selenoprotein in human plasma (Hill et al., 1996). Although its specific role within the plasma is unknown, Wilson and Tappel (1993) reported that selenoprotein P binds to cell membranes. Also, the temporal association between selenoprotein P levels and protection against oxidative damage, liver necrosis, and lipid peroxidation suggests that this protein serves an antioxidant role for cell membranes (Burk and Hill, 1994).

Goldthioglucose (GTG) was observed to induce obesity in a toxicological study of albino mice treated with the compound (Brecher and Waxier, 1949). The principal explanation for this phenomenon is that GTG enters cells of the ventromedial hypothalmus through the standard glucose entry pathway (Debons et al., 1977) and induces lesions damaging neurons in the satiety area (Brown and Viles, 1986), leading to uncontrolled appetite. However, uncontrolled appetite is not the sole determinant of weight gain in GTG-treated subjects, which become obese compared to a control group even if food intake is comparable (Miyada et al., 1987).

Interestingly, GTG has been independently shown to inhibit selenoenzymes (Chaudiere and Tappel, 1984), including 5'DI and glutathione peroxidase in rats (Berry et al, 1991) and thioredoxin reductase (Hill et al., 1997).

There is a clear need for a better understanding of the relationship between obesity and observed metabolic deficiencies and for therapeutic agents capable of correcting the relevant defect which underlies obesity. A better understanding of the mechanisms by which metabolism is controlled and body weight is maintained can result in drugs and treatments for patients who are overweight or underweight. Weight maintenance and efficiency of caloric intake would also be of interest to healthy individuals who wish to modify their weight as well as to growers of livestock who wish to improve efficiency of their operations.

SUMMARY OF THE INVENTION The present invention relates generally to metabolic defects and the causes of obesity or other weight disorders. The present inventors have established that changes in selenoprotein activity result in changes in body weight, and more particularly, that obese individuals have decreased selenoprotein activity at comparable body weights with lean individuals. This nexus has allowed development of a screening assay to identify pharmaceutical agents useful for modulating body weight, e.g., for treating obesity, causing weight reduction or causing weight gain. One advantage of this assay is that it can be conducted quickly and in bulk so it speeds identification of pharmaceutical agents useful for modulating body weight.

Selenoproteins play a role in regulating certain metabolic processes. Defects in those processes result in obesity or other weight-related disorders. Moreover, laboratory animals genetically predisposed to obesity exhibit defects in metabolic processes which require selenoproteins. For example, fatty Zucker rats (fa/fa) are deficient in activities related to 5'deiodinase and glutathione peroxidase.

Knowing a deficiency in selenium metabolism affects selenoprotein activity in general, one can screen for substances which raise the level of activity of any selenoprotein to identify pharmcologically active substances useful for treating obesity or reducing body weight. Similarly, one can screen for substances which depress the level of activity of any selenoprotein to identify pharmacologically active substances useful for increasing body weight or increasing metabolic efficiency. Accordingly, the invention relates to a method for identifying a pharmaceutical agent useful for modulating body weight. In one embodiment of the invention, the method comprises assaying for selenoprotein activity in the presence and absence of a test substance, determining whether the presence of the test substance increases or decreases selenoprotein activity relative to its absence, and identifying any test substance which increases or decreases selenoprotein activity as a pharmaceutical agent useful for modulating body weight. Examples of preferred selenoproteins which can be used in the invention are 5'-deiodinase, glutathione peroxidase, selenoprotein P and thioredoxin reductase. According to the invention, selenoprotein activity can be measured in a cell, a cell extract, a body fluid, serum, plasma, a tissue or a tissue extract. Selenoprotein activity can be measured using a purified or partially-purified selenoprotein or a selenoprotein in a cell, a cell extract, a body fluid, serum, plasma, a tissue or a tissue extract. The source of the cell, cell extract, body fluid, serum, plasma, tissue or tissue extract can be an organ, examples of which include liver and lung. In various embodiments of the invention, the selenoprotein may be in crude form, or be purified or partially-purified. Moreover, the selenoprotein activity which is assayed can be a protein activity, or the level of expression of the selenoprotein.

In an embodiment of the invention, the method identifies test substances which cause an increase in selenoprotein activity as a pharmaceutical agent useful for reducing weight gain. In another embodiment of the invention, the method identifies test substances which cause a decrease in selenoprotein activity as a pharmaceutical agent useful for inducing weight gain.

The invention is further embodied by a method for identifying a pharmaceutical agent useful for modulating body weight by administering a test substance to an animal, assaying an organ, tissue, cell, body fluid, or an extract thereof, from the animal for selenoprotein activity, determining whether the test substance increases or decreases selenoprotein activity relative to selenoprotein activity assayed in a control animal in a comparable manner, and identifying any test substance which increases or decreases selenoprotein activity as a pharmaceutical agent useful for modulating body weight. The various further embodiments of this method, such as the selenoprotein, assayed tissue and the like, are the same as in the method wherein the effect of the test substance on the selenoprotein activity is assessed directly.

The invention also includes pharmaceutical agents identified by any of the above methods.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 depicts the results of assays measuring GPx activity in the livers of lean and fatty Zucker rats. The rate of increase of GPx activity with body weight for lean rats is more than twice that of fatty rats. For lean rats, the equation for GPx activity is given by GPx = 240.02 + 4.24 BW; For fatty rats, the equation is GPx = 333.66 + 2.06 BW.

Fig. 2 depicts the results of assays measuring GPx activity in lung tissue of lean and fatty Zucker rats. Lean rats demonstrate levels of GPx activity that increase with body weight. Fatty rats demonstrate no such increase. For lean rats, the equation for GPx activity is given by GPx = -11.79 + 0.72 BW; For fatty rats, the equation is GPx = 32.21 + 0.1 BW.

Fig. 3 depicts feed efficiency in SWR, AKR, C3H/HeJ and DBA mice. The study measured weight gain resulting from a high fat diet. Fig. 4 depicts the relationship between feed efficiency and GPx activity in SWR, AKR, C3H/HeJ and DBA mice. Mice which were increasingly resistant to weight gain displayed higher levels of GPx activity.

Fig. 5 sets forth a list of some well known selenoproteins and other biological compounds which are responsive to the indicated selenoproteins.

DETAILED DESCRIPTION OF THE INVENTION The relationship between obesity and the level of selenoprotein activity is demonstrated in animal models. This relationship permitted development of a method which can be used to test substances for the ability to modulate the activity of selenoproteins and determine which of those substances act as pharmaceutical agents useful for modulating body weight. Activators of selenoprotein activity are useful for treating obesity and reducing weight gain, whereas inhibitors of selenoproteins are useful for reduction of energy utilization and enhancement of appetite, for example, to enable weight gain or maintenance of weight with the intake of fewer calories. Such drugs can also be useful in the alleviation of wasting associated with certain diseases (e.g., AIDS).

The activity associated with a selenoprotein can be modulated in a variety of ways. For convenience and as used herein, the term "selenoprotein activity" includes both protein activity (including enzymatic activities) and levels of expression of the selenoprotein. Activators and inhibitors of selenoproteins as used herein are substances which increase or decrease the protein or enzymatic activity associated with one or more selenoproteins which are present in a cell or tissue or body fluid or extract thereof or which alter the amount of selenoprotein present, e.g., by changing its level of expression. Selenoproteins are produced by incorporation of selenocysteine as the protein is translated, and inhibitors or activators which affect selenocysteine availability and incorporation into proteins can be identified by the methods of the invention. Selenoprotein activity can be modulated by substances which increase or decrease the level of expression or the amount of the selenoprotein that is present. Selenoprotein activity can be modulated by activators and inhibitors which alter the function of a selenoprotein, e.g., by binding blocking an active site or modifying the conformation of the selenoprotein to enhance or reduce its activity. These are meant as examples, but do not limit the ways in which selenoprotein activity may be modulated according to the invention. The present method for identifying pharmaceutical agents useful for modulating body weight includes assaying for the activity of a selenoprotein from an animal or cell treated with a test substance, and comparing the activity to that of the selenoprotein from an animal or cell which has not been treated with the test substance. Assays for identifying therapeutics of the invention also include direct assay of selenoprotein activity using a pure or partially-purified selenoprotein or a crude mixture containing a selenoprotein. Test substances which increase or decrease selenoprotein activity are selected as useful agents for modulating body weight.

Selenoproteins of the invention include, but are not limited to glutathione peroxidase (GPx), selenoprotein P, selenoprotein W, iodithyronine deiodinase (5'DI), thioredoxin reductase (TRR), mitochondrial capsule selenoprotein (MCS),

[NiFeSej-hydrogenase and formate dehydrogenase. (See Fig. 5) Any selenoprotein- associated activity can be assayed.

An assay measuring glutathione peroxidase activity, whether from an animal, a cell, a pure protein and the like, is a preferred assay. For example, assay kits for GPx are commercially available. There is no specific requirement for the source of the enzymatic activity. However, preferred sources are likely to be those which enable large numbers of substances to be screened simultaneously. Intact cells or tissues can be used since they allow the identification of activators and inhibitors of activities of selenoproteins which act indirectly. For example, a test substance which induces increased expression of 5'deiodinase can be identified by an assay using a cell extract which supports at least transcription and possibly translation depending on what is detected (e.g., mRNA, enzyme activity, or amount of protein). Similarly, to identify test substances which affect selenocysteine incorporation, the assay system needs active protein translation capability. Such assays can be obtained from intact cells or tissues and are known in the art.

8 Various rat, mouse and human tissues and cell lines known to express selenoproteins can be used to assay test substances to identify activators and inhibitors of selenoprotein activity. For example, Dreher et al. (1997) provides a method to examine various tissues and cell lines for expression of selenoprotein transcripts. As reported in Dreher et al., cytosolic GPx mRNA transcripts were detected in kidney, spleen, heart, liver and lung of rats, and corresponded to measurements of GPx activity. Human heart, liver and lung tissues also exhibited strong cytosolic GPx signals. Liver and lung tissue of both humans and rats displayed selenoprotein P transcripts. Of the tissues tested, hepatic tissue showed the broadest repertoire of selenoprotein transcripts. Human cell lines were found to express cytosolic GPx

(thyroid carcinoma) and selenoprotein P (HepG2 and HTh74 thyroid cells).

If the test substance is administered to an animal, the selenoprotein activity can be assayed in or from a tissue, body fluid, including plasma or serum, or an extract of any of these. For example, in screening for activity of a selenoprotein, one can compare the results of an assay which includes a test substance with a parallel assay where the test substance is not present. In another embodiment, the test substance can be added to cells or administered to an animal and samples taken at successive time points to determine activation or inhibition of selenoprotein activity over time. To assay for 5'DI, cells or tissues are harvested and sonicated in 100 mM potassium phosphate, 1 mM EDTA, pH 6.9 (PE buffer) containing 25 mM DTT, 5 - 150 μg of cell sonicate protein, 0.2 nM ¹²⁵I-3,3',5'-triiodothyronine (rT3), varying concentrations of unlabeled rT3 and 10 mM DTT in PE buffer in a final volume of 300 μl. The reaction is incubated for 30 min. at 37 °C and I^" release is quantitated (Berry, 1992). Increased I^" release corresponds to an increase in 5'DI activity.

Glutathione peroxidase activity can be assayed using the BIOXYTECH® GPx-340™ Assay (OXIS International, Portland, OR) using, for example, a cell or tissue homogenate. The GPx-340 assay is an indirect measure of the activity of GPx. Oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by GPx, is recycled to its reduced state by the enzyme glutathione reductase (GR). Reduction of GSSG is accompanied by the oxidation of NADPH to NADP+ leading to a decrease in absorbance at 340nm (A340).

In the BIOXYTECH® assay, for example, cell or tissue homogenate is added to a solution containing glutathione, glutathione reductase, and NADPH, with or without the test substance. The enzyme reaction is initiated by adding the substrate, tert-butyl hydroperoxide and the A340 is recorded. The rate of decrease in the A340 is directly proportional to the GPx activity in the sample. The assay reaction mixture is: 1 mM Glutathione, > 0.4 U/mL Glutathione reductase, 0.2 mM NADPH, 0.22 mM tert-Butyl Hydroperoxide, pH 7.6 ± 0.05 at 23 °C. Alternatively, the plasma form of GPx can be measured by an enzyme-linked immunoassay (ELISA). Such an assay is useful for measuring GPx activity in the tissues or other body components of animals treated with or without a test substance. In this assay, samples are incubated in the wells of a microtiter plate, which have been coated with antibodies specific for human plasma-GPx (pl-GPx). Polyclonal antibodies obtained by using a synthetic antigen and purified by affinity chromatography can also be used for this purpose and are available in kit form (pl-GPx-EIA™, OXIS International, Portland, OR). In this regard, any antibody specific for GPx, whether monoclonal or polyclonal, can be used in an ELISA to detect GPx levels. Likewise, other selenoproteins can be detected using an ELISA with an antibody specific for the particular selenoprotein.

To assay for thioredoxin reductase activity, cell or tissue extracts are prepared using a buffer containing 250 mM sucrose, 20 mM N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid, and 1 mM EDTA (pH 7.4). 100 μl of the extract (protein content 0.1 - 0.6 mg) is added to 1 ml of assay mixture, with or without the test substance (as appropriate), in a cuvette. The assay mixture contains 100 mM potassium phosphate

(pH 7.0), 10 mM EDTA, 2 mg/ml DTΝB, and 0.2 mg/ml ΝADPH. The change in absorption at 412 nm is monitored. A reference cuvette containing assay mixture and extraction buffer is monitored in parallel to correct for oxidation of the substrate by air. To screen large numbers of samples, volumes can be reduced and assays performed in microtiter plates.

10 It is also useful to monitor amounts of selenoproteins which are produced in the presence and absence of a test substance. Amounts of selenoproteins can be determined by radioimmunoassay (RIA) or by radioimmunoprecipitation. For example, selenoproteins can be labeled in growing cells supplemented with ⁷⁵SeO₃ or in animals injected with small doses of ⁷⁵SeO₃. ⁷⁵Se-labelled selenoproteins can then be captured on microtiter plates coated with the appropriate antibody preparation and the quantity of bound ⁷⁵Se determined. For example, Yang et al. (1987) describes the creation of monoclonal antibodies specific for selenoprotein P and use of a competitive RIA to demonstrate that selenium-deficient rats have less than 10% as much selenoprotein P in their plasma as control rats.

It is well within the ability of one of ordinary skill to generate polyclonal or monoclonal antibodies to one or more selenoproteins of choice, for use in immunoassays. In this regard, any type of immunoassay capable of specifically detecting changes in the amounts of a selenoprotein is contemplated by the invention. To determine whether decreased levels of selenoproteins are associated with obesity, animal models were employed. Zucker rats carrying two copies of a recessive fa allele have a genetic predisposition to obesity. Plasma T3 concentrations in these rats was significantly reduced (Table 2). T3 concentrations in fa/fa Zucker rats were determined to be one third of that found in normal (lean) Fa/Fa or Fa/fa counterparts, even when the obese rats had a body weights which averaged 50% higher.

GPx activity was measured for liver and lung tissue. For both fatty and lean rats, GPx activity in liver (Fig. 1) increased with body weight. However, comparing the increase in GPx activity between fatty and lean rats as a function of body weight, it was found that the increase was twice as much in lean rats. The results obtained from lung tissue are in Fig. 2. GPx activity measured in lung tissue of fatty rats displayed essentially no increase as a function of body weight, although a clear increase was measured in lean rats.

Assays were performed to investigate GPx activity in four inbred strains of mice. To provide a basis for making this comparison, feed efficiency was determined

11 for each of the mouse strains. Feed efficiency is a measure of weight gained per calorie consumed. Higher feed efficiency means that, for a given caloric intake, greater body weight is attained. Mouse strains were ordered according to feed efficiency (Fig. 3) and levels of GPx activity were measured before and after feeding the mice a controlled diet to increase their weight over a 7 week period. GPx activity was highest for SWR mice, which display the lowest relative feed efficiency and lowest for DBA mice, which display the highest relative feed efficiency (Fig. 4). The relationship for mice displaying distinct intermediate relative feed efficiencies (AKR and C3H/HeJ) was consistent with the results for SWR and DBA mice. The GPx activity measured in 4 week old mice predicts the subsequent rate of body weight gain for a given diet.

Throughout this application, various publications, patents, and patent applications have been referred to. The teachings and disclosures of these publications, patents, and patent applications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which the present invention pertains.

It is to be understood and expected that variations in the principles of invention herein disclosed may be made by one skilled in the art and it is intended that such modifications are to be included within the scope of the present invention.

12 EXAMPLE 1 Comparison of Glutathione Peroxidase (GPx) Activities in Lean and Fatty Zucker Rats.

a) Materials and Methods

Animals - Lean (Fa/Fa or Fa/fa) and fatty (fa/fa) female Zucker rats, 28-30 days of age, were obtained from Charles River Laboratories (Wilmington, MA). The animals were maintained on a standard lab chow diet. All specimens appeared healthy with no apparent developmental, physiological, or behavioral abnormalities. The rats were shipped live to PelFreez Biologicals (Rogers, AR) for tissue collection.

Tissue Collection - Blood samples were collected by cardiac puncture into heparinized glass tubes. Liver, lung, skeletal muscle, and kidneys were perfused with heparinized saline and quick-frozen in liquid nitrogen. Blood samples and rat organs were shipped on wet ice and dry ice, respectively, to the University of Rochester Medical Center (Rochester, NY).

Tissue Preparation and Storage - Blood was centrifuged at 4 °C for 10 minutes at approximately 8500 x g and the plasma collected and stored at -20 °C for further assays. The cellular pellet was washed in 10 volumes of 4 °C buffer (50 mM TRIS-HC1, pH7.5, 5 mM EDTA, 1 mM dithiothreitol) and recentrifuged. The supernatant was removed and the pellet lysed by adding 4 volumes of 4 °C deionized water. The lysates were then centrifuged at 8500 x g at 4 °C for 10 minutes and the clarified supernatant was transferred to tubes and stored at -70 °C until assayed.

To prepare solid tissues, a representative sample was weighed to estimate the tissue volume, and the tissue was placed in 5 volumes of 4 °C buffer (50 mM TRIS-HC1 pH7.5, 5 mM EDTA, 1 mM dithiothreitol). Using a tissue homogenizer

("SDT Tissumizer", Tekmar Company, Cincinnati, OH) the tissue was macerated at a setting between 30-40 (of 100) for 10 seconds, allowed to cool undisturbed on ice to dissipate any generated heat, and again homogenized as described. If necessary, a third homogenization was performed. Specimens were always kept on ice during the homogenization. The homogenates were then transferred to the appropriate tubes and

13 centrifuged at 5 °C for 60 minutes at approximately 120,000 x g (Beckman 50.1 TI rotor, 40,000 RPM). Supematants were collected, avoiding the whitish film covering the tube top. Supematants were kept on ice and frozen at -70 °C immediately after preparation. Buffers were made fresh each day, with dithiothreitol added immediately before chilling on ice.

Plasma T3 Determinations - IMx Microparticulate Enzyme Immunoassay assay kits (Abbott, Abbott Park, IL) were used to determine total plasma T3 concentrations. Enzyme Assay - BIOXYTECH GPx-340 assay kits for glutathione peroxidase were purchased from Oxis International, Inc. (Portland, OR). The assay protocol was performed as recommended by the manufacturer using a Milton Roy Spectronic 1201 spectrometer. To assure linearity, several dilutions of the samples were assayed prior to running the actual assay. Protein Determination - Protein concentrations were determined by the

Coomassie Brilliant Blue G-250 colorimetric assay.

b) Results Plasma T3 Determinations - As shown in table 2, plasma T3 was over three times more abundant in lean mice compared to fatty mice.

Table 2

Lean (Fa/?) Fatty (fa/fa)

Number of observations 15 13

Average body weight (g) 71.65 110.14*

Average T3 concentration (ng/dl) 76.67 24.23 *

t-tests, * pθ.001

The T3 results are consistent with reduced 5'DI activity in fa/fa rats as suggested in several prior studies (Katzeff et al., 1993; Young et al., 1984; Mclntosh et al., 1989).

14 GPx Activity - Zucker rats of each phenotype were analyzed for liver GPx activity. The results are shown in the Fig. 1. The results indicate that GPx activity in liver tissue of lean rats is related to body weight (B W) by the equation GPx = 240.02 + 4.24 BW where GPx activity is measured in mU/mg protein and body weight is measured in grams. One unit of GPx will catalyse the oxidation of 1 mmol of reduced glutathione per minute at pH 7.0 at 25 °C. The corresponding relationship in fatty rats is given by the equation GPx = 333.66 + 2.06 BW. The results indicate that fatty rats have reduced GPx activity versus lean when compared at similar body weights. In an similar manner, lung tissue was assessed for GPx activity. The results are shown in Fig. 2. GPx activity in lung tissue of lean rats is related to body weight by the equation GPx = -11.79 + 0.72 BW. The corresponding relationship in fatty rats is GPx = 32.21 + 0.01 BW. Again, the results indicate that fatty rats have reduced GPx activity versus lean rats when compared at similar body weights.

15 EXAMPLE 2 Comparison of Glutathione Peroxidase (GPx) Activities Among Mice Having Different Susceptibilities to Obesity

a) Materials and Methods

Ten mice of each strain (AKR, DBA, C3H and SWR) at 4 weeks of age, were obtained from Jackson Laboratories (Bar Harbor, ME). The animals were maintained on a standard lab chow diet. All specimens appeared healthy with no apparent developmental, physiological, or behavioral abnormalities. The mice were shipped live to PelFreez Biologicals (Rogers, AR) for tissue collection.

Tissue collection, preparation, storage, GPx assays, and protein determinations were performed as described in Example 1.

b) Results Four mouse strains were selected based on a recent review that examined feed efficiency when a high fat diet was provided (West et al., 1992). Calculation of feed efficiency was used as a measure of strain susceptibility to dietary obesity. SWR, AKR, C3H and DBA mice 5 to 12 weeks old were provided with a high fat diet. The feed efficiency, given as percent change in body weight per 1000 kcal of dietary intake is depicted in Fig. 3.

The relationship between GPx activity in 4 week old AKR, DBA, C3H and SWR mice and the subsequent weight gain at weeks 5 to 12 following high fat feeding was then studied. The results from SWR mice, which have the lowest feed efficiency and the greatest resistance to weight gain under high fat feeding, were used as a baseline for comparing hepatic GPx activity. Fig. 4 depicts the relationship between feed efficiency and GPx activity for the four mouse strains. Hepatic GPx activity is consistently and proportionately lower in mice with increased susceptibility to obesity.

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Claims

WE CLAIM:

1. A method for identifying a pharmaceutical agent useful for modulating body weight which comprises: assaying for selenoprotein activity in the presence and absence of a test substance; determining whether the presence of said test substance increases or decreases selenoprotein activity relative to the absence of said test substance; and identifying any test substance which thereby increases or decreases selenoprotein activity as a pharmaceutical agent useful for modulating body weight.

2. The method of Claim 1 wherein said selenoprotein is 5'-deiodinase, glutathione peroxidase, selenoprotein P or thioredoxin reductase.

3. The method of Claim 1 wherein said activity is measured in a cell, a cell extract, a body fluid, serum, plasma, a tissue or a tissue extract.

4. The method of Claim 1 wherein said activity is measured using a purified or partially-purified selenoprotein or a selenoprotein in a cell, a cell extract, a body fluid, serum, plasma, a tissue or a tissue extract.

5. The method of Claim 3 or 4 wherein said cell, cell extract, body fluid, serum, plasma, tissue or tissue extract is obtained from an organ.

6. The method of Claim 5 wherein said organ is liver or lung.

7. The method of Claim 2 wherein selenoprotien is purified or partially- purified.

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8. The method of Claim 1 wherein said activity is protein activity of said selenoprotein.

9. The method of Claim 1 wherein said activity is the level of expression of said selenoprotein.

10. The method of Claim 1 wherein said activity increases and modulating body weight results in reducing weight gain.

11. The method of Claim 1 wherein said activity decreases and modulating body weight results in inducing weight gain.

12. A method for identifying a pharmaceutical agent useful for modulating body weight which comprises: administering a test substance to an animal; assaying an organ, tissue, cell, body fluid, or an extract thereof, from said animal for selenoprotein activity; determining whether said test substance increases or decreases selenoprotein activity relative to selenoprotein activity assayed in a control animal in a comparable manner; and identifying any test substance which thereby increases or decreases selenoprotein activity as a pharmaceutical agent useful for modulating body weight.

13. The method of Claim 12 wherein said selenoprotein is 5'-deiodinase, glutathione peroxidase, selenoprotein P or thioredoxin reductase.

14. The method of Claim 12 wherein said activity is protein activity of said selenoprotein.

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15. The method of Claim 12 wherein said activity is the level of expression of said selenoprotein.

16. The method of Claim 12 wherein said organ is liver or lung.

17. The method of Claim 12 wherein said activity increases and modulating body weight results in reducing weight gain.

18. The method of Claim 12 wherein said activity decreases and modulating body weight results in inducing weight gain.

19. A method for identifying a pharmaceutical agent useful for treating obesity or inducing weight loss which comprises: assaying for selenoprotein activity in the presence and absence of a test substance; determining whether the presence of said test substance increases selenoprotein activity relative to the absence of said test substance; and identifying any test substance which thereby increases selenoprotein activity as a pharmaceutical agent useful for treating obesity or inducing weight loss.

20. The method of Claim 19 wherein said selenoprotein is 5'-deiodinase, glutathione peroxidase, selenoprotein P or thioredoxin reductase.

21. The method of Claim 20 wherein selenoprotein is purified or partially- purified.

22. The method of Claim 19 wherein said activity is measured in a cell, a cell extract, a body fluid, serum, plasma, a tissue or a tissue extract.

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23. The method of Claim 19 wherein said activity is measured using a purified or partially-purified selenoprotein or a selenoprotein in a cell, a cell extract, a body fluid, serum, plasma, a tissue or a tissue extract.

24. The method of Claim 19 wherein said activity is protein activity of said selenoprotein.

25. The method of Claim 19 wherein said activity is the level of expression of said selenoprotein.

26. A method for identifying a pharmaceutical agent useful for treating obesity or inducing weight loss which comprises: administering a test substance to an animal; assaying an organ, tissue, cell, body fluid, or an extract thereof, from said animal for selenoprotein activity; determining whether said test substance increases selenoprotein activity relative to selenoprotein activity assayed in a control animal in a comparable manner; and identifying any test substance which thereby increases selenoprotein activity as said pharmaceutical agent.

27. The method of Claim 26 wherein said selenoprotein is 5'-deiodinase, glutathione peroxidase, selenoprotein P or thioredoxin reductase.

28. The method of Claim 26 wherein said activity is protein activity of said selenoprotein.

29. The method of Claim 26 wherein said activity is the level of expression of said selenoprotein.

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30. The method of Claim 26 wherein said organ is liver or lung.

31. A pharmaceutical agent useful for modulating body weight identified by any one of the the methods of Claims 1, 12, 19 or 26.

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