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WO1999055904A1 - Test de criblage d'agents therapeutiques permettant de moduler le poids corporel - Google Patents

Test de criblage d'agents therapeutiques permettant de moduler le poids corporel Download PDF

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WO1999055904A1
WO1999055904A1 PCT/US1999/009550 US9909550W WO9955904A1 WO 1999055904 A1 WO1999055904 A1 WO 1999055904A1 US 9909550 W US9909550 W US 9909550W WO 9955904 A1 WO9955904 A1 WO 9955904A1
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selenoprotein
activity
test substance
tissue
cell
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Hanan Polansky
Jeffrey D. Schraver
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SCI PHARMACEUTICALS Inc
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SCI PHARMACEUTICALS Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the invention relates to methods for screening pharmaceutical agents to find useful therapeutics for modulating body weight.
  • changes in selenoprotein activity result in metabolic changes associated with obesity and other weight disorders.
  • drugs which modulate selenoprotein activity are useful for treating obesity and other weight related disorders.
  • 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.
  • post- obese 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).
  • 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.
  • Type I iodothyronine 5'-deiodinase 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.
  • 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.
  • GSH glutathione
  • Selenocysteine is the catalytic moiety of GPx (Spallholz and Boylan, 1991).
  • GPx 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).
  • GTG Goldthioglucose
  • GTG has been independently shown to inhibit selenoenzymes (Clustrated and Tappel, 1984), including 5'DI and glutathione peroxidase in rats (Berry et al, 1991) and thioredoxin reductase (Hill et al., 1997).
  • 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.
  • the invention relates to a method for identifying a pharmaceutical agent useful for modulating body weight.
  • 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.
  • preferred selenoproteins which can be used in the invention are 5'-deiodinase, glutathione peroxidase, selenoprotein P and thioredoxin reductase.
  • 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.
  • the selenoprotein may be in crude form, or be purified or partially-purified.
  • the selenoprotein activity which is assayed can be a protein activity, or the level of expression of the selenoprotein.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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),
  • GPx glutathione peroxidase
  • selenoprotein P selenoprotein W
  • iodithyronine deiodinase 5'DI
  • TRR thioredoxin reductase
  • MCS mitochondrial capsule selenoprotein
  • an assay measuring glutathione peroxidase activity is a preferred assay.
  • 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.
  • 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).
  • the assay system needs active protein translation capability. Such assays can be obtained from intact cells or tissues and are known in the art.
  • 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.
  • Dreher et al. (1997) provides a method to examine various tissues and cell lines for expression of selenoprotein transcripts.
  • 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 thyroid carcinoma
  • selenoprotein P HepG2 and HTh74 thyroid cells
  • 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.
  • 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.
  • 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 ⁇ g of cell sonicate protein, 0.2 nM 125 I-3,3',5'-triiodothyronine (rT3), varying concentrations of unlabeled rT3 and 10 mM DTT in PE buffer in a final volume of 300 ⁇ l.
  • 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-340TM 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).
  • 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.
  • the plasma form of GPx can be measured by an enzyme-linked immunoassay (ELISA).
  • ELISA enzyme-linked immunoassay
  • samples are incubated in the wells of a microtiter plate, which have been coated with antibodies specific for human plasma-GPx (pl-GPx).
  • pl-GPx antibodies specific for human plasma-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-EIATM, OXIS International, Portland, OR).
  • any antibody specific for GPx whether monoclonal or polyclonal, can be used in an ELISA to detect GPx levels.
  • other selenoproteins can be detected using an ELISA with an antibody specific for the particular selenoprotein.
  • 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 ⁇ l 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
  • Amounts of selenoproteins can be determined by radioimmunoassay (RIA) or by radioimmunoprecipitation.
  • RIA radioimmunoassay
  • selenoproteins can be labeled in growing cells supplemented with 75 SeO 3 or in animals injected with small doses of 75 SeO 3 .
  • 75 Se-labelled selenoproteins can then be captured on microtiter plates coated with the appropriate antibody preparation and the quantity of bound 75 Se determined.
  • 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.
  • any type of immunoassay capable of specifically detecting changes in the amounts of a selenoprotein is contemplated by the invention.
  • 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.
  • GPx activity in liver (Fig. 1) increased with body weight.
  • 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.
  • 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.
  • 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.
  • tissue homogenizer 50 mM TRIS-HC1 pH7.5, 5 mM EDTA, 1 mM dithiothreitol.
  • Plasma T3 Determinations - IMx Microparticulate Enzyme Immunoassay assay kits 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
  • Plasma T3 Determinations As shown in table 2, plasma T3 was over three times more abundant in lean mice compared to fatty mice.
  • 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).
  • GPx 32.21 + 0.01 BW.
  • mice of each strain 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.
  • GPx activity in 4 week old AKR, DBA, C3H and SWR mice was then studied.
  • 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.
  • Thioredoxin reductase activity is decreased by selenium deficiency. Biochem. Biophys. Res. Commun. 234:293-295

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Abstract

La présente invention concerne des procédés permettant de cribler des agents thérapeutiques afin de découvrir ceux qui peuvent être utilisés afin de moduler le poids corporel. En particulier, des modifications de l'activité de la sélénoprotéine entraînent des changements métaboliques associés à l'obésité et à d'autres dérèglements du poids. La présente invention permet d'obtenir des médicaments modulant l'activité de la sélénoprotéine, qui peuvent être utilisés pour traiter l'obésité et d'autres affections liées au poids.
PCT/US1999/009550 1998-04-29 1999-04-29 Test de criblage d'agents therapeutiques permettant de moduler le poids corporel Ceased WO1999055904A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8304401B2 (en) 2008-09-02 2012-11-06 Trustees Of Dartmouth College Compositions and methods for decreasing type III deiodinase activity to modulate adiposity and blood glucose levels

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4849346A (en) * 1987-02-12 1989-07-18 Regents Of The University Of Minnesota Method for determining thioredoxin reductase activity
US5594104A (en) * 1995-01-31 1997-01-14 Eli Lilly And Company Anti-obesity proteins

Patent Citations (2)

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US4849346A (en) * 1987-02-12 1989-07-18 Regents Of The University Of Minnesota Method for determining thioredoxin reductase activity
US5594104A (en) * 1995-01-31 1997-01-14 Eli Lilly And Company Anti-obesity proteins

Non-Patent Citations (2)

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Title
FLEGAL K M, ET AL.: "DIETARY SELENIUM AND CADMIUM INTERRELATIONSHIPS IN GROWING SWINE", THE JOURNAL OF NUTRITION, AMERICAN SOCIETY FOR NUTRITION, US, vol. 110, no. 06, 1 January 1980 (1980-01-01), US, pages 1255 - 1261, XP002919563, ISSN: 0022-3166 *
GOTZSCHE L S B-H, ET AL.: "THE INFLUENCE OF GROWTH HORMONE AND THYROXINE ON IODOTHYRONINE DEIODINASE ACTIVITY IN THE LIVER, KIDNEY AND BROWN ADIPOSE TISSUE IN HYPOPHYSECTOMIZED RATS", ACTA ENDOCRINOLOGICA., SCANDINAVIAN UNIVERSITY PRESS, OSLO., SE, vol. 125, no. 02, 1 January 1991 (1991-01-01), SE, pages 219 - 226, XP002919564, ISSN: 0001-5598 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8304401B2 (en) 2008-09-02 2012-11-06 Trustees Of Dartmouth College Compositions and methods for decreasing type III deiodinase activity to modulate adiposity and blood glucose levels

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WO1999055904A1 (fr) Test de criblage d'agents therapeutiques permettant de moduler le poids corporel

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