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WO2005045054A2 - Procedes pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse - Google Patents

Procedes pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse Download PDF

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WO2005045054A2
WO2005045054A2 PCT/EP2004/012555 EP2004012555W WO2005045054A2 WO 2005045054 A2 WO2005045054 A2 WO 2005045054A2 EP 2004012555 W EP2004012555 W EP 2004012555W WO 2005045054 A2 WO2005045054 A2 WO 2005045054A2
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fat
genes
body mass
substance
expression
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WO2005045054A3 (fr
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Gudrun Brockmann
Ulla Renne
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FORSCHUNGSINSTITUT fur DIE BIOLOGIE LANDWIRTSCHAFTLICHER NUTZTIERE
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FORSCHUNGSINSTITUT fur DIE BIOLOGIE LANDWIRTSCHAFTLICHER NUTZTIERE
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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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates in particular to methods for identifying active substances for influencing the body mass and the fat deposits.
  • Obesity is a major health risk factor for type II diabetes, coronary heart disease and metabolic diseases.
  • WHO World Health Organization
  • BMI Body Mass Index
  • Obesity results from the storage of excess energy from food.
  • the physiological causes of obesity can be one or more changes in appetite behavior, nutrient metabolism, heat development or energy distribution.
  • Body weight and appetite are essentially regulated by neuropeptides released by the hypothalamus in response to signals from fat, muscle, the liver and the gastrointestinal tract.
  • the hypothalamic responses can activate the sympathetic nervous system that innervates the internal organs.
  • the brain also controls energy expenditure via the hypothalamic-pituitary-thyroid axis.
  • the extremely obese phenotype is often accompanied by a disorder of hoostasis, which is regulated by hormones related to obesity, which can lead to hyperleptinemia and hyperinsulinemia, for example.
  • Obesity is a complex trait that reflects the effect of a gene network and is affected by food, age, gender, and exercise.
  • Evidence of a genetic impact on obesity came from twin studies.
  • the inheritance of fat mass has been estimated to be around 40-70%, and environmental factors contribute to around 26% of the phenotype variance.
  • QTL quantitative trait loci
  • PRKAG3 protein kinase, AMP-activated, gamma 3 non-catatlytic subunit
  • genes affecting body mass and body composition have been identified in monogenic mouse models. The fundamental importance of these genes for the regulation of body mass has been demonstrated in humans and animals.
  • the genes identified include naturally mutated genes (e.g. in the leptin or leptin receptor gene) and genetically modified genes in transgenic models (e.g. adipocyte-specific gene expression of hydroxysteroid 11-beta dehydrogenase 1) or in knockout models (e.g. the Insulin receptor, overview by Butler and Cone; Trends Genet. 17 (2001) 50-54) with altered growth and fat deposits.
  • the mutated gene usually leads to the complete failure of the function of the encoded protein.
  • each base can be mutated, for example using ENU (ethylnitrosourea; ethylnitrosourea), whereby less drastic changes in the function of genes can also be generated (Hrabe de Angelis et al., Nat. Genet. 25 (2000) 444-447; Nolan et al ., Nat. Genet. 25 (2000) 440-443).
  • ENU ethylnitrosourea
  • mutants are only recognized on a screen if the difference to the baseline is large enough. The identification of such mutations requires repeated backcrosses to show that the observed effect is due to a gene before different methods are combined to find the mutated gene.
  • a polygenic animal model (mouse model) was used, which - first generally expressed - is characterized in that amplifies in animals in specific tissues (fat, hypothalamus), a set of genes', and a set of genes is reduced is expressed, so that the proteins encoded by these genes are produced in an increased or decreased proportion in different tissues compared to average animals.
  • tissue model a polygenic animal model
  • polymorphisms polymorphisms
  • mice lines (referred to as DU6 and DU6i) used in the context of the present invention are distinguished by an above-average body weight and fat accumulation
  • the differentially expressed genes identified in the context of the present invention represent genes which control body weight and obesity at the end of the juvenile development phase.
  • the animals were fed ad libitum with a breeding diet that had 12.5 MJ / kg metabolic energy and an average content of 22.5% raw protein, 5% raw fat, 4.5% raw fiber, 6.5 % Raw ash, 13.5% water, 48% N-free extract, vitamins, trace elements, amino acids and minerals (Altromin diet 1314, Germany).
  • the genes code for proteins that play a role in metabolism, signal transduction, cell division, the cytoskeleton and immune responses.
  • the following 77 genes were significantly increased (+) or reduced (-) transcribed in the obese animals examined compared to control animals in epididymal fat tissue:
  • Table 1A Different expressed genes in the epididymal adipose tissue of male animals of the lines DU6 and DU6i compared to DUKs and DBA / 2 at the age of 42 days under standard housing and feeding conditions.
  • Table 2A below contains 10 differentially expressed genes from Table 1A above.
  • Table 2B contains 4 differentially expressed genes from Table IB above. These 10 genes are located in chromosome areas, for which an influence on body mass and fat accumulation has been proven in genetic coupling studies. Such areas of chromosomes are also referred to as quantitative trait locus or 'quantitative trait locus' (QTL).
  • QTL quantitative trait locus
  • the coupling analyzes for mapping the QTL were carried out in cross-sectional populations between the selected lines DU6 x DUKs (Brockmann, GA; Haley, CS; Renne, U .; Knott, SA; Schwerin, M.
  • Table 2B List of the 4 differently expressed genes in QTL regions for growth and fat accumulation of the polygenic mouse model (hypothalamus):
  • This gene has polymorphisms in the promoter (see Table 3B) Increased transcript levels of Prkca, the gene coding for protein kinase C alpha, were found in the selected animals. The gene position corresponds to a QTL for increased body weight and fat accumulation in the selected mouse lines.
  • the protein is involved in G-protein and Wnt signaling pathways that regulate cell growth and differentiation.
  • PRKCA is an intracellular signaling protein that catalyzes the phosphorylation of substrate proteins and is ubiquitous. When activated, the protein changes its position from the cytosol into cellular membranes to develop its physiological activity.
  • the PRKCA Cl domain is a diacylglycerol / phorbol ester binding region. Complete enzyme activity requires diacylglycerol, phospholipid and calcium. Diacylglycerol is produced as an intermediate in the biosynthesis of fatty acids and is likely to be present in an increased number in the selected mouse lines.
  • Hlf2 Hl histone family, member 2
  • Histones are basic core proteins that are responsible for the nucleosome structure of the chromosome fibers in eukaryotes.
  • the linker histone Hl interacts with linker DNA between nucleosomes and causes the compression of chromatin into higher ordered structures.
  • the gene ⁇ fc2 which is increased expressed in the selected animals, encodes adenylate kinase 2, which is a mitochondrial enzyme that catalyzes the phosphorus transfer between ATP and AMP. As such, it is involved in the regulation of energy metabolism in tissues. So far, knockout models for adenylate kinase have not been associated with reduced body weight.
  • the cDNA frequency of the gene Cox8 is reduced in the selected animals.
  • This gene which codes for the cytochrome c oxidase subunit VIII (COX subunit VIII), is another gene which is involved in the maintenance of energy homeostasis. Cytochrome c is the terminal enzyme of the respiratory chain that causes electron transfer from cytochrome c to molecular oxygen.
  • the COX subunit VIII is a core-encoded polypeptide.
  • the function of the core-encoded subunits is not yet known, although it has been suggested that they could be involved in modulating the catalytic function. Changes in the activity of Cox8 can affect the efficiency of energy distribution.
  • KLKBP kallikrein binding protein, also known as serpin
  • KLKBP serine (or cysteine) proteinase inhibitor. It binds to kallikrein and inhibits its action. It has recently been shown in rat models that KLKBP is a potential pathogenic factor in diabetic retinopathy and can play a role in animal development and growth. In the dwarf rat, the Klkbp gene is induced by growth hormone (Hatcher et al., FASEB J 13 (1999) 1839-1844). KLKBP also has a possible function in vascular biology. Transgenic mice that overexpress kallikrein are chronically hypotensive.
  • the Hfe and Igh-Ia genes which encode the hemochromatose protein and heavy chain Ia of immunoglobulin, are involved in the immune response. Both genes are up-regulated in the mouse lines selected for high body weight.
  • the mouse hemochromatosis protein regulates the duodenal uptake of transferin-bound iron from plasma.
  • hemochromatosis a disease that results from a mutation in the Hfe gene, iron ingested from food causes increased iron accumulation in the liver, heart and pancreas. Insulin resistance associated with excessive accumulation of iron in the liver has been observed with diabetes mellitus II (Roblin et al., Diabetes Metab 28 (2002) 335-339). So far, a direct effect of the Hfe gene on obesity has not been demonstrated. Hfe knockout mice were not examined for changes in body weight and composition.
  • the hemochromatose protein is an integral membrane protein with domains for immunoglobulin and class I histocompatibility antigens alpha 1 and alpha 2.
  • the gene Pctpl which codes for the factor similar to phosphatidylcholine transfer protein, is significantly downregulated compared to control lines.
  • Phosphatidylcholines comprise the most general class of phospholipids in eukaryotic cells. These insoluble molecules are separated by a highly specific membrane Phosphatidylcholine transfer protein (PC-TP), which belongs to the superfamily of steroidogenic acute regulatory protein-related transfer (START) domain, transfers (Roderick et al. Nat. Struct. Biol. 9 (2002) 507-511). The same protein was also identified as a serologically defined colon cancer antigen 28, suggesting its possible role in growth regulation.
  • PC-TP membrane Phosphatidylcholine transfer protein
  • minichromosome maintenance proteins a family of six conserved polypeptides that occur in the nucleus of all eukaryotes, are essential for the initiation of DNA replication.
  • the mRNA transcript amounts of Mcmdl are significantly reduced in the selected animals compared to non-selected control animals.
  • Mcmdl minichromosome maintenance deficient 7; minichromosome maintenance proteins
  • the MCM7 protein is a ' direct target of the MYCN transcription factor. Therefore, the amount of Mcmdl transcripts can be a direct measure of cell division activity. From these observations it is concluded that the cell division rate in epididymal adipose tissue in lean control animals is much higher than in comparison to selected, fat animals.
  • Serine proteases such as Serp ⁇ na3n, take on important functions, including in digestion, blood clotting or the
  • Inhibitors irreversibly inhibit these activities Imitation of the three-dimensional structure of the normal substrate of the protease [http://users.rcn.com/ j kimball .ma.ultranet / BiologyPages / S / Serine_Proteases .html]. Serpins may also play a role in development [Inglis, JD et al. Isolation of two cDNAs encoding novel alpha 1-antichymotrypsin-like proteins in a murine chondrocytic cell line, 1991, Gene 106 (2), 213-220].
  • Table 4 Allocation of the gene ID numbers from tables 3 A and 3B to the gene names
  • Table 6 Allocation of the gene ID numbers from table 5 to the gene names
  • the 10 genes that both express differently in adipose tissue and are located in those regions of the chromosome (see Table 2A) that have shown an influence on body mass and fat accumulation are genes that are highly likely to be the genetic causes of the increased There are fat deposits in the selection line.
  • the other genes from the list of the 77 genes (adipose tissue, see Table IA) and the 23 genes (hypothalamus, see Table IB) are rather secondarily regulated genes that show increased or reduced gene activity due to the genetically fixed differences. They are involved in all probability 'in the metabolism, the regulation of energy balance and other control circuits.
  • Table IA anterior-derived genes
  • Table IB hypothalamus
  • the present invention therefore relates to a method for identifying active substances for influencing body mass and fat deposits, in which a substance to be investigated is administered to non-human mammals and its influence on the expression of the following genes is determined:
  • the substance is an active substance influencing the body mass and the fat accumulation if the expression of one or more of the genes is changed by administration of the substance.
  • the aforementioned method is also referred to as a 'screening method'.
  • the invention further relates to a method for producing a pharmaceutical composition for influencing the body mass and the amount of fat, in which one of the aforementioned Carries out a method for identifying active substances for influencing the body mass and the fat accumulation and formulating the identified active substance into a pharmaceutical composition suitable for administration.
  • the active ingredient is suitable for influencing the body mass and the fat deposits to limit (reduce) the fat deposits.
  • a substance to be examined is administered to non-human mammals and its influence on the expression of the aforementioned genes is determined, the substance being a (reducing) active substance which limits the body mass and the amount of fat if the substance is administered by administration of the substance Expression of one or more of the genes labeled '(-)' in Tables 1A and IB is increased and / or the expression of one or more genes labeled '(+)' in Tables 1A and IB is decreased.
  • the aforementioned method is also referred to as the 'screening method'.
  • the invention thus further relates to a method for producing a pharmaceutical composition for influencing the body mass and the fat accumulation, in which one carries out a aforementioned method for the identification of active substances for lowering (reducing) the body mass and the fat accumulation, and the identified active substance becomes one for administration formulated suitable pharmaceutical composition
  • the invention also makes it possible to identify active substances which are suitable for increasing the fat deposits by administering a substance to be examined to non-human mammals and determining their influence on the expression of the aforementioned genes, the substance being is an active substance which increases the body mass and the fat accumulation, if the expression of one or more of the substances in Table 1A and IB is increased at '(+)' identified genes and / or the expression of a plurality of or in Tables 1A and IB '(-)' Gene marked decreased "is
  • the above procedure is also referred to as 'screening method'. ,
  • the invention thus furthermore relates to a method for producing a pharmaceutical composition for influencing the body mass and the fat accumulation, in which one carries out a aforementioned method for identifying active substances for increasing the body mass and the fat accumulation and the identified active substance is converted into a pharmaceutical composition suitable for administration formulated.
  • the genes marked with '(-)' are preferably the genes marked with '(-)' in Tables 2A and 2B, i.e. Cox8, Pctpl, Mcdm7, Serpina3n and / or the sequence 4930506L07Rik.
  • the genes marked with '(+)' are preferably the genes marked with '(+)' in Tables 2A and 2B, i.e. Prkca, Hlf2, Ak2, Klkbp, Hfe, Igh-Ia, NA, Ly ⁇ a and / or the sequence 2810037C14Rik.
  • Particularly preferred are those of the aforementioned genes for which polymorphisms in the promoter region or in the 3 'flanking region have been detected in the animals selected for high body mass and high fat content.
  • the non-human mammals * are preferably rodents, in particular mice.
  • the invention also includes the use of non-human mammals, particularly rodents or mice, for use in the aforementioned screening process.
  • the present invention thus makes it possible to use the active ingredients identified by the aforementioned screening methods for the production of pharmaceutical compositions
  • Influencing body mass and fat deposits for Reduce body mass and fat deposits or to increase body mass and fat deposits.
  • the invention further relates to pharmaceutical compositions which contain one or more active substances which influence, increase or decrease the expression of the genes mentioned above in the aforementioned manner.
  • the active compounds can be used either individually or in combination with one another and, if appropriate, can be formulated together with pharmaceutically acceptable auxiliaries and / or carriers.
  • active substances which influence, or decrease the expression of the above-mentioned genes in the aforementioned manner are used to influence the body mass and the fat deposits, to lower the body mass and the fat deposits or. Increased body mass and fat deposits provided.
  • SEQ ID NO. corresponds to the number specified under code number 400 in the sequence listing according to WIPO Standard ST.25.
  • Figure 1 Sequence comparisons between DU6, DU ⁇ i, DBA / 2, DUKs and ENSEMBL for the genes listed in Tables 3A and 3B.
  • Figure 2 Sequence comparisons between DU6, DU ⁇ i, DBA / 2, DUKs and ENSEMBL for the genes listed in Table 5.
  • FIG. 3 A: Chromosome map of the mouse with the QTL positions identified in the crossing DU ⁇ i x DBA / 2 and the genes differentially expressed in adipose tissue; on the right side of each chromosome, the QTL regions are shown as bars in different shades of gray.
  • QTLs are abbreviated as follows: BW * for body weight, Lepq * for leptin, Igflq * for insulin-like growth factor 1, Igfbp * for insulin-like growth factor binding protein, Afw * for abdominal fat weight, Mwq * for muscle weight.
  • B Chromosome map of the mouse with the QTL positions identified in the crossing DU ⁇ i x DBA / 2 and the genes differentially expressed in the hypothalamus; on the right side of each chromosome, the QTL regions are shown as bars in different shades of gray.
  • QTLs are abbreviated as follows: BW * for body weight, Lepq * for leptin, Igflq * for insulin-like growth factor 1, Igfbp * for insulin-like growth factor binding protein, Afw * for abdominal fat weight, Mwq * for muscle weight.
  • the loci of C85523 (no symbol) and L20450 (Zfp97) are not known.
  • DBA stands for DBA / 2
  • FIG. 5 Differences in body weight on day 42 between CSSs and DBA / 2 mice in different backcross generations (BC). Asterisks indicate different levels of significant differences between the CSS and DBA / 2 animals: * P ⁇ 0.05, ** P> 0.01, *** P ⁇ 0.001. In generations BC4 and BC5 not all CSS were generated and therefore no data is available for these lines.
  • FIG. 6 Exemplary profile of IGFBP in serum samples from selected F 2 progeny; The example shows the large differences in IGFBP concentrations between F 2 animals. The bands were visualized by phospho-imaging and quantified using the ImageQuaNT software as described in the material and method section (example section). Serum levels of distinct IGFBPs were measured semi-quantitatively (Box 1: IGFBP-3; Box 2: IGFBP-2; Box 33: IGFBP-4; Box 4: IGF binding affinity between 28 and 30 kDa; S: Standard serum, that was included in each run to normalize the different blots).
  • Box 1 IGFBP-3
  • Box 2 IGFBP-2
  • Box 33 IGFBP-4
  • Box 4 IGF binding affinity between 28 and 30 kDa
  • S Standard serum, that was included in each run to normalize the different blots.
  • FBN The basis of all mouse lines of the Institute for the Biology of Agricultural Livestock (FBN) is the heterogeneous breeding line Fzt: DU, which at the beginning of the 1970s through systematic crossing of 4 breeding lines (NMRI orig., Han: NMRI, CFW, CF1) and 4 inbred lines (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) was bred and is kept in an effective population size of 200 animal pairs.
  • NMRI orig., Han: NMRI, CFW, CF1 4 breeding lines
  • CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln 4 inbred lines
  • mice lines from the FBN were used for the studies on mapping genetic factors for growth and fat accumulation:
  • the animals used for the analysis were phenotypically characterized in terms of body weight, abdominal fat weight and the weight of the liver, kidneys and spleen.
  • FIG. 3 shows the chromosome map of the mouse with the QTL positions identified in the crossing DU ⁇ i x DBA / 2.
  • Body mass (g) 1 Bw5 36 (25; 51) 1.30 0.31 -0.94 0.49 5.36 2 Bw6 56 1.25 0.34 0.54 0.50 5.1 4 Bw7 59 (34; 72) 0.88 0.33 -0.85 0.48 2.81 5 Bw8 42 0.73 0.32 0.98 0.51 3.0 5 Bwl3 81 (73; 89) 1.45 0.30 0.25 0.42 5.96 7 Bwl4 28 (23; 33) 2.34 0.33 -0.94 0.51 12.31 11 Bw4 55 (36; 65) 1.32 0.31 -0.28 0.45 4.91 11 Bwl6 14 (6; 17) 1.06 0.31 - 0.81 0.43 3.92 12 Bw9 17 (0; 50) 0.77 0.29 -0.35 0.43 2.05 13 BwlO 47 (33; 61) 1.42 0.40 -0.48 0.63 3.35 13 Bwl5 10 (3; 16) -1.23 0.39 -0.90 0.50 3.84 15 Bwll 6 1.11 0.37 -0.59 0.58
  • Muscle mass (mg) 1 Mwq 35 (11; 97) 13 4 -9 7 3.18 7 Mwql 29 (20; 34) 27 4 -11 7 9.55 11 Mwq2 10 (0; 18) 11 4 -14 6 3.13 11 Mwq3 59 ( 50; 65) 17 4 -13 6 5.86 12 Mwq4 20 (14; 26) 19 4 -9 6 6.23 13 Mwq 10 (0; 21) -140 5 -6 7 2.32 13 Mwq5 43 (33; 52) 26 6 - 9 8 5 a Most likely location is given as the distance from the centromere. Where the confidence intervals have been estimated, they are in brackets after the estimated one
  • the QTL effects are given as a reduction in the sum of the matching squares in the model with 1 QTL compared to the model with 0 QTL.
  • Insulin (pmol / 1) 4 Insql 61 (49; 72) 52.76 14.1 16.4 20.9 4.19 7 I sq 32 (23; 56) 42.4 13.8 -42.2 20.8 3.64 15 Insq 16 (0; 38) 31.3 14.3 -43.6 24.1 2.32
  • IGF-I (ng / ml) 10 Igßql 38 (32; 45) 0.056 0.007 -0.011 0.010 14.98 18 Igßq2 39 (26; 54) 0.034 0.008 0.002 0.013 5.34 2 igßq 88 (57; 100) 0.013 0.009 0.041 0.016 2.58 5 igßq 18 (0; 39) 0.020 0.008 -0.018 0.012 2.47 15 Igflq 56 (47; 65) 0.019 0.007 -0.008 0.009 2.48
  • IGF-binding protein-3 (RDV C ) 5 I p3ql 58 (48; 68) 0.106 0.029 0.123 0.047 5.58 10 I ⁇ p3q2 46 (37; 51) 0.272 0.033 -0.048 0.049 16.73 a Most likely location is given as the distance from the centromere. Where the confidence intervals have been estimated, they are in brackets after the estimated one
  • Offspring were weaned on the 21st day.
  • the animals were fed ad libitum with a breeding diet that had 12.5 MJ / kg metabolic energy and an average content of 22.5% raw protein, 5% raw fat, 4.5% raw fiber, 6.5 % Raw ash, 13.5% water, 48% N-free extract, vitamins, trace elements, amino acids and minerals (Altro in-Diet 1314, Germany).
  • the body weight on day 42 in the selected line DU ⁇ (58.3 ⁇ 6.1g) was more than twice as high as that of the arbitrarily paired control DUKs (28.7 + 2.3g) (BÜNGER, L., U RENNE, and G.
  • the QTL analysis was performed using pedigrees of the F 2 crossing design. These were created by crossing four males of the high growth line DU ⁇ with four females of the control line DUKs. By repeated pairing of the parents and subsequent pairing of the Fi descendants of the same parents, large family trees with a total of 54 Fi descendants and 715 F 2 descendants were created. The pairing was first performed at 10 weeks of age and repeated after 6 weeks; pairing was performed in all family trees at the same time. The structure of the individual family trees is summarized in Table 1. The largest family tree (identity number 8) with 341 F 2 progeny was selected for the genome study. The quantitative measurements taken during the QTL analysis concerned body weight, abdominal fat and the weight of the liver, kidney and spleen of all F 2 individuals.
  • the measured abdominal fat was the gonadal fat in the males and the fat of the perimetrium in females.
  • the ratio of abdominal fat to body weight was defined as the percentage of abdominal fat.
  • the phenotypic data were always recorded at 42 days of age; this is the time for the selection decision in all generations. This age corresponds to the end of the juvenile phase of ontogeny. Marker analysis:
  • the distribution of marker alleles between the lines for 84 loci was examined.
  • the markers were selected so that they were distributed over all chromosomes, the distance between the markers averaging 16.6 cM and the largest interval being 42 cM.
  • the markers were selected for their high variability between suitable inbred lines from the mouse genome database (MGD). MapPair primers of the mouse were obtained from Research Genetics (Huntsville, AL, USA). The Dl IBhm * primers were a gift from Thomas Boehm (Nehls et al. 1995 YAC / Pl contigs defining the location of 56 microsatellite markers and several genes across a 3.4 cM interval on mouse Chromosome 11. Mamm. Genome &: 321-331 ). D9Fbnl was developed by Rainer Theory Book in our institute.
  • DNA from mouse tail tissue samples was extracted using the QIAmp tissue kit (Qiagen, Germany) by selectively binding DNA to an ion exchange matrix after digestion Proteinase K extracted.
  • the DNA was amplified with Taq polymerase (Appligene, France), using a modified standard PCR protocol (Dietrich et al. 1992 A genetic map of the mouse suitable for typing intraspecific crosses. Genetics 131: 423-447): 20 ng of template DNA was used in a total reaction volume of 10 ⁇ l.
  • the DNA was at 25 ° C for 15 seconds at 94 ° C, 1 minute at 55 ° C and 1 minute 70 ° C amplified.
  • the DNA was automatically distributed for the PCR using the Biomek 2000 system (Beckman).
  • the amplifications were carried out in 96-well microtiter plates in a PCR system 9600 (Perkin Elmer).
  • the PCR products were dried, redissolved in 3 ⁇ l formamide loading buffer and separated on 40 cm long, 6% polyacrylamide gels under denaturing conditions. The gels were stained with silver nitrate (Riesner et al.
  • the parental marker alleles were loaded on separate lanes on each gel and served as the standard for the genotyping of the Fi and F 2 progeny. All genotyping results were evaluated twice by independent individuals and runs with discrepancies were repeated.
  • the data were obtained by multiple regression, as developed for the analysis of crossings between outbred lines (Haley et al. 1994 Mapping quantitative trait loci in crosses between outbred lines using least Squares. Genetics 136: 1195-207).
  • the standard interval mapping model with a single QTL was applied to the coupling group.
  • the estimates obtained were (as described by Zeng (1993 Theoretical basis for Separation of multiple linked gene effects in mapping quantitative trait loci. Proc. Natl. Sei. USA 90: 10972-10976) and Jansen (1993 Interval mapping of multiple quantitative trait loci. Genetics 135: 205-211) proposed) revised by adapting the genetic background effects to the coupling groups.
  • the genetic background effects were gradually included as co-factors, starting with the locus with the greatest estimated effect, followed by the locus with the second greatest effect, until no further QTLs could be detected at the specified level of significance.
  • a background effect was always excluded from the analysis when its own position was analyzed.
  • the Sexchromosom was divided a pseudo-autosomal region and an X specific area '.
  • the body weight as a co-variant in the analysis of the abdominal fat-weight and in the analysis was the percentage abdominal fat-fraction included 'in order to investigate the relationship between the two properties.
  • the indicated level is equivalent to the level at which a false positive result is expected in a genome examination. In a genome of 20 independent pairs of chromosomes, this is approximately equivalent to the use of the chromosomal 5% significance level, so that on average one of these chromosomes is significant in a study of the entire genome at this level.
  • the chromosome-wide levels vary between the chromosomes depending on their length and the markers they contain.
  • mice line DU6i which was generated by inbreeding over four generations from the line DU ⁇ selected for high body weight.
  • the commercially available inbred mouse line DBA / 2 (Harlan Netherlands, Horst) was used as a contrast line.
  • the breeding line DU ⁇ was selected over 78 generations for high body weight at the age of 42 days (Bünger et al. 1990, loc. Cit.).
  • the line DU ⁇ comes from original crosses of four base populations (NMRI orig., Han: NMRI CFW, CFl) and four inbred populations (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) of the Research Institute for Biology , agricultural livestock, Dummerstorf, Germany (student 1985, loc. cit.).
  • the animals were fed ad libitum with a breeding diet that had 12.5 MJ / kg metabolic energy and an average content of 22.5% raw protein, 5% raw fat, 4.5% raw fiber, 6.5% raw Ash, 13.5% water, 48% N-free extract, vitamins, trace elements, amino acids and minerals (Altromin-Diet 1314, Germany) included.
  • Family tree design 17 males of the DU ⁇ i
  • the QTL analysis was performed using a pedigree of the F 2 crossing design. These were established by crossing a female of the high growth inbred line DU ⁇ i with a male of the contrast line DBA / 2. Repeated mating of the parents and subsequent mating within the subfamilies of 12 pairs of the Fi progeny created a large family tree with a total of 411 F2 progeny. The pairing was first performed at 10 weeks of age and repeated after 6 weeks.
  • the phenotypic data were recorded at 42 days of age; this is the age of selection in all generations.
  • the quantitative measurements taken during the QTL analysis concerned the weight of body (BW), abdominal fat (AFW), muscle (MW), liver (LW), kidney (KW) and spleen (SW) as well as the serum concentration of Leptin, insulin and insulin-like growth factor 1 (IGF-I).
  • F 2 animals that had not fasted were killed between 9 a.m. and 12 noon. The animals were decaptured in order to obtain the largest possible volume of blood serum for the analysis of the physiological parameters.
  • the measured abdominal fat was the testicular fat in the males and the fat of the perimetrium in the females.
  • the ratio of abdominal fat to body weight was defined as the percentage of abdominal fat (AFP).
  • the weight of the quadriceps which includes the rectus femulus muscle, the vastus intermedicus muscle, the vastus lateralis muscle, and the vastus medialis muscle, was recorded in a representative manner
  • IGF-I insulin-like growth factor I
  • Serum IGFBPs from 208 males and 161 females were determined by Western ligand blot analysis (Hossenlopp, P., Seurin, D., Segovia-Quinson, B., Hardouin, S., and Binoux, M. (1986) Analysis of serum insulin-like growth factor binding proteins using western blotting: use of the method for titration of the binding proteins and competitive binding studies. Anal. Biochem. 154, 138-143).
  • Serum samples were 1: 5 with sample buffer [50 mM Na 2 HP0, pH 7.0; 1% (w / v) sodium dodecyl sulfate (SDS); 50% (w / v) glycerin], boiled for 5 minutes and electrophoretically separated on an SDS-polyacrylamide gel (5% collecting gel / 12% separating gel) using the Mini Protean II TM system (Biorad, Kunststoff, Germany). The separated proteins were transferred to a nitrocellulose membrane (Millipore, Eschborn, Germany). The blots were blocked with 1% fish gelatin and incubated with [ 125 I] IGF-II (10 6 cpm per blot).
  • Binding proteins were visualized on a phosphor imager Storm (Molecular Dynamics, Krefeld, Germany) and quantified using the ImageQuaNT software (Molecular Dynamics). The signal intensities were corrected for the background and based on identical standards different blots normalized ( Figure 6).
  • the serum levels of the IGFBPs are given as relative densitometric values (RDV) compared to the standard.
  • the sum of all IGFBPs was quantified and defined as total IGF binding capacity.
  • IGFBP-2 is regulated differently in mice selected for high body weight and inhibits the body growth of transgenic mice (Hoeflich, A., Wu, M., Mohan, S., Föll, J., Wanke, R., Froehlich, T., Arnold, GJ, Lahm, H., Kolb, HJ, and Wolf, E. (1999) Overexpression of insulin-like growth factor- binding protein-2 in transgenic mice reduces postnatal body weight gain. Endocrinology 140, 5488-5496), the amount of IGFBP-2 was calculated relative to the total IGF binding capacity in the serum (relIGFBP-2), taking into account the variation in the total serum IGFBP levels between different mouse strains.
  • IGF-1 insulin-like growth factor I (IGF-I) was determined by means of an ELISA in the serum after acid-ethanol extraction as previously described (Kratzsch, J., Blum, WF, Schenker, E., Keller, E., ceremoniesis, G., Haustein, B., Ventz , M., and Rotzsch, W. 1993. Measurement of insulin-like growth factor I (IGF-I) in normal adults, patients with liver cirrhosis and acromegaly: experience with a new enzyme immunoassay. Exp. Clin. Endocrinol. 101: 144-149).
  • the markers were selected for their high variability between suitable inbred lines from the mouse genome database (MGD, MOUSE GENOME DATABASE, Mouse Genome Informatics, The Jackson Laboratory, Bar Harbor, Main. World Wide Web (URL: http: // www .informatics.jax.org /) June, 1998).
  • MapPair primers of the mouse were obtained from Research Genetics (Huntsville, AL, USA). Over 630 microsatellite markers were tested for informative parent alleles before the parents and F 2 were genotyped for 93 loci covering all chromosomes with an average distance of 14.1 cM. The marker map is shown in Table 10.
  • mice DU ⁇ and their inbred derivative DU6i were used as contrast lines for the present experiments.
  • the internal control line of the selection experiment DUKs and the commercially available inbred strain DBA / 2 were used as contrast lines for the present experiments.
  • the same mouse lines were used for QTL mapping. used in crossed populations (Brockmann et al. 1998 loc. cit., Brockmann et al. 2000 loc. cit., Brockmann et al. 2001 loc. cit.).
  • the selection lines DU ⁇ , DU6i and the control line DUKs originate from original crosses of four base populations (NMRI orig., Han: NMRI, CFW, CFl) and 4 inbred populations (CBA / Bln, AB / Bln, C57BL / Bln, XVII / Bln) of the Research Institute for the Biology of Farm Animals, Dummerstorf, Germany (student 1985 loc. Cit.).
  • the breeding line was DU ⁇ selected for over 100 generations for high body weight at the age of -42 days (Bünger et al. 1990 loc. cit.). The age of 42 days was the age of selection in all generations. This age corresponds to the end of the juvenile phase of ontogeny. After 42 days, both the animals have the. Selection lines as well as those of the unselected contrast lines completed the period of rapid growth and are post-adolescent.
  • the animals were fed ad libitum with a breeding diet containing 12.5 MJ / kg metabolic energy and an average content of 22.5% raw protein, 5.0% raw fat, 4.5% raw fiber, 6 , 5% raw ash, 13.5% water, 48% N-free extract, vitamins, trace elements, amino acids and minerals (diet 1314; Altromin, Germany).
  • Epididymal adipose tissue was collected from males who had not fasted between 9:00 a.m. and 12:00 p.m. The animals were 42 days old. After decapitation, the epididymal fat was immediately collected and weighed. The same amounts of tissue from 20 animals were snap frozen in each mouse line in liquid nitrogen and stored at -80 ° C. until the RNA was prepared.
  • RNA samples were prepared for each of the four mouse strains DU ⁇ , DU ⁇ i, DUKs and DBA / 2.
  • RNA isolation equal amounts of frozen tissue samples from 20 individuals were pooled and homogenized under liquid nitrogen. The union was used to reduce the individual variability that is high in rearing populations.
  • Total RNA was obtained from pooled tissue samples according to the Chomczynski and Sacchi acid-guanidinium-thiocyanate-phenol-chloroform extraction protocol (Chomczynski and Sacchi 1987 Single- step method of RNA isolation by acid guanidinum thiocyanate-phenol-chloroform extraction. Analytical biochem. 162: 156-159), which was modified by CLONTECH (CLONTECH Laboratories GmbH, Heidelberg, Germany).
  • the supernatant was transferred to a fresh reaction vessel and mixed with twice the volume of saturated phenol (100 g phenol, 15% v / v glycerol, 100 mM sodium acetate (pH 4.0), 18.6% v / v deionized water). After vortexing for 1 minute and incubating on ice for 5 minutes, 1/5 volume of chloroform was added to the sample, the sample was shaken and placed on ice for a further 5 minutes. The sample was then centrifuged to separate the phases. The upper, aqueous phase was transferred to a fresh reaction vessel. The phenol-chloroform extraction was repeated twice. Finally, the RNA was precipitated by adding a volume of isopropanol to the aqueous phase.
  • RNA had A260 / A280 rates of 1.8 or higher.
  • the Affymetrix GeneChips MullK A and B were used as oligonucleotide microarrays for the analysis of 13,069 probe groups which represented over 11,000 mouse-specific genes and ESTs (expressed sequence tags) (Affymetrix, Santa Clara, CA, USA).
  • ESTs expressed sequence tags
  • the samples were prepared in accordance with the recommendations of the Affymetrix user manual.
  • First strand synthesis was performed by a T7 (dT) 24 primer and SuperScript II Reverse Transcriptase (Gibco BRL Life Technologies) using a 10 mg total RNA sample.
  • the synthesis of the second strand was carried out according to the SyperScript Choice System (Gibco BRL Life Technologies) using DNA polymerase I from E. coli, ligase from E. coli and RNaseH. Smoothing the fragment ends was carried out using 'T4 polymerase performed.
  • An in vitro transcription reaction was used to incorporate biotin-11-CTP and biotin-16-UTP into the cRNA probe (BioArray HighYield RNA Transcript Labeling Kit, Enzo).
  • the fragmented cDNA was hybridized overnight at 45 ° C to ensure the quality of the probe.
  • "Spikes" were introduced into the hybridization mixture and test-2 arrays (Affymetrix) were hybridized the day before.
  • Washing was performed using the GeneChip Fluidics station (Affymetrix) according to the manufacturer's protocol. Staining was performed using R-phycoerythrin streptavidin (Molecular Probes) followed by an antibody amplification procedure using a biotinylated anti-streptavidin antibody (Vector Laboratories) and goat IgG (Sigma-). The scanning was carried out with a resolution of 3 ⁇ m, an excitation wavelength of 488 nm and an emission wavelength of 570 nm using the GeneArray scanner (Hewlett Packard). data analysis
  • the expression levels were calculated using the programs "GeneChip Expression Analysis Software-System Data Mining Tool (Version 1.2.)” And “Microarray Suite (Version 4.0.1.)” From Affymetrix.
  • the hybridization signals on each GeneChip were scaled using all probe groups to minimize the differences in the overall signal intensities between two arrays, which allows a more reliable detection of biologically relevant changes in the sample.
  • Affymetrix's "decision matrix” was used to evaluate existing, barely existing and nonexistent amounts of a gene in a tested RNA.
  • the normalized gene expression signals were then compared in pairs between GeneChips, either with RNA from a selected mouse line or with The hybridizations of the GeneChips with DU ⁇ and DU6i probes as selected mouse lines and DUKs and DBA / 2 as unselected mouse lines were used as a kind of replica.
  • the crosswise comparisons DU ⁇ versus DUKs and DU ⁇ i versus DUKs on the one hand and DU6 versus DBA / 2 and DU ⁇ i versus DUKs on the other hand were used as replicate comparisons. Genes that were detected as positive hybridization signals on at least one of the GeneChips were used for the gene expression comparison.
  • a group of consensus genes was selected from the four paired comparisons between the two selected (DU ⁇ and DU ⁇ i) and the two unselected mouse lines (DUKs and DBA / 2), which included all genes that were differentially expressed in all four comparisons. Expression differences between the mouse lines are given as an x-fold increase (positive values) or x-fold decrease (negative values) of the gene expression in the selected mouse line in comparison with the expression in the unselected mouse lines.
  • Mapping genes are given as an x-fold increase (positive values) or x-fold decrease (negative values) of the gene expression in the selected mouse line in comparison with the expression in the unselected mouse lines.
  • All genes which included the pool of differentially expressed genes in epididymal adipose tissue between large and fat selected and non-selected mouse lines, were generated using the sequence information of gene-specific oligonucleotides on the Affymetrix GeneChips, as well as the TIGR, LocusLink, Ensembl- and the human-mouse homology database mapped in silico.
  • the mapping position was accepted when a specific single hit was found in the various databases and the results from the human-mouse homology database were consistent with the other results.
  • a radiation hybrid mapping was performed using the Mouse Hamster Radiation Hybrid (RH) panel (ResGen, Huntsville, AL, USA).
  • RH Mouse Hamster Radiation Hybrid
  • the results from the hybridization of the Affymetrix GeneChips with the combined samples of the selected or non-selected mouse lines were used for transcripts of selected differentially expressed, positional candidate genes in individual RNA samples of the epididymal adipose tissue of 5 males aged 42 days per Line controlled. These 5 animals were different from the animals used for the pooled samples for hybridization of the GeneChip.
  • real-time flourimetry was carried out during the PCR using the FastStart DNA Master SYBR Green I Kit in a LightCycler (Röche Diagnostics GmbH, Mannheim, Germany).
  • RNA of each individual was extracted using the RNeasy Kit (QIAGEN, Hilden, Germany) by a method comprising tissue homogenization and selective RNA adsorption on silica gel membranes in the presence of a high salt concentration buffer system.
  • the RNA was quantified spectrophotometrically at 260 nm and additionally quantified by densitometry of RNA on ethidium bromide-stained agarose gels. 1 ⁇ g total RNA was used for the RT-PCR in a total volume of 10 ⁇ l per reaction. RT-PCR was performed in parallel for total RNA of adipose tissue from all 20 animals (5 animals per line).
  • the cDNA synthesis was carried out in the presence of 50 ng oligo-dT (12-18) primers and 10 units M-MLV RT (H) reverse transcriptase (Promega Corp., WI, USA) according to the manufacturer's recommendations.
  • M-MLV RT H reverse transcriptase
  • Gene-specific primers for expression analysis were generated from the sequence regions that corresponded to the sequence positions of the oligonucleotides that represented the gene on the MUllK-GeneChips.
  • the primer sequences for the gene-specific transcript amplification and the PCR conditions are listed in Table 11 (11A: adipose tissue; 11B: hypothalamus.)
  • the individual expression data of the genes tested were normalized using the expression level of the RPS29 gene, which was found to be non-differentially regulated in our mouse model
  • Table 11A Primer for real-time PCR analysis of gene transcript quantities (adipose tissue)
  • J03482 201 89 491 gtgagcaagggcatcctggt (48) 691 ggtcttcttggccgcctttt (49)
  • the DNA sequences of the genes differentially expressed in adipose tissue were determined using the primers shown in Tables 12A (adipose tissue, promoter area) and 12C (adipose tissue, 3'- untranslated area).
  • the DNA sequences of the genes differentially expressed in the hypothalamus were determined using the primers shown in Tables 12B (hypothalamus, promoter area).
  • genomic DNA sequences were first extracted from the Ensembl database. For the investigations of the promoter area, we exported the genomic sequences from 1000 bp upstream to 500 bp downstream of the transcription start point of each gene of interest. For the 3 'untranslated regions of the selected candidate genes, 1000 bp downstream of the last exon were extracted from the Ensembl database. In the extracted genomic DNA sequences, specific primers for the sequencing of the promoter or the 3 'untranslated regions were derived.
  • Forward primer should be approx. 1000 bp. upstream of the transcription start point, reverse primer should be in the 1st exon.
  • Location of the primers in the 3 'untranslated region Forward primer should be in the last exon, reverse primer approx. 1000 bp. lie downstream of the last exon.
  • the forward primers and reverse primers are also referred to below as F and R primers, respectively.
  • Length of the PCR product 1200-1300 bp.
  • Length of the primer approx. 20 nt (min. 20 nt, max. 25 nt)
  • Melting temperature (T m ) of the primer optimal 65 ° C Difference in the annealing temperature between forward and reverse primer: max. 1 ° C
  • an additional primer lying within the first PCR fragment was derived for the subsequent sequencing in order to bridge the gap.
  • Genomic Target DNA was isolated from the mouse tail using the DNeasy Tissue Kit from Qiagen (Hilden, Germany) in accordance with the manufacturer's protocol. The DNA concentration and quality were determined using the photometer from Pharmacia Biotech (Freiburg, Germany). The measurement was carried out at 280 nm. For subsequent PCR, the DNA concentration was adjusted to 20 ng / ⁇ l with AE buffer (elution buffer) from the DNeasy tissue kit from Qiagen. PCR conditions
  • PCRs were carried out with the specific primers derived for the target regions and the isolated genomic DNA from mouse tail in order to amplify the promoter sections or 3′-untranslated regions of interest, so that sufficient material was available for the subsequent sequencing.
  • genomic DNA 1.0 ⁇ l (20 ng / ⁇ l) 10X reaction buffer 1.0 ⁇ l dNTP-Mix 0.8 ⁇ l (lOpmol / ⁇ l) MgCl 2 0.2 ⁇ l (25mmol / ⁇ l) Primer F 0.5 ⁇ l (lOpmol / ⁇ l) Primer R 0.5 ⁇ l (lOpmol / ⁇ l)
  • a goat of the DU ⁇ i line was mated to several females of the control line DBA / 2 for the generation of the CSS. Repeated backcrossings to the inbred line DBA / 2 serially transferred one chromosome of the selected line to the genetic background of the recipient line.
  • the entire genome was covered with 148 informative genetic microsatellite markers.
  • the body weight in the inbred line DU ⁇ i was 3.6 times higher than in DBA / 2 mice.
  • the body weight in the CSS decreased with each back-crossing to the DBA / 2 strain.
  • Body weight differences on day 42 between CSSs and DBA / 2 mice in different backcross generations (BC) are shown in Figure 5 at the time of generation BC4 and BC5. Not all CSS were generated at the same time in these generations, so no data is shown for some lines. After eight generations of back-crossing, significant effects of the DU ⁇ i chromosomes in terms of body weight were observed for chromosomes 1, 3, 6, 7 and 14 at the age of 21 days.
  • Chromosomes 2, 4, 8, 11, 14, 15, 16 and 17 showed effects for weight gain between the 21st and 42nd day. Chromosomes 2, 5, 11 and 17 were crucial for weight gain between days 42 and 63. The greatest effect on body weight was observed on chromosome 5, whereby this Effect was significant at every age. The data confirm the results obtained by coupling analyzes in cross-over populations.

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L'invention concerne des procédés pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse.
PCT/EP2004/012555 2003-11-07 2004-11-05 Procedes pour identifier des principes actifs ayant une influence sur la masse corporelle et l'accumulation de graisse Ceased WO2005045054A2 (fr)

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