WO2004044241A2 - Use of fish larvae as a screening model - Google Patents

Use of fish larvae as a screening model Download PDF

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Publication number
WO2004044241A2
WO2004044241A2 PCT/EP2003/012706 EP0312706W WO2004044241A2 WO 2004044241 A2 WO2004044241 A2 WO 2004044241A2 EP 0312706 W EP0312706 W EP 0312706W WO 2004044241 A2 WO2004044241 A2 WO 2004044241A2
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Prior art keywords
accordance
fish
larva
target gene
larvae
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PCT/EP2003/012706
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French (fr)
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WO2004044241A3 (en
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Matthias Austen
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DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung
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Priority to AU2003288055A priority Critical patent/AU2003288055A1/en
Publication of WO2004044241A2 publication Critical patent/WO2004044241A2/en
Publication of WO2004044241A3 publication Critical patent/WO2004044241A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/4603—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates from fish

Definitions

  • This present invention concerns the use of fish larvae, in particular zebrafish larvae or medaka larvae, as a model organism for investigating metabolism diseases, such as obesity, and associated diseases as well as for the development of corresponding prevention and/or treatment strategies.
  • This present invention particularly concerns a procedure for the identification and/or characterization of genes and/or gene products or active substances which affect energy homeostasis as well as a pharmaceutical composition comprising genes, gene products or active substances identified by using the procedure in accordance with this present invention for the prevention and/or treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • This present invention furthermore concerns a procedure for determining the fat content of fish larvae, in particular of zebrafish larva
  • Obesity is defined as an increase in body fat content as a result of a positive energy balance and can be caused by a number of different factors, such as, for example, genetic, metabolic, biochemical, psychological, and behavioral factors.
  • obesity may occur as the direct result of excessive food intake, as a psychosomatic symptom, in the case of metabolism diseases as well as rare hereditary syndromes.
  • overweight patients additionally often suffer from accompanying effects, such as fat liver cells, fatty liver, and secondary fat metabolism disorders with increased serum lipid protein concentrations.
  • obesity- mediated diseases such as hypertonia, diabetes mellitus, gout, fat distribution disorders, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, some types of cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • the object of this present invention was therefore to provide a novel procedure for the identification and/or characterization of genes, gene products, or active substances which affect energy homeostasis.
  • this is achieved through the use of fish larvae in different stages of development, in particular zebrafish larvae or medaka larvae, as a model organism for the investigation of the homeostatic system for regulating body weight.
  • Fish larvae in particular the zebrafish larvae Danio rerio or the medaka larvae Oryzias latipes, because of their small size, the possibility of in vitro fertilizations, a large number of offspring (50-300 eggs per week), the development of offspring outside of the mother animals, the short development times of the offspring (within the first 24 hours after fertilization, essentially all organs are already being developed; fish larvae hatch after 3 days, and sexual maturity is reached after 3-4 months) and the possibility to observe the development of organs and tissues directly in living, transparent fish larvae, exhibit significant advantages with respect to prior-art vertebrate model organisms, such as C.
  • a first object of this present invention therefore concerns a procedure for the identification and/or characterization of genes or/and gene products which affect energy homeostasis, comprising:
  • Step (a) of the procedure in accordance with this present invention comprises the provision of a fish larva in which the activity of the target gene or/and target gene product which is assumed to affect energy homeostasis is modulated.
  • the activity of a target gene or/and target gene product of the fish larva is modulated on the gene level, transcript level or/and gene product level.
  • Suitable procedures for modulating the activity of a target gene or/and target gene product on the gene level, transcript level or/and gene product level are sufficiently known to those in the art, such as the activation or inhibition of transcription of the target gene, stabilization or destabilization of the mRNA derived from the target gene, activation or inhibition of translation of the mRNA derived from the target gene, activation or inhibition of splicing of the mRNA derived from the target gene or/and activation or inhibition of the target gene product.
  • the activity of the target gene is modulated through activation and inhibition of transcription of the target gene by means of regulatory proteins, such as transcription factors, which, in interaction with the RNA polymerase, initiate or intensify the transcription of the target gene, or repressors, which, through reversible binding to signal structures of the target gene, such as, for example, operator sequences, prevent a transcription of the target gene.
  • regulatory proteins such as transcription factors, which, in interaction with the RNA polymerase, initiate or intensify the transcription of the target gene, or repressors, which, through reversible binding to signal structures of the target gene, such as, for example, operator sequences, prevent a transcription of the target gene.
  • the activity of the target gene is modulated through stabilization or destabilization of the mRNA derived from the target gene by means of regulatory proteins or ribozymes.
  • regulatory proteins covers both proteins which, for example through the formation of complexes with the mRNA, lead to a stabilization of the same, as well as proteins which, through internal separation of the mRNA, degradation from the 5' end of the mRNA or 3'-5'-exonucleolytic degradation, lead to a destabilization and/or a reduction of the mRNA.
  • ribozymes represents a technical term that is sufficiently known to those in the art and does not need any detailed explanation herein.
  • the activity of the target gene is modulated through activation or inhibition of translation of the mRNA derived from the target gene by means of regulatory proteins, antisense or/and RNAi molecules.
  • regulatory proteins covers both proteins which initiate or intensify translation of the mRNA, such as proteins which, through regulation of the phosphorylation of the initiations and elongation factors, lead to activation of the translation of the mRNA, as well as proteins which block the translation of the mRNA.
  • antisense molecules represents a technical term that is sufficiently known to those in the art and therefore does not require a detailed definition herein.
  • Preferred antisense molecules are short DNA, RNA or nucleic acid analog fragments (such as, for example, PNAs, LNAs, phosphorothiote oligonucleotides, morpholino oligonucleotides, 2-fluoro-RNAs or mixed compounds thereof) with a nucleic acid sequence of at least 10 nucleotides which are complementary to a partial area of the mRNA derived from the target gene.
  • the antisense molecules have a length of 15 to 30 nucleotides and, more preferably, 20 to 25 nucleotides.
  • RNAi molecules is another technical term that is familiar to those in the art and shall therefore not be explained in detail herein.
  • RNAi molecules are double-string RNA molecules with a length of at least 10 base pairs, preferably at least 18 base pairs, and particularly preferably at least 20 base pairs. Particularly preferably, the RNAi molecules have a length between 18 and 28 base pairs and, more preferably, a length between 20 and 23 base pairs.
  • the RNAi molecules can either be manufactured synthetically or in a vector-based manner in the target cells (Elbashir et al., Nature 411 : 494-498, 2001 ; Sui et al., Proc. Natl. Acad. Sci. USA 99: 5515-5520, 2002). The sequence of the RNAi molecules is selected in such a manner that it corresponds to specific sequence areas of the mRNA derived from the target gene.
  • the activity of the target gene is modulated through activation or inhibition of splicing of the ⁇ mRNA derived from the target gene.
  • Procedures for the activation or inhibition of splicing of mRNA are sufficiently known to those in the art and shall therefore not be explained in more detail herein.
  • the activity of the target gene product is modulated through interaction of the target gene product with an activator, inhibitor and/or antibody.
  • activator comprises proteins, protein fragments, peptides, protein substrates, protein cofactors as well as small organic molecules, which lead to stabilization of the target gene product and/or to an increase in the activity of the target gene product.
  • inhibitor comprises proteins, protein fragments, peptides or small organic molecules, which lead to a destabilization of the target gene product and/or to a decrease or inhibition of the activity of the target gene product.
  • this present invention also covers the reduction or inhibition of the activity of the target gene product by binding a target gene product-specific antibody.
  • antibody is a common technical term and shall therefore not be explained in detail herein.
  • the above regulatory enzymes, repressors, ribozymes, antisense or RNAi molecules, activators, inhibitors and/or antibodies are transferred through transformation into the respect target cells of the fish larva, e.g. cells of a specific organ or tissue.
  • the transformation can be carried out directly or by using expression vectors in accordance with prior-art procedures, e.g. through calcium phosphate coprecipitation, lipofetechnisch, electroporation, particle bombardment, or viral infection.
  • Suitable expression vectors comprise both viral systems, such as retroviral (Lin et al., Science 265: 666- 669, 1994) or adenoviral systems, as well as different bacterial plasmides.
  • Such vectors are sufficiently known to those in the art (Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press).
  • larvae can be transferred through microinjection during the early (preferably 1-8 cell stage) embryonic stage.
  • This also applies to the transfer of transgenes, for example to the expression of RNAi-mediating short RNAs or for the expression of other RNAs (coding or non-coding) (Stuart et al., Development 103: 403-412, 1998; Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th ed., Univ.
  • Step (b) of the procedure in accordance with this present invention involves the measurement of the content of at least one metabolic parameter of the fish larva.
  • Preferred metabolic parameters of the fish larva are metabolic parameters which are related with the homeostatic system for the regulation of body weight, such as body fat, glycogen or/and blood sugar.
  • the body fat content of the fish larva is measured. This is preferably performed by adding a lipophilic coloring agent for the fish larva, and by quantitatively determining the coloring agent that is stored proportionally with the fat content of the fish larva.
  • Examples for possible lipophilic coloring agents which can be used to determine the body fat content of the fish larva include Oil Red O (1-([4- (Xylylazo)-xylyl]azo)-2-naphthol), Sudan Black B (C29H24N6), Sudan IV (C24H20N40), Sudan Red 7B (C24H21 N5): the lipophilic coloring agent Oil Red O is particularly preferred.
  • Oil Red O is particularly preferred.
  • the quantitative determination of the coloring agent stored proportionally with the fat content of the fish larva can be performed in accordance with prior-art procedures known to those in the art, such as, for example, visual or/and photometric determination of the stored coloring agent.
  • the quantitative determination of the stored coloring agent in the case of the lipophilic coloring agent Oil Red O be performed through the extraction of the fat-bound coloring agent in a suitable solvent, such as isopropanol, and photometric determination of the extracted coloring agent occurs at a wavelength of 525 nm.
  • Step (b) of the procedure in accordance with this present invention in addition to or instead of the body fat content of the fish larva, the glycogen content of the fish larva is measured.
  • Suitable procedures for determining the glycogen contents of fish larvae are sufficiently known to those in the art.
  • the blood sugar level of the fish larva is determined. Suitable procedures and measuring devices for determining the blood sugar level of fish larvae are sufficiently known to those in the art.
  • the blood sugar level of the fish larva is determined by using a measuring device which requires a very low blood volume (approx. 1 ⁇ l), for example the device OneTouch Ultra (LifeScan Inc., Milpitas, USA).
  • the blood of the fish larvae is obtained, for example, by opening the tail artery in a physiological buffer solution, followed by light centrifugation.
  • Step (c) of the procedure in accordance with this present invention comprises the determination of the extent of the impact on energy homeostasis of the fish larva.
  • the extent of the impact on energy homeostasis of the fish larva is determined by comparing the content of at least one metabolic parameter of the fish larva wherein the activity of a target gene or/and target gene product which is assumed to affect energy homeostasis is modulated that has been measured in Step (b) of the procedure with that of a reference fish larva and subsequent analysis of the data obtained.
  • the target gene or/and target gene product is a gene or/and gene product which is responsible for the activation and/or upward regulation of the homeostatic system for the regulation of body weight.
  • the target gene or/and target gene product is a gene or/and gene product which is responsible for inhibition and/or downward regulation of the homeostatic system for the regulation of body fat content.
  • the procedure in accordance with this present invention therefore does not only make it possible to identify and/or characterize genes or/and gene products which regulate energy homeostasis in healthy humans, but also genes or/and gene products which are responsible for the onset of diseases concerning the regulation of body weight, such as obesity, and associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • the procedure in accordance with this present invention is therefore conducted by using genetically modified fish larvae, e.g. transgenic fish larvae, or fish larvae that have been modified through mutation or insertion.
  • the fish larva is the zebrafish larva Danio rerio.
  • the zebrafish larva can, in the procedure in accordance with this present invention, used at any stage of development for the identification and/or characterization of genes or/and gene products which affect energy homeostasis.
  • the age of the fish larva is 8 days, particularly preferably 8 to 14 days.
  • the fish larva is the medaka larva Oryzias latipes.
  • a second object of this present invention concerns a procedure for the identification and/or characterization of active substances which affect energy homeostasis, comprising:
  • Step (d) the determination of the extent of the impact on energy homeostasis of the fish larva.
  • Step (a) of the procedure in accordance with this present invention comprises the provision of a fish larva.
  • the fish larva is the zebrafish larva Danio rerio, or the medaka larva Oryzias latipes, already described above.
  • the procedure in accordance with this present invention can also be performed with genetically modified fish larvae.
  • Step (b) of the procedure in accordance with this present invention comprises the administration of a test substance which is assumed to affect energy homeostasis to the fish larva.
  • test substances in particular peptides, aptamers and low-molecular organic molecules are suited.
  • the test substance is chosen from a substance library.
  • Step (c) of the procedure in accordance with this present invention comprises the measurement of the content of at least one metabolic parameter of the fish larva.
  • this is at least one metabolic parameter, as already described above, a metabolic parameter of the homeostatic system for the regulation of body weight, such as body fat, glycogen, and blood sugar.
  • the measurement of the content of at least one metabolic parameter of the fish larva is carried out as already described above.
  • Step (d) of the procedure in accordance with this present invention comprises the determination of the extent of the impact on energy homeostasis of the fish larva and is carried out in a manner that is substantially similar to that already described above.
  • the procedures in accordance with this present invention can be carried out in any conventional standard laboratory in a quick, simple, and inexpensive manner and without requiring major technical systems to identify and/or characterize genes and/or gene products and/or active substances which affect energy homeostasis.
  • zebrafish larvae Danio rerio are used in the procedure in accordance with this present invention, the extent of the impact of the homeostatic system on the regulation of body weight can be observed directly on living fish, e.g. through a visible increase or decrease of the fat content of the studied fish larva compared with a reference fish larva.
  • a third object of this present invention pertains to a pharmaceutical composition, comprising a gene or/and gene product identified by using the above-described procedure in accordance with this present invention or an active ingredient identified by using the above-described procedure in accordance with this present invention and, optionally pharmaceutically compatible carriers, diluents, and auxiliary substances.
  • the pharmaceutical composition in accordance with this present invention may be present in a form that can be administered topically, parenterally, intravenously, intramuscularly, subcutaneously or transdermally and can be manufactured by using well-known, conventional, state-of-the-art procedures.
  • the pharmaceutical composition in accordance with this present invention is produced in the form of a pill or as an intravenous injection.
  • the pharmaceutical composition in accordance with this present invention is used for the prevention or/and treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • diseases in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • the pharmaceutical composition in accordance with this present invention is administered to patients suffering from one of the above diseases in a quantity that is sufficient to alleviate the symptoms associated with this disease, prevent further propagation of the disease, fully cure the disease and/or prevent reoccurrence of the disease.
  • the quantity of the pharmaceutical composition to be administered depends on several factors, such as, for example, selection of the gene, gene product, or active substance to be administered (specificity, efficacy, etc.), type of administration (pill, injection, etc.), type and extent of the disease as well as the age, weight, and general condition of the patient, and can be easily determined by those in the art by considering the above factors.
  • the pharmaceutical composition in accordance with this present invention is administered topically, parenterally, intravenously, intramuscularly, subcutaneously or transdermally.
  • the pharmaceutical composition is administered in the form of a pill or as an intravenous injection.
  • a fourth object of this present invention concerns the use of a gene and/or gene product or active substance identified by using the above-described procedure in accordance with this present invention for the prevention and/or treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • diseases in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
  • a fourth object of this present invention concerns the procedure for determining the fat content of fish larvae used in a preferred embodiment of the above procedure, comprising: (a) the addition of a lipophilic coloring agent for the fish larva, and
  • Step (a) of the procedure in accordance with this present invention comprises the addition of the lipophilic coloring agent to the fish larva. This is preferably carried out by killing off of the fish larvae, incubating the dead fish larvae in a coloring solution comprising the lipophilic coloring agent, and removing the non-fat-bound lipophilic coloring agent.
  • Suitable lipophilic coloring agents which can be used in the procedure in accordance with this present invention comprise the coloring agents already identified above.
  • Step (b) comprises the quantitative determination of the coloring agent stored proportionally with the fat content of the fish larva and is carried out in a manner that is substantially similar to that already described above.
  • the procedure in accordance with this present invention is used to determine the fat content in zebrafish larvae Danio rerio or medaka larvae Oryzias latipes. Considering that the zebrafish larvae are transparent at the beginning of their development, this species of fish therefore makes it particularly easy to determine the fat content.
  • the zebrafish larvae are at least 8 days old, in particular 8 to 14 days.
  • Th e procedure in accordance with this present invention is the first procedure which has been described so far for determining the fat content of fish larvae. Prior-art procedures for the measurement of triglycerides, such as the standard enzyme FS, do not work with fish larvae.
  • Figure 1 shows the coloring of the fatty tissue of zebrafish larvae Danio rerio in different stages of development with Oil Red O.
  • C 9 days, fish larvae were fed starting on the fifth day
  • D 9 days, fish larvae were not fed.
  • the fish larvae were killed through incubation in an ice-cold tricaine solution.
  • a coloring solution was prepared from 60 % isopropanol/Oil Red O and 40 % PBS. This coloring solution was left alone for 10 minutes; afterwards, insoluble color particles were removed via centrifugation over a period of 15 minute. The dead fish larvae were incubated for 20 minutes in the coloring solution, and the coloring solution was removed to the extent that this was possible.
  • the nonfat-bound coloring agent was removed through incubation in 60 % isopropanol-PBS, 40 % isopropanol-PBS, and 20 % isopropanol-PBS for 4 minutes each.
  • the fish larva were washed again 2x for 10 minutes each in PBS-T.
  • the coloring agent bound in the larva was afterwards extracted by using isopropanol over a period of 15 minutes.
  • 500 ⁇ l isopropanol were used for the extraction.
  • the supernatant of the extraction was measured in a photometer at 525 nm.
  • the discolored larvae were once again washed twice by using PBS-T. Afterwards, they were homogenized, for example by using a FastPep device manufactured by the company Savant, and their protein content was measured. A corrected/relative fat value was therefore obtained by dividing the absorption value measured at 525 nm by the protein content.
  • both individual as well as the entire incubations can be performed in container inserts with a mesh bottom, such as Netwell by the company Costar, with a mesh width of 0.75 ⁇ m.

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Abstract

This present invention concerns the use of fish larvae, in particular zebrafish larvae or medaka larvae, as a model organism for investigating metabolism diseases, such as obesity and associated diseases, as well as for the development of corresponding prevention and/or treatment strategies. This present invention concerns, in particular, a procedure for the identification and/or characterization of genes and/or gene products or active substances which affect energy homeostasis as well as a pharmaceutical composition comprising genes, gene products or active substances identified by using the procedure in accordance with this present invention for the prevention and/or treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea. Furthermore, this present invention concerns a procedure for determining the fat content of fish larvae, in particular of zebrafish larvae or medaka larvae.

Description

Use of Fish Larvae as a Screening Model
Description
This present invention concerns the use of fish larvae, in particular zebrafish larvae or medaka larvae, as a model organism for investigating metabolism diseases, such as obesity, and associated diseases as well as for the development of corresponding prevention and/or treatment strategies. This present invention particularly concerns a procedure for the identification and/or characterization of genes and/or gene products or active substances which affect energy homeostasis as well as a pharmaceutical composition comprising genes, gene products or active substances identified by using the procedure in accordance with this present invention for the prevention and/or treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea. This present invention furthermore concerns a procedure for determining the fat content of fish larvae, in particular of zebrafish larvae or medaka larvae.
Today, obesity is one of the most common metabolism diseases worldwide. Although in Western industrialized nations, as much as every third person is considered overweight today, so far, relatively little is known about this disease. Obesity is defined as an increase in body fat content as a result of a positive energy balance and can be caused by a number of different factors, such as, for example, genetic, metabolic, biochemical, psychological, and behavioral factors. For example, obesity may occur as the direct result of excessive food intake, as a psychosomatic symptom, in the case of metabolism diseases as well as rare hereditary syndromes. ln addition to the increase in fatty tissue, overweight patients additionally often suffer from accompanying effects, such as fat liver cells, fatty liver, and secondary fat metabolism disorders with increased serum lipid protein concentrations. In addition, many patients suffer from so-called obesity- mediated diseases, such as hypertonia, diabetes mellitus, gout, fat distribution disorders, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, some types of cancer, such as, for example, cancer of the genital organs, and sleep apnea.
Although in the past, several genes have been described which impact the homeostatic system for regulating body weight, such as leptin or the peroxisome proliferator-activated receptor-gamma coactivator, the actual molecular mechanisms and/or molecules which affect obesity or the regulation of body weight are not known.
The object of this present invention was therefore to provide a novel procedure for the identification and/or characterization of genes, gene products, or active substances which affect energy homeostasis.
In accordance with this present invention, this is achieved through the use of fish larvae in different stages of development, in particular zebrafish larvae or medaka larvae, as a model organism for the investigation of the homeostatic system for regulating body weight. Fish larvae, in particular the zebrafish larvae Danio rerio or the medaka larvae Oryzias latipes, because of their small size, the possibility of in vitro fertilizations, a large number of offspring (50-300 eggs per week), the development of offspring outside of the mother animals, the short development times of the offspring (within the first 24 hours after fertilization, essentially all organs are already being developed; fish larvae hatch after 3 days, and sexual maturity is reached after 3-4 months) and the possibility to observe the development of organs and tissues directly in living, transparent fish larvae,, exhibit significant advantages with respect to prior-art vertebrate model organisms, such as C. elegans, Drosophila melanogaster, and Mus musculus. In particular, within the scope of the studies conducted in respect of this present invention, a novel procedure for determining the fat, glycogen and/or blood sugar content in fish larvae, in particular zebrafish larvae or medaka larvae, was developed which is suitable for use in genetic functional analysis as well as for the analysis of the effect of pharmacologically active substances.
A first object of this present invention therefore concerns a procedure for the identification and/or characterization of genes or/and gene products which affect energy homeostasis, comprising:
(a) the provision of a fish larva wherein the activity of a target gene or/and target gene product which is assumed to affect energy homeostasis is modulated,
(b) the measurement of the content of at least one metabolic parameter of the fish larva, and
(c) the determination of the extent of the impact on energy homeostasis of the fish larva.
Step (a) of the procedure in accordance with this present invention comprises the provision of a fish larva in which the activity of the target gene or/and target gene product which is assumed to affect energy homeostasis is modulated. Preferably, the activity of a target gene or/and target gene product of the fish larva is modulated on the gene level, transcript level or/and gene product level. Suitable procedures for modulating the activity of a target gene or/and target gene product on the gene level, transcript level or/and gene product level are sufficiently known to those in the art, such as the activation or inhibition of transcription of the target gene, stabilization or destabilization of the mRNA derived from the target gene, activation or inhibition of translation of the mRNA derived from the target gene, activation or inhibition of splicing of the mRNA derived from the target gene or/and activation or inhibition of the target gene product. In a preferred embodiment of this present invention, the activity of the target gene is modulated through activation and inhibition of transcription of the target gene by means of regulatory proteins, such as transcription factors, which, in interaction with the RNA polymerase, initiate or intensify the transcription of the target gene, or repressors, which, through reversible binding to signal structures of the target gene, such as, for example, operator sequences, prevent a transcription of the target gene.
In another preferred embodiment of this present invention, the activity of the target gene is modulated through stabilization or destabilization of the mRNA derived from the target gene by means of regulatory proteins or ribozymes. In this context, the term "regulatory proteins" covers both proteins which, for example through the formation of complexes with the mRNA, lead to a stabilization of the same, as well as proteins which, through internal separation of the mRNA, degradation from the 5' end of the mRNA or 3'-5'-exonucleolytic degradation, lead to a destabilization and/or a reduction of the mRNA. The term "ribozymes" represents a technical term that is sufficiently known to those in the art and does not need any detailed explanation herein.
In another preferred embodiment of this present invention, the activity of the target gene is modulated through activation or inhibition of translation of the mRNA derived from the target gene by means of regulatory proteins, antisense or/and RNAi molecules. As used herein, the term "regulatory proteins" covers both proteins which initiate or intensify translation of the mRNA, such as proteins which, through regulation of the phosphorylation of the initiations and elongation factors, lead to activation of the translation of the mRNA, as well as proteins which block the translation of the mRNA. The term "antisense molecules" represents a technical term that is sufficiently known to those in the art and therefore does not require a detailed definition herein. Preferred antisense molecules are short DNA, RNA or nucleic acid analog fragments (such as, for example, PNAs, LNAs, phosphorothiote oligonucleotides, morpholino oligonucleotides, 2-fluoro-RNAs or mixed compounds thereof) with a nucleic acid sequence of at least 10 nucleotides which are complementary to a partial area of the mRNA derived from the target gene. Preferably, the antisense molecules have a length of 15 to 30 nucleotides and, more preferably, 20 to 25 nucleotides. The term "RNAi molecules" is another technical term that is familiar to those in the art and shall therefore not be explained in detail herein. Preferred RNAi molecules are double-string RNA molecules with a length of at least 10 base pairs, preferably at least 18 base pairs, and particularly preferably at least 20 base pairs. Particularly preferably, the RNAi molecules have a length between 18 and 28 base pairs and, more preferably, a length between 20 and 23 base pairs. The RNAi molecules can either be manufactured synthetically or in a vector-based manner in the target cells (Elbashir et al., Nature 411 : 494-498, 2001 ; Sui et al., Proc. Natl. Acad. Sci. USA 99: 5515-5520, 2002). The sequence of the RNAi molecules is selected in such a manner that it corresponds to specific sequence areas of the mRNA derived from the target gene.
In another preferred embodiment of this present invention, the activity of the target gene is modulated through activation or inhibition of splicing of the ■mRNA derived from the target gene. Procedures for the activation or inhibition of splicing of mRNA are sufficiently known to those in the art and shall therefore not be explained in more detail herein.
In another preferred embodiment of this present invention, the activity of the target gene product is modulated through interaction of the target gene product with an activator, inhibitor and/or antibody. The term "activator", as used herein, comprises proteins, protein fragments, peptides, protein substrates, protein cofactors as well as small organic molecules, which lead to stabilization of the target gene product and/or to an increase in the activity of the target gene product. By contrast, the term "inhibitor", as used herein, comprises proteins, protein fragments, peptides or small organic molecules, which lead to a destabilization of the target gene product and/or to a decrease or inhibition of the activity of the target gene product. Furthermore, this present invention also covers the reduction or inhibition of the activity of the target gene product by binding a target gene product-specific antibody. The term "antibody" is a common technical term and shall therefore not be explained in detail herein.
Preferably,the above regulatory enzymes, repressors, ribozymes, antisense or RNAi molecules, activators, inhibitors and/or antibodies are transferred through transformation into the respect target cells of the fish larva, e.g. cells of a specific organ or tissue. The transformation can be carried out directly or by using expression vectors in accordance with prior-art procedures, e.g. through calcium phosphate coprecipitation, lipofektion, electroporation, particle bombardment, or viral infection. Suitable expression vectors comprise both viral systems, such as retroviral (Lin et al., Science 265: 666- 669, 1994) or adenoviral systems, as well as different bacterial plasmides. Such vectors are sufficiently known to those in the art (Maniatis et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press). In addition, larvae can be transferred through microinjection during the early (preferably 1-8 cell stage) embryonic stage. This also applies to the transfer of transgenes, for example to the expression of RNAi-mediating short RNAs or for the expression of other RNAs (coding or non-coding) (Stuart et al., Development 103: 403-412, 1998; Westerfield, M., The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio), 4th ed., Univ. of Oregon Press, Eugene, 2000; Thermes et al., Mech. Dev.: 91 , 2002; Medaka homepage http://biol1.bio.nagoya-u.ac.jp: 8000/; Sui et al., 2002, supra).
Step (b) of the procedure in accordance with this present invention involves the measurement of the content of at least one metabolic parameter of the fish larva. Preferred metabolic parameters of the fish larva are metabolic parameters which are related with the homeostatic system for the regulation of body weight, such as body fat, glycogen or/and blood sugar. In a particularly preferred embodiment of this present invention, in Step (b) of the procedure in accordance with this present invention, the body fat content of the fish larva is measured. This is preferably performed by adding a lipophilic coloring agent for the fish larva, and by quantitatively determining the coloring agent that is stored proportionally with the fat content of the fish larva. Examples for possible lipophilic coloring agents which can be used to determine the body fat content of the fish larva include Oil Red O (1-([4- (Xylylazo)-xylyl]azo)-2-naphthol), Sudan Black B (C29H24N6), Sudan IV (C24H20N40), Sudan Red 7B (C24H21 N5): the lipophilic coloring agent Oil Red O is particularly preferred. The quantitative determination of the coloring agent stored proportionally with the fat content of the fish larva can be performed in accordance with prior-art procedures known to those in the art, such as, for example, visual or/and photometric determination of the stored coloring agent. It is particularly preferred that the quantitative determination of the stored coloring agent in the case of the lipophilic coloring agent Oil Red O be performed through the extraction of the fat-bound coloring agent in a suitable solvent, such as isopropanol, and photometric determination of the extracted coloring agent occurs at a wavelength of 525 nm.
In another preferred embodiment of this present invention, in Step (b) of the procedure in accordance with this present invention, in addition to or instead of the body fat content of the fish larva, the glycogen content of the fish larva is measured. Suitable procedures for determining the glycogen contents of fish larvae are sufficiently known to those in the art.
In another preferred embodiment of this present invention, in Step (b) of the procedure in accordance with this present invention, in addition to or instead of determining the body fat content or/and glycogen contents of the fish larva, the blood sugar level of the fish larva is determined. Suitable procedures and measuring devices for determining the blood sugar level of fish larvae are sufficiently known to those in the art. Preferably, the blood sugar level of the fish larva is determined by using a measuring device which requires a very low blood volume (approx. 1μl), for example the device OneTouch Ultra (LifeScan Inc., Milpitas, USA). The blood of the fish larvae is obtained, for example, by opening the tail artery in a physiological buffer solution, followed by light centrifugation.
Step (c) of the procedure in accordance with this present invention comprises the determination of the extent of the impact on energy homeostasis of the fish larva. Preferably, the extent of the impact on energy homeostasis of the fish larva is determined by comparing the content of at least one metabolic parameter of the fish larva wherein the activity of a target gene or/and target gene product which is assumed to affect energy homeostasis is modulated that has been measured in Step (b) of the procedure with that of a reference fish larva and subsequent analysis of the data obtained. For example, in case the measurement of the body fat content of a fish larva wherein the activity of a target gene or/and target gene product which is assumed to affect energy homeostasis is modulated, yields, compared with a reference fish larva, an increase in fat content, the target gene or/and target gene product is a gene or/and gene product which is responsible for the activation and/or upward regulation of the homeostatic system for the regulation of body weight. In case the measurement of the body fat content, however, yields a decrease of the fat content, the target gene or/and target gene product is a gene or/and gene product which is responsible for inhibition and/or downward regulation of the homeostatic system for the regulation of body fat content. In case a comparison of the measurements, however, determines that both fish larvae have a comparable body fat content, the target gene or/and target gene product are not involved in regulating the homeostatic system for the regulation of body weight. The procedure in accordance with this present invention therefore does not only make it possible to identify and/or characterize genes or/and gene products which regulate energy homeostasis in healthy humans, but also genes or/and gene products which are responsible for the onset of diseases concerning the regulation of body weight, such as obesity, and associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea. Preferably, in accordance with this present invention, the procedure in accordance with this present invention is therefore conducted by using genetically modified fish larvae, e.g. transgenic fish larvae, or fish larvae that have been modified through mutation or insertion.
In a particularly preferred embodiment of this present invention, the fish larva is the zebrafish larva Danio rerio. The zebrafish larva can, in the procedure in accordance with this present invention, used at any stage of development for the identification and/or characterization of genes or/and gene products which affect energy homeostasis. Preferably, however, the age of the fish larva is 8 days, particularly preferably 8 to 14 days.
In another preferred embodiment of this present invention, the fish larva is the medaka larva Oryzias latipes.
In addition to the above-described procedures for the identification and/or characterization of genes or/and gene products which affect energy homeostasis, a second object of this present invention concerns a procedure for the identification and/or characterization of active substances which affect energy homeostasis, comprising:
(a) the provision of a fish larva,
(b) the administration of a test substance which is assumed to affect energy homeostasis to the fish larva,
(c) the measurement of the content of at least one metabolic parameter of the fish larva, and
(d) the determination of the extent of the impact on energy homeostasis of the fish larva. Step (a) of the procedure in accordance with this present invention comprises the provision of a fish larva. Preferably, the fish larva is the zebrafish larva Danio rerio, or the medaka larva Oryzias latipes, already described above. In addition to wild-type fish larvae, pursuant to this present invention, however, the procedure in accordance with this present invention can also be performed with genetically modified fish larvae.
Step (b) of the procedure in accordance with this present invention comprises the administration of a test substance which is assumed to affect energy homeostasis to the fish larva. As test substances, in particular peptides, aptamers and low-molecular organic molecules are suited. To permit an efficient search for active substances, in accordance with one preferred embodiment of this present invention, the test substance is chosen from a substance library.
Step (c) of the procedure in accordance with this present invention comprises the measurement of the content of at least one metabolic parameter of the fish larva. Preferably, this is at least one metabolic parameter, as already described above, a metabolic parameter of the homeostatic system for the regulation of body weight, such as body fat, glycogen, and blood sugar. The measurement of the content of at least one metabolic parameter of the fish larva is carried out as already described above.
Step (d) of the procedure in accordance with this present invention comprises the determination of the extent of the impact on energy homeostasis of the fish larva and is carried out in a manner that is substantially similar to that already described above.
By using fish larvae in accordance with this present invention for investigating the homeostatic system in terms of regulation of body weight, for the first time, procedures are provided which permit the simple, quick, inexpensive and highly specific identification and/or characterization of genes, gene products or active substances which affect energy homeostasis and which are related with the onset of metabolism diseases, such obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea. In view of the numerous benefits of fish larva described above, in particular the zebrafish larva Danio rerio or the medaka larva Oryzias latipes, as a model organism compared with prior art model systems, the procedures in accordance with this present invention can be carried out in any conventional standard laboratory in a quick, simple, and inexpensive manner and without requiring major technical systems to identify and/or characterize genes and/or gene products and/or active substances which affect energy homeostasis. In case, in a preferred embodiment of this present invention, zebrafish larvae Danio rerio are used in the procedure in accordance with this present invention, the extent of the impact of the homeostatic system on the regulation of body weight can be observed directly on living fish, e.g. through a visible increase or decrease of the fat content of the studied fish larva compared with a reference fish larva.
A third object of this present invention pertains to a pharmaceutical composition, comprising a gene or/and gene product identified by using the above-described procedure in accordance with this present invention or an active ingredient identified by using the above-described procedure in accordance with this present invention and, optionally pharmaceutically compatible carriers, diluents, and auxiliary substances. The pharmaceutical composition in accordance with this present invention may be present in a form that can be administered topically, parenterally, intravenously, intramuscularly, subcutaneously or transdermally and can be manufactured by using well-known, conventional, state-of-the-art procedures. Preferably, the pharmaceutical composition in accordance with this present invention is produced in the form of a pill or as an intravenous injection.
The pharmaceutical composition in accordance with this present invention is used for the prevention or/and treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
Preferably, the pharmaceutical composition in accordance with this present invention is administered to patients suffering from one of the above diseases in a quantity that is sufficient to alleviate the symptoms associated with this disease, prevent further propagation of the disease, fully cure the disease and/or prevent reoccurrence of the disease. The quantity of the pharmaceutical composition to be administered depends on several factors, such as, for example, selection of the gene, gene product, or active substance to be administered (specificity, efficacy, etc.), type of administration (pill, injection, etc.), type and extent of the disease as well as the age, weight, and general condition of the patient, and can be easily determined by those in the art by considering the above factors.
The pharmaceutical composition in accordance with this present invention is administered topically, parenterally, intravenously, intramuscularly, subcutaneously or transdermally. Preferably, the pharmaceutical composition is administered in the form of a pill or as an intravenous injection.
A fourth object of this present invention concerns the use of a gene and/or gene product or active substance identified by using the above-described procedure in accordance with this present invention for the prevention and/or treatment of diseases, in particular metabolism diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
A fourth object of this present invention concerns the procedure for determining the fat content of fish larvae used in a preferred embodiment of the above procedure, comprising: (a) the addition of a lipophilic coloring agent for the fish larva, and
(b) quantitative determination of the coloring agent stored proportionally with the fat content of the fish larva.
Step (a) of the procedure in accordance with this present invention comprises the addition of the lipophilic coloring agent to the fish larva. This is preferably carried out by killing off of the fish larvae, incubating the dead fish larvae in a coloring solution comprising the lipophilic coloring agent, and removing the non-fat-bound lipophilic coloring agent. Suitable lipophilic coloring agents which can be used in the procedure in accordance with this present invention comprise the coloring agents already identified above.
Step (b) comprises the quantitative determination of the coloring agent stored proportionally with the fat content of the fish larva and is carried out in a manner that is substantially similar to that already described above.
In a particularly preferred embodiment of the invention, the procedure in accordance with this present invention is used to determine the fat content in zebrafish larvae Danio rerio or medaka larvae Oryzias latipes. Considering that the zebrafish larvae are transparent at the beginning of their development, this species of fish therefore makes it particularly easy to determine the fat content. Preferably, the zebrafish larvae are at least 8 days old, in particular 8 to 14 days. Th e procedure in accordance with this present invention is the first procedure which has been described so far for determining the fat content of fish larvae. Prior-art procedures for the measurement of triglycerides, such as the standard enzyme FS, do not work with fish larvae.
Below, this present invention shall be explained in more detail in Figure 1 and in the following Example.
Figure 1 shows the coloring of the fatty tissue of zebrafish larvae Danio rerio in different stages of development with Oil Red O.
A: 27 hours,
B: 5 days,
C: 9 days, fish larvae were fed starting on the fifth day, D: 9 days, fish larvae were not fed.
Example: Determination of Fat Content with Oil Red O
First, the fish larvae were killed through incubation in an ice-cold tricaine solution. To color the fish larvae with Oil Red O, first, a coloring solution was prepared from 60 % isopropanol/Oil Red O and 40 % PBS. This coloring solution was left alone for 10 minutes; afterwards, insoluble color particles were removed via centrifugation over a period of 15 minute. The dead fish larvae were incubated for 20 minutes in the coloring solution, and the coloring solution was removed to the extent that this was possible. The nonfat-bound coloring agent was removed through incubation in 60 % isopropanol-PBS, 40 % isopropanol-PBS, and 20 % isopropanol-PBS for 4 minutes each. Afterwards, the fish larva were washed again 2x for 10 minutes each in PBS-T. The coloring agent bound in the larva was afterwards extracted by using isopropanol over a period of 15 minutes. For every 20 larvae, 500 μl isopropanol were used for the extraction. The supernatant of the extraction was measured in a photometer at 525 nm. After the extraction, the discolored larvae were once again washed twice by using PBS-T. Afterwards, they were homogenized, for example by using a FastPep device manufactured by the company Savant, and their protein content was measured. A corrected/relative fat value was therefore obtained by dividing the absorption value measured at 525 nm by the protein content.
To simplify the test design, both individual as well as the entire incubations can be performed in container inserts with a mesh bottom, such as Netwell by the company Costar, with a mesh width of 0.75 μm.
Note: to fully exclude differences in the quantity of food given per incubation vessel and, consequently, errors during the interpretation of the test, in the case of genetic screens after and/or during the overexpression of a gene, after a pretreatment with a chemical substance, after injection of antisense oligonucleotides or after a RNAi treatment, coincubation with control larvae carrying a genetic marker, for example through overexpression of a fluorescent protein such as GFP, an enzyme that is easy to detect by means of a color reaction, such as β-galactosidase, or a changed pigmentation, can be carried out. Differences in ambient conditions can then be excluded as causes for difference in the fat content of the fish larvae.

Claims

Claims
1. A method for the identification or/and characterization of genes or/and gene products which affect energy homeostasis, comprising:
(a) providing a fish larva wherein the activity of a target gene or/and a target gene product which is assumed to affect energy homeostasis is modulated,
(b) measuring the content of at least one metabolic parameter of the fish larva, and
(c) determining the extent of the impact on energy homeostasis of the fish larva.
2. The method in accordance with Claim 1 , characterized in that the target gene or/and target gene product is modulated on the gene level, transcript level or/and gene product level.
3 The method in accordance with Claim 1 or 2, characterized in that the target gene of the fish larva is modulated by activation or inhibition of the transcription of the target gene, stabilization or destabilization of the mRNA derived from the target gene, activation or inhibition of the translation of the mRNA derived from the target gene or/and activation or inhibition of splicing of the mRNA derived from the target gene.
4. The method in accordance with Claim 3, characterized in that the transcription of the target gene is activated or inhibited by regulatory proteins or/and repressors.
5. The method in accordance with Claim 3, characterized in that the mRNA derived from the target gene is stabilized or destabilized by regulatory proteins or/and ribozymes.
6. The method in accordance with Claim 3, characterized in that the mRNA derived from the target gene is activated or inhibited by regulatory proteins, antisense molecules or/and RNAi-molecules.
7. The method in accordance with Claim 1 or 2, characterized in that the target gene product of the fish larva is modulated by interaction with an activator, inhibitor or/and antibody.
8. The method for the identification and/or characterization of active substances which affect energy homeostasis, comprising
(a) providing a fish larva,
(b) administering a test substance which is assumed to affect energy homeostasis to the fish larva,
(c) measuring the content of at least one metabolic parameter of the fish larva, and
(d) determining the extent of the impact on energy homeostasis of the fish larva.
9. The method in accordance with Claim 8, characterized in that the test substance is selected from a substance library, comprising peptides, aptamers, and low-molecular organic molecules.
10. The method in accordance with any one of Claims 1 through 9, characterized in that the at least one metabolic parameter of the fish larva is body fat , glycogen or/and blood sugar.
11. The method in accordance with any one of Claims 1 through 10, characterized in that the body fat content of the fish larva is measured.
12. The method in accordance with Claim 11 , characterized in that the body fat content of the fish larva is measured by adding a lipophilic coloring agent and quantitatively determining the coloring agent that is stored proportionally with the fat content of the fish larva .
13. The method in accordance with Claim 12, characterized in that the lipophilic coloring agent is Oil Red 0, Sudan black B, Sudan IV, or Sudan Red 7B.
14. The method in accordance with Claim 12 or 13, characterized in that the quantitative determination of the stored coloring agent is carried out by visual or/and photometric means.
15. The method in accordance with any one of Claims 1 through 14, characterized in that the extent of the impact on energy homeostasis of the fish larva is determined by comparing the content of the at least one metabolic parameter of the fish larva with that of a reference fish larva and analysing of the data obtained.
16. The method in accordance with any one of Claims 1 through 15, characterized in that the fish larva is a zebrafish (Danio rerio) larva or a medaka (Oryzias latipes) larva.
17. The method in accordance with Claim 16, characterized in that the fish larva is at least 8 days, in particular 8 to 14 days old.
18. A pharmaceutical composition, comprising a gene or/and gene product identified by using the method in accordance with any one of Claims 1 through 17 or an active substance identified by using the method in accordance with any one of Claims 1 through 17 and, optionally, pharmaceutically compatible carriers, diluents, and auxiliary substances.
19. The pharmaceutical composition in accordance with Claim 18 for the prevention or/and treatment of diseases, in particular metabolic diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example, cancer of the genital organs, and sleep apnea.
20. Use of the gene or/and gene product or active substance identified by using the method in accordance with any one of Claims 1 through 17 for the prevention and/or treatment of diseases, in particular metabolic diseases, such as obesity, and other diseases concerning the regulation of body weight as well as associated diseases, such as eating disorders, cachexia, diabetes mellitus, high blood pressure, coronary heart disease, hypercholesterolemia, dyslipidemia, osteoarthritis, gallstone disorders, cancer, such as, for example.procedure cancer of the genital organs, and sleep apnea.
21. A method for determining the fat content of fish larvae, comprising
(a) adding a lipophilic coloring agent to the fish larva under conditions wherein the coloring agent is deposited in the fat tissue of the fish larvae proportionally with the fat content of the fish larvae, and
(b) quantitatively determining the coloring agent, that is deposited in the fat tissue of the fish larvae .
22. The method in accordance with Claim 21 , characterized in that the lipophilic coloring agent is Oil Red O, Sudan black B, Sudan IV or Sudan Red 7B.
23. The method in accordance with Claim 21 or 22, characterized in that the coloring agent is quantitatively determined by means of visual or/ procedure and photometric determination.
24. The method in accordance with any one of Claims 21 through 23, characterized in that the fish larva is a zebrafish (Danio rerio) larva or a medaka (Oryzias latipes) larva.
25. The method in accordance with any of Claims 21 through 24, characterized in that procedure the fish larva is at least 8, in particular 8 through 14 days old.
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KR101371361B1 (en) 2012-11-29 2014-03-07 한국과학기술연구원 Method for quantifying fat and fatty oil using sudan iv

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