JPWO2007010999A1 - Lipid metabolism and sugar metabolism modified animal by KRAP gene mutation, modification method and modification agent - Google Patents

Abstract

A novel drug that can contribute to the prevention and / or treatment of obesity and / or diabetes, a transgenic animal that can be used for the purpose of elucidating the pathology of obesity and / or diabetes, and a nucleic acid molecule that can be used for the production thereof provide. One embodiment of the present invention is a transgenic non-human animal that has reduced or lacked expression of the KRAP gene.

Description

  The present invention relates to a nucleic acid molecule capable of increasing energy consumption, increasing insulin sensitivity, preventing or eliminating obesity, and / or causing abnormal blood hormone levels in animals, and a drug containing such a nucleic acid. The present invention relates to a transgenic animal containing a molecule, increased energy consumption, increased insulin sensitivity, prevention or elimination of obesity, and / or a method for inducing abnormal blood hormone levels.

  To date, several genes involved in mammalian obesity are known. Animals that exhibit anti-obesity or obesity have been produced by modifying these genes positively or negatively. Examples of such modified target genes include leptin (FGF), neuropeptide Y (neuropeptide Y), angiopoietin-related growth factor, which can function systemically as an endocrine signal factor. ), Transcriptional regulators PGC1α (PPAR gamma coactivator 1α), CBP (CREB binding protein), SRC-1, TIF2, RXRα (retinoid X receptor α), and intracellular lipids that function by varying gene expression over a wide range Metabolism-related enzymes ACC2 (acetyl CoA carboxyylase 2), SCD1 (stealyl CoA desaturase 1) Intracellular signaling regulators SHIP2, PTP1B, S6 kinase, SOCS-6, include those encoding various protein species, such as mitochondrial function protein UCP (uncoupling protein). Many of these proteins have functions that are not well understood or are multifunctional (that differ depending on the cell in which they are expressed, or that have different effects depending on the interacting proteins). In particular, c-Cbl was originally studied as a cancer-related gene, but it controls various signals in the cell depending on the expression cell and situation, and this deficient mouse has increased energy consumption, anti-obesity, and glucose metabolism change. It is known to show. In addition, transgenic mice with enhanced expression of FGF19 and FGF19-administered mice (FGF is a humoral factor that acts remotely in the blood) show an anti-obesity effect by FGP19, which can be used to treat obesity and diabetes. It attracts attention from a viewpoint (nonpatent literature 1-31).

  A KRAP (Ki-ras-induced acting-interacting protein) gene has recently been identified as one of cancer-related genes that are up-regulated by activated Ki-ras (Non-patent Document 32). The protein encoded by this gene was known to have a coiled-coil region, but its physiological expression site and function were unknown.

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Detective section of saliva in trangenic m acking aquaporin-5 water channels. J Biol Chem. 1999 Jul 16; 274 (29): 20071-4 Enerback S, Jacobson A, Simpson EM, Guerra C, Yamashita H, Harper ME, Kozak LP. Mice racking mitochondria uncoupling protein are cold-sensitive but not obese. Nature. 1997 May 1; 387 (6628): 90-4. Tomlinson E, Fu L, John L, Hultgren B, Huang X, Renz M, Stephan JP, Tsai SP, Powell-Braxton L, French D, Stewart TA. Transgenic rice expressing human fibroblast growth factor-19 display increased metabolic rate and decreased adiposity. Endocrinology. 2002 May; 143 (5): 1741-7. Fu L, John LM, Adams SH, Yu XX, Tomlinson E, Renz M, Williams PM, Soriano R, Corpuz R, Moffat B, Vandlen R, Simons L, Foster J, St. J Fibroblast growth factor 19 increases metabolites rate and reverses dietary and leptin-defective diabets. Endocrinology. 2004 Jun; 145 (6): 2594-603. Klaman LD, Boss O, Peroni OD, Kim JK, Martino JL, Zabolotny JM, Moghal N, Lubkin M, Kim YB, Sharpe AH, Sticker-BronGh. Incremented energy expenditure, decremented adiposity, and tissue-specific insulative sensitivity in protein-tyrosine phosphatase 1B-definitive rice. Mol Cell Biol. 2000 Aug; 20 (15): 5479-89. Inokuchi J, Komiya M, Baba I, Naito N, Sazazuki T, Shirawa S .; Deregulated expression of KRAP, a novel gene encoding actin-interacting protein, in human colon cancer cells. J Hum Genet (2004) 49: 46-52.

  INDUSTRIAL APPLICABILITY The present invention can be used for novel drugs that can contribute to the prevention and / or treatment of obesity and / or diabetes, transgenic animals that can be used for the purpose of elucidating the pathology of obesity and / or diabetes, and the production thereof An object is to provide nucleic acid molecules and the like.

  The present inventors have found that KRAP is distributed in a large amount in the liver and pancreas due to physiological expression, and ubiquitously exists in many organs and tissues other than the heart and skeletal muscle. Furthermore, the present inventors have found that animals in which the KRAP gene has been disrupted exhibit changes in lipid metabolism and sugar metabolism and become animals with a small amount of fat, thereby completing the present invention.

The present invention
[1] A transgenic non-human animal with reduced or lacked expression of the KRAP gene;
[2] The transgenic non-human animal according to [1] above, which contains a KRAP gene disrupted by homologous recombination;
[3] The transgenic non-human animal according to the above [1], which has a modified lipid metabolism and / or sugar metabolism;
[4] The transgenic non-human animal according to the above [1], which exhibits increased energy consumption, increased insulin sensitivity, and / or abnormal blood hormone levels;
[5] An isolated mutant KRAP gene nucleic acid molecule capable of homologous recombination with a KRAP gene in the genome and capable of reducing or eliminating the expression of the KRAP gene in a non-human animal cell;
[6] The nucleic acid molecule according to [5], wherein the mutation is a deletion of at least one exon;
[7] The nucleic acid molecule according to [6], wherein the mutation is a deletion of a region containing at least one of exons 11 to 16;
[8] A vector comprising the nucleic acid molecule of any one of [5] to [7] above;
[9] The vector according to [8] above, which is an expression vector for producing a transgenic animal or gene therapy;
[10] A host cell into which the vector of [8] or [9] has been introduced;
[11] An isolated interfering nucleic acid having a sequence that is substantially the same or substantially complementary to at least a part of the KRAP gene or mRNA and that can inhibit the expression of the KRAP gene in a cell, tissue, or individual molecule;
[12] The interfering nucleic acid molecule according to [11], wherein at least a part is double-stranded;
[13] A binding protein that specifically binds to a KRAP protein or a part thereof and can inhibit the function of the KRAP protein in a cell, tissue, or individual;
[14] A lipid metabolism and / or sugar metabolism modifier comprising the interfering nucleic acid molecule or binding protein according to any one of [11] to [13];
[15] The lipid metabolism and / or sugar metabolism-modifying agent according to the above [14], which is an energy consumption enhancer, an obesity prevention or elimination agent, or an insulin sensitivity enhancer;
[16] A method of inducing alteration of lipid metabolism and / or sugar metabolism in a mammalian cell, tissue or non-human animal by inhibiting the expression or function of a KRAP gene or protein;
[17] The method according to [16] above, wherein the alteration of lipid metabolism and / or sugar metabolism is increased energy consumption, prevention or elimination of obesity, increased insulin sensitivity, and / or abnormal blood hormone levels;
[18] an interfering nucleic acid molecule having a sequence that is substantially identical or substantially complementary to at least a portion of a KRAP gene or mRNA, and capable of inhibiting the expression of the KRAP gene in a cell, tissue, or individual, and / or The step of inhibiting the expression of the KRAP gene using a binding protein that specifically binds to the KRAP protein or a part thereof and can inhibit the function of the KRAP protein in a cell, tissue or individual [16] Or the method according to [17],
I will provide a.

  According to the administration of the nucleic acid molecule or the like of the present invention or the method of the present invention, by inhibiting the expression or function of the KRAP gene or protein, artificially alters lipid metabolism and sugar metabolism and increases energy consumption in animals. Can further prevent or eliminate obesity and / or prevent or treat diabetes. In particular, according to the present invention, obesity caused by a meal such as ingestion of a high fat diet can be suppressed. On the other hand, adverse effects due to inhibition of KRAP gene or protein expression or function have not been found.

  The transgenic animal of the present invention is characterized in that lipid metabolism and sugar metabolism are modified, and particularly, as a result of increased energy consumption, the body is smaller and the amount of fat is smaller than that of the wild type. Therefore, it is useful as a model animal in studies on body weight control and obesity mechanisms, sugar metabolism, etc., for example, studies for elucidation of pathological conditions such as obesity and diabetes.

FIG. 1 is a diagram showing a comparison of amino acid sequences of KRAP gene products of human (upper) and mouse (lower). The underlined part is the coiled coil motif.
FIG. 2 shows targeted disruption of the mouse KRAP gene. Panel (A): A region of the mouse KRAP gene (Wild-type locus), including exons 8-17, is shown schematically with associated restriction enzyme sites. Panel (B): shows a targeting vector containing a neomycin (neo) cassette. The corresponding positional relationship with the genomic gene (A) intended for homologous recombination is indicated by a dotted line. Panel (C): shows a mutant allele predicted to be generated by homologous recombination. Panel (D) Left: Southern blot photograph using tail DNA digested with EcoRV. Center: Northern blot photograph showing specific deletion of KRAP mRNA in mutant (− / −) liver compared to wild type (+ / +). Below is β-actin. Right: Western blot photograph showing specific deletion of KRAP protein in mutant (− / −) liver compared to wild type (+ / +). Below is β-tubulin.
[FIG. 3] is a graph showing the growth curves (vertical axis: body weight, horizontal axis: weeks of age) for wild type (Wt), heterozygote (Ht) and KRAP knockout mice (Ko) (average ± se; respectively). n = 5-14 / group). Left: male, right: female.
FIG. 4 shows a comparison of organ weights for wild type (Wt) and KRAP knockout mice (Ko). Mean ± se for 20-week-old males (n = 9), vertical axis is the percentage of tissue weight / body weight. WAT = white adipose tissue, liver = liver, BAT = brown adipose tissue, kidney = kidney, heart = heart.
FIG. 5 is a photograph showing HE-stained images of tissue sections of wild-type (Wt) and KRAP knockout mice (Ko) prepared after fasting a 22-week-old male fed a normal diet for 16 hours. is there. WAT = white adipose tissue, BAT = brown adipose tissue, Liver = liver. Scale bar is 50 μm.
FIG. 6 shows the relative amounts of liver triglycerides and glycogen for wild-type (Wt) and KRAP knockout mice (Ko) in the fed state. Left: triglyceride, right: glycogen.
FIG. 7 is a graph showing a comparison of food intake (KRAP + / + (n = 7), KRAP − / − (n = 9)) per mouse individual (left) or normalized by body weight (right). Values are mean ± se. An asterisk indicates a significant difference of P <0.05, and “NS” indicates no significant difference compared to a wild-type mouse.
FIG. 8 shows the daily energy consumption profile (panel (A)) and respiratory quotient (RQ) (panel (B)) for 22-week-old male mice under normal diet. Values are mean ± se (KRAP + / + (n = 7), KRAP − / − (n = 9)). The asterisk indicates a significant difference of P <0.05. Panel (C): A measure of total energy consumption per body weight.
FIG. 9 is a diagram showing the results of a glucose tolerance test. Vertical axis: blood glucose concentration, horizontal axis: time after injection. A 16-week-old male mouse maintained on a normal diet was used. KRAP + / + (n = 10), KRAP − / − (n = 10) (mean ± se). An asterisk indicates a significant difference of P <0.01.
FIG. 10 shows the results of an insulin sensitivity test. Left: blood glucose concentration, right: blood glucose concentration expressed as a percentage with the time of injection taken as 100%. An asterisk indicates a significant difference (*: P <0.05; **: P <0.01).
FIG. 11 shows blood glucose levels of KRAP + / + (black column), KRAP − / − (white column) and KRAP +/− (shaded column) mice under feeding.
FIG. 12 shows blood ketone body levels of KRAP + / + (black column) and KRAP − / − (white column) mice under fed (fed) and fasting for 15 hours (15h fast). . The vertical axis represents the blood ketone body concentration.
FIG. 13 is a diagram showing weight adjustment when a high fat diet is given. Panel (A): Weight curve of mouse, Panel (B): Weight gain in 4 weeks, Panel (C): Graph showing weight gain in 4 weeks divided by weight at start, Panel (D) : Weight division display of WAT weight. An asterisk indicates a significant difference (*: P <0.001; **: P <0.01).
FIG. 14 is a diagram showing the amount of food intake during a period when a high fat diet was given. Left: Amount of food consumed per day (g), Right: the weight divided by weight. An asterisk indicates a significant difference (*: P <0.05; **: P <0.001).
FIG. 15 is a diagram showing energy consumption when a high fat diet is given. An asterisk indicates a significant difference (*: P <0.05).
FIG. 16 is a diagram showing the results of Northern blotting on the expression of several genes in tissues. Tissue: Liver = liver, Skeletal muscle = skeletal muscle, WAT = white adipose tissue, BAT = brown adipose tissue; animal: W = wild type (+ / +), K = KRAP knockout mouse (− / −); gene: ACC1 = Acetyl-CoA carboxylase 1, ACC2 = acetyl-CoA carboxylase 2, ACO = acyl-CoA oxidase, UCP2 = uncoupled protein 2, β-actin = β-actin, LPL = lipoprotein lipase, HSL = hormone-sensitive lipase, PPARγ = Peroxisome proliferator-activated receptor γ, Leptin = leptin, aP2 = adipocyte fatty acid binding protein, 36B4 = acidic ribosomal phosphorylated protein P0, UCP1 = uncoupled protein 1, PGC1α = PPARγ-coactin Beta 1α. For liver and skeletal muscle, data for two wild type and KRAP knockout mice are shown.
FIG. 17 is a diagram showing the measurement results of spontaneous momentum. WT = wild type, KO = KRAP knockout mouse. Panel (A): The vertical axis represents the number of movement actions counted, and the horizontal axis represents time and light / dark conditions. Panel (B): Shows total counts under “dark” (dark) and “light” (bright) conditions.
FIG. 18 is a diagram showing the influence on the liver when a high fat diet is given. WT = wild type, KO = KRAP knockout mouse. Panel (A): A photograph showing the appearance of the liver. Panel (B): It is a figure which shows a tissue triglyceride content.

In the present invention, the “KRAP gene” includes all homologs and alleles encoding KRAP that can be expressed as a polypeptide. Such genes are known for at least mice and humans (eg, FIG. 1), and a desired cDNA library is screened under stringent conditions using probes designed based on such known sequences. Can be identified. “Stringent conditions” means, for example, hybridization conditions at 42 ° C. in a solution of 50% formamide, 120 mM Na ₂ HPO ₄ , 7% SDS, 1 mM EDTA, 250 mM NaCl, and equivalent stringency conditions. Say.

  For example, the coding region of the mouse genomic gene (SEQ ID NO: 1 in the sequence listing) registered as Genbank accession number NC — 000068 or the full length of the mouse cDNA (mRNA) registered as accession number AB120565 (SEQ ID NOs: 2 and 3) 60% or more in the homology search using the BLAST search of NCBI (National Center for Biotechnology Information) for the full length (SEQ ID NOs: 4 and 5) of human cDNA (mRNA) registered as Genbank accession number AB116937 Nucleotide identity or greater than 50% amino acid identity, preferably greater than 80% nucleotide identity or greater than 70% amino acid identity Which, more preferably those having a 90% nucleotide identity or 80% amino acid identity or greater is included in the gene encoding the "KRAP" protein with respect to the present invention.

  “Wild-type” KRAP gene refers to the above-mentioned mouse or human KRAP gene registered in Genbank, or a natural-type KRAP gene having substantially the same expression and function as those KRAP genes. Say.

  The “mutant” KRAP gene is quantitative or qualitative in that a part of the nucleotide sequence is modified as compared with the KRAP gene (particularly a natural type or wild type gene) obtained as described above. Refers to any KRAP gene that results in different KRAP gene expression.

  In the present invention, mutant KRAP that retains homology or identity to the extent that homologous recombination with the KRAP gene in the genome is possible, and can reduce or eliminate the expression of the KRAP gene in non-human animal cells. Genes are used. Examples of such mutant KRAP genes include, but are not limited to, those having at least one exon deletion and those having a mutation that causes a frame shift. Examples of the deletion include those in which the mutation is present in the C-terminal half region of the KRAP gene, more specifically, deletion of a region containing at least one of exons 11 to 16, exons 11 to 16 The region containing all of the can be missing. Alternatively, the defect can be, for example, about 5 to 95%, 20 to 80%, or 35 to 70% of the coding region. The deletion of the coding region may be small as long as it results in disruption of the KRAP function, and may be wide as long as a coding region and / or a non-coding region capable of homologous recombination remain.

  By inserting such a mutant KRAP gene nucleic acid molecule into an appropriate vector, a vector for gene transfer, for example, a vector for homologous recombination can be prepared. Preferably, the nucleic acid molecule is DNA and the vector comprises a mutant KRAP gene nucleic acid molecule and a selectable marker gene (eg, a neo gene conferring G418 resistance).

  In producing the transgenic animal of the present invention, such a vector is introduced into a host cell (for example, mouse blastocyst) by a general method. Methods for producing a transgenic animal are well known to those skilled in the art, and those skilled in the art can arbitrarily select a known method and appropriately employ it for producing the transgenic animal of the present invention.

  The transgenic animal of the present invention should be confirmed by a standard method that the expression of the KRAP gene or protein is significantly reduced or absent compared to that in the host animal (having the wild type KRAP gene) used for production. Can be specified by. Furthermore, for example, lipid metabolism (especially increased energy consumption), sugar metabolism (especially increased insulin sensitivity), and fluctuations in various blood hormone concentrations related to these are confirmed using the assay methods described later in the Examples. The transgenic animal of the present invention can also be specified.

  The transgenic animal of the present invention can be used as a model animal in various experiments for the purpose of elucidating the pathogenesis of diseases and metabolic syndrome including obesity, diabetes and the like and further treating it. Furthermore, it can also be used for elucidating the function of the KRAP gene itself.

  According to the present invention, ex vivo gene therapy is also possible by using a vector containing the mutant KRAP gene as described above. That is, in general gene therapy, a functional gene is introduced in vitro into a cell derived from a treatment target containing a defective gene, and the genetically manipulated cell is returned to the treatment target. The invention includes a step of introducing the mutant KRAP gene as described above in vitro into a cell derived from a treatment subject containing a functional KRAP gene, and a step of returning the genetically engineered cell to the treatment subject. Gene therapy vectors used in such methods are also within the scope of the present invention. For such gene therapy vectors, vectors such as adenoviruses, retroviruses and herpesviruses are used, and their production methods are well known to those skilled in the art.

  The present invention provides methods of inducing alterations in lipid and / or sugar metabolism in mammalian cells, tissues or non-human animals by inhibiting the expression or function of KRAP genes or proteins. In order to inhibit the expression or function of a KRAP gene or protein, for example, an interfering nucleic acid molecule designed on the basis of the base sequence of the KRAP gene (eg antisense RNA; short containing a double-stranded part capable of causing RNA interference) (For example, 18-200 bases) RNA, double-stranded nucleic acid containing DNA and RNA, siRNA, miRNA, etc.), a binding protein molecule that specifically binds to KRAP protein (where “protein” is a polypeptide Can be used including, for example, polyclonal or monoclonal antibodies, various fragments of antibodies, humanized antibodies, etc.). Methods for designing and producing these various interfering nucleic acid molecules and methods for preparing binding protein molecules such as antibodies or antibody fragments are well known to those skilled in the art.

  These interfering nucleic acid molecules and / or binding protein molecules may be used alone or in combination as lipid metabolism and / or sugar metabolism modifiers, such as energy expenditure enhancers, obesity prevention or elimination agents, or insulin sensitivity enhancers. In combination with an appropriate carrier that is physiologically acceptable, and administered to an animal, effects such as increased energy consumption, prevention or elimination of obesity, or increased insulin sensitivity can be obtained. Physiologically acceptable carriers and formulation methods that can be used for such purposes are well known to those skilled in the pharmaceutical arts. The appropriate dosage may vary within a wide range depending on the animal species, individual body weight, age, sex, general health condition, etc., but those skilled in the art will appropriately determine the appropriate animal by selecting and experimenting with it. can do.

1. Construction of KRAP targeting vector Construction of the targeting vector will be described with reference to FIG. From the 129 / SV mouse genomic library (Stratagene), genomic DNA of the KRAP gene was isolated using a mouse KRAP cDNA probe (base numbers 2962 to 3759 in SEQ ID NO: 1 in the sequence listing). First, an XhoI-SalI fragment was prepared from isolated genomic DNA and placed as the 3 'arm into the SalI site of pBluescript SK. Second, an XbaI-EcoRI fragment was prepared from the isolated genomic DNA and inserted as a 5 'arm into the XbaI-EcoRI site. Third, the pGKneo cassette was placed in the EcoRI site. Finally, the diphtheria toxin A fragment cassette was inserted into the SalI site with XhoI-SalI. In this way, as shown in FIG. 2 panels (A) and (B), a 7.2 kb EcoRI-XhoI fragment containing the KRAP gene exons 11 to 16 (55% of the coding region) (sequence of the sequence listing) Base number 20598-28867 in No. 1 was replaced with a pGKneo cassette having the opposite transcription direction, and a targeting vector was constructed, and the 5 ′ and 3 ′ arms of this targeting construct each had 2.3 kb (SEQ ID No. 1 in the Sequence Listing). And 6.4 kb (base numbers 27876 to 34348 in SEQ ID NO: 1 in the sequence listing), and the diphtheria toxin A fragment cassette (DTA-A) is adjacent to the 3 ′ genomic arm. This targeting vector It was linearized with SalI.

2. Preparation of KRAP knockout mouse The linearized targeting vector was introduced into embryonic stem (ES) cells by electroporation, and the recombinant was selected with G418 on feeder cells (embryonic fibroblasts). This mutant embryonic stem cell was microinjected into a blastocyst (blastocyst) of a C57BL / 6 mouse, and the resulting male chimera was crossed with a C57BL / 6 mouse. Heterozygous (KRAP +/−; sometimes abbreviated as “Ht”) mice were mated to obtain KRAP knockout (KRAP − / −; sometimes abbreviated as “Ko”) mice.

  The observed genotype ratio of the offspring was consistent with the expected Mendelian ratio. Thus, it was shown that disruption of the KRAP gene did not affect embryogenesis. KRAP (− / −) mice were fertile.

  Genomic DNA extracted from the tail of the animal was digested with EcoRV, and an external probe (350 bp XbaI fragment, base numbers 15786-16140 in SEQ ID NO: 1 in the sequence listing; FIG. 2, panel (C) indicated by “Probe A”) Hybridized. The wild type (KRAP + / +; sometimes abbreviated as “Wt”) is a 21.0 kb restriction fragment, the mutant (− / −) is an 8.0 kb fragment, and both are heterozygous (− / +) Fragments were detected (Fig. 2, left photo of panel (D)).

  Furthermore, homologous recombinants were confirmed using a neo probe, and it was confirmed that there was only one integration site.

  Total RNA was extracted from wild type (+ / +) and mutant (− / −) livers according to a conventional method, and Northern blotting was performed using nucleotide numbers 2962 to 3759 in SEQ ID NO: 1 in the sequence listing as probes. (FIG. 2 (D) center). In KRAP (− / −), KRAP mRNA was specifically deleted (β-actin used as an internal control showed equivalent expression).

Wild type (+ / +) and mutant (− / −) liver lysates were prepared and Western blots were performed. Mouse tissues were sonicated in RIPA buffer [20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease inhibitor cocktail (Roche)]. This solution was centrifuged at 15,000 rpm for 30 minutes to obtain an extract, and the amount of protein was measured using a protein assay reagent (Bio-Rad). These samples were developed by SDS-PAGE and transferred to a nitrocellulose membrane. The blot was blocked and affinity purified in blocking buffer (0.1% Tween 20, 5% non-fat skim milk, 50 mM Tris-HCl, 150 mM NaCl, 0.1% NaN ₃ , pH 7.5). Incubated with KRAP antibody at 4 ° C. overnight (dilution ratio 1: 2,000). This was developed with HRP-conjugated anti-rabbit IgG goat secondary antibody and ECL (Amersham Pharmacia).

The anti-KRAP antibody used for detection was prepared as follows. Recombinant human KRAP (amino acids 1039 to 1246 in SEQ ID NO: 4 in the sequence listing) was expressed as a bacterial fusion protein using the pGEX6P-1 vector (Pharmacia) according to the instructions. The fusion protein was soluble in non-denaturing buffer and purified using glutathione-Sepharose 4B (Amersham). This recombinant KRAP protein was injected into rabbits and boosted to give antiserum. The antiserum was purified using an affinity column prepared by crosslinking the recombinant protein to CNBr-activated Sepharose 4B (Amersham).
The results are shown in FIG. 2 (D) right). In KRAP (− / −), the KRAP protein was specifically deleted (β-tubulin used as an internal control showed equivalent expression).

3. Measurement of body weight change Animals were maintained in a temperature controlled facility (23 ° C.) with a 12 hour light / dark cycle. Mice were given optional access to a standard rodent diet (CE-2 (Clea Japan); hereinafter sometimes referred to as “normal diet”) and water. Body weights were recorded weekly for KRAP + / +, +/−, and − / − animals obtained by mating heterozygous mice.

  The results are shown in FIG. The body weight of KRAP − / − pups was not significantly different from litter KRAP + / + and +/− mice at least within 5 days after birth. When fed a normal diet, KRAP − / − mice weighed less than both + / + and +/− mice. This difference occurred at 3 weeks of age for both male (FIG. 3, left) and female (FIG. 3, right).

4). Organ Weight Measurement Male KRAP + / + (n = 9) and KRAP − / − (n = 9) mice matched in age (20 weeks of age) were euthanized and epididymal white adipose tissue (WAT) Wet weights of liver, scapular brown adipose tissue (BAT), kidney and heart were measured.

  The results are shown in FIG. The relative weight of WAT and liver (g / g body) was lower in KRAP − / − mice than in KRAP + / + mice. On the other hand, no significant difference was observed for BAT, kidney and heart.

5). Morphological Observation WAT, BAT and liver of 22 week old male mice were obtained 16 hours after fasting. These tissue fragments were fixed in 3.7% formaldehyde in PBS, dehydrated in ethanol and embedded in paraffin. Sections were deparaffinized, rehydrated, stained with hematoxylin and eosin (HE), and observed with a light microscope.

  The results are shown in FIG. KRAP-/-adipocytes in HE-stained WAT and BAT (appearing white) were clearly reduced in size compared to KRAP + / + adipocytes. In lipid staining of KRAP − / − mice, almost no lipid vacuoles were observed.

6). Measurement of Tissue Triglyceride and Glycogen Content The triglyceride content of the liver of 20-week-old male mice under standard feeding conditions of a normal diet was determined by Folch et al. (Folch J, Lees M, Sloane Stanley GH., A simple method for the isolation). and purification of total lipids from human tissues, J Biol Chem. 1957 May; 226 (1): 497-509) and assayed using the enzymatic triglyceride E test (Wako Pure Chemical Industries). For measurement of liver glycogen, livers from the same mouse were homogenized in 3% (w / w) perchloric acid and the homogenate was incubated with amyloglucosidase at 40 ° C. for 2 hours to hydrolyze glycogen. Next, the resulting glucose residue was measured using an enzymatic glucose CII test (Wako Pure Chemical Industries).

  The results are shown in FIG. Morphological observations suggest that KRAP-/-liver has a low triglyceride content, but in fact, liver tissue triglyceride extraction and measurement showed that KRAP-/-liver was KRAP + / + liver. And 40% less triglycerides. On the other hand, there was no significant difference between the two genotypes in the liver glycogen content.

7). Measurement of food intake Male mice at 22 weeks of age were individually placed in a metabolic cage. Mice were provided with a normal diet that weighed in advance for 24 hours. Body weight was measured before the start of the test. After the end of the 24-hour period, the remaining food including food waste was weighed, and the amount of food intake per day (g / day / kg-body weight) was calculated.
The results are shown in Table 1 and FIG.

  The body weight of KRAP + / + mice was significantly heavier than KRAP − / − mice (P <0.001). Food intake was significantly higher in KRAP − / − mice than in KRAP + / + mice when food consumption was adjusted for body weight. These results indicate that the delay in weight gain in KRAP − / − mice is not due to food intake.

8). Study of energy metabolism Male wild-type mice (22 weeks old) (n = 7) and KRAP-/-(n = 9) mice matched for age for individual measurement of energy metabolism were individually treated in a metabolic cage for 1 day. (Ichikawa M, Fujita Y, Effects of nitrogen and energy meta-both 1 of N, and 1 of the weights of the world. , Kanai S, Ichimaru Y, Funakoshi A, Miyasaka K., The diurnal rhythm of energy expe diture differs between obese and glucose-intolerant rats and streptozotocin-induced diabetic rats, J Nutr.2000 Oct; 130 (10): 2562-7). Automatic _O 2 -CO ₂ analyzer (NEC Medical System, model IH26, Tokyo, Japan) was used to measure oxygen consumption and carbon dioxide production amount exhaled continuously. The energy consumption per hour and per day was calculated.

  The results are shown in FIG. The daily energy consumption is greater in KRAP − / − mice than in KRAP + / + mice (P <0.01), and in KRAP − / − mice, 2929.3 ± 163.1 kJ / kg body weight, KRAP + / + mice. It was 2257.2 ± 152.2 kJ / kg body weight. Regarding respiratory quotient (RQ) during metabolic measurement, there was no significant difference between both genotypes (P = 0.37), 0.90 ± 0.03 for KRAP + / + mice, and 0.88 for KRAP − / − mice. ± 0.02. These results indicate that KRAP deficiency resulted in increased energy consumption. The source of energy used for combustion in KRAP-/-mice was normal, at least on the normal diet. Measurements on food intake and energy expenditure showed that metabolic turnover was clearly enhanced by KRAP deficiency in mice.

9. Intraperitoneal glucose tolerance test and insulin sensitivity test Animals fasted overnight were subjected to an intraperitoneal glucose tolerance test using a dose of 2 g / kg body weight of glucose. The insulin sensitivity test was performed by injecting insulin at a dose of 0.75 U / kg body weight to animals fasted for 3 hours. Tail vein blood was collected at 15, 30, 60, 90 and 120 minutes after injection and used for glucose quantification using Medisense Xtra (Abbott Japan).

  The results are shown in FIGS. At 60, 90 and 120 minutes after glucose administration, glucose levels in KRAP − / − mice were significantly lower compared to KRAP + / + mice (FIG. 9). Also, in the insulin sensitivity test, insulin injection resulted in significantly lower glucose levels in KRAP − / − mice compared to KRAP + / + mice (FIG. 10).

  These results indicate that KRAP − / − mice are more glucose tolerant and insulin sensitive compared to KRAP + / + mice maintained on a normal diet.

10. Clinical Biochemical Measurements Blood was collected from the retroorbital sinus vein under normal feeding conditions. The blood was kept on ice until centrifuged (3,000 g, 15 minutes, 4 ° C.), serum was collected and stored at −70 ° C. until used for analysis. Insulin, leptin, adiponectin and thyroxine (T4) concentrations in serum were measured using ELISA kits (Shibayagi Co., R & D systems, Otsuka pharmaceutical Co., Endocrinetech. Co., respectively). Triglycerides, total cholesterol and non-esterified free fatty acids (NEFA) were measured by enzymatic assay (Wako Pure Chemical Industries). Glucose and ketone bodies were quantified using Medisense Xtra using tail vein blood.
The results are shown in Tables 2 and 3 and FIGS.

  Expressed as mean ± se for 15-week-old male mice (n = 10) and 34-week-old female mice (n = 8-9) of the indicated genotypes fed a normal diet.

  Expressed as mean ± se for 15-week-old male mice (n = 10) and 34-week-old female mice (n = 11) of the indicated genotypes fed a normal diet.

  Serum cholesterol and free fatty acid levels in KRAP + / + and KRAP − / − mice fed a normal diet were not significantly different. Serum triglyceride levels tended to be lower in KRAP − / − mice, although no statistically significant difference was observed. KRAP − / − mice showed mild hypoglycemia and reduced circulating insulin levels.

  Ketone bodies are produced by breaking down fat as an energy source in starvation. In KRAP − / − mice, a low value that is the same as that of the wild type under non-fasting indicates that it is not ketoacidosis (no abnormality like starvation). Moreover, since the ketone body concentration increased under fasting, it was suggested that lipolysis and conversion of NEFA to ketone bodies were not abnormal in KRAP − / − mice.

  Since leptin is a circulating hormone that increases fat loss independent of reduced food intake, serum levels of this hormone were measured. Leptin was actually reduced in KRAP − / − mice.

  Adiponectin is a fat-derived hormone and is thought to have a role in insulin tolerance and diabetes associated with obesity. Adiponectin was increased in KRAP + / + mice. KRAP − / − mice showed increased energy consumption, but their serum thyroxine (T4) levels were lower than KRAP + / + mice. Moreover, the fact that the formation of ketone bodies in the fed state was comparable to the wild type supported that KRAP-/-mice had no abnormalities such as starvation, which was observed when normalized for body weight. Consistent with / − mice eating higher than KRAP + / +.

11. High fat whose weight was adjusted to male KRAP + / + (n = 7) and KRAP − / − (n = 6) mice whose age of control was matched (23 weeks old) when fed a high fat diet Dietary feed (“High Fat Diet 32”, crude fat content about 32%, ratio of fat-derived calories to total calories about 60%; Japan Claire) was given for 4 weeks. These mice were raised on a normal diet until 23 weeks of age. The body weight was measured every two days, the weight of the remaining food including food waste was measured every two days, and the amount of food intake per day (g / day / kg-body weight) was estimated. After the period of 4 weeks on the high fat diet, these mice were subjected to the same energy metabolism measurement as described above.

  The results are shown in FIGS. FIG. 13, panel (A) is a mouse body weight curve, panel (B) is a weight gain in 4 weeks, panel (C) is a graph showing the weight gain in 4 weeks divided by weight at the start, Panel (D) is the weight percent of the epididymal white adipose tissue (WAT) weight. From this result, even when a high-fat diet was given to the mice of the present invention, the amount of weight gain per body weight was significantly less than that of the wild type, and thus the mutation in the KRAP gene caused obesity caused by diet. It is clear that it has a preventive effect.

  FIG. 14 shows the amount of food intake during the period when the high fat diet was given. On the left is the amount of food consumed per day (g), and on the right is the weight divided by weight. It can be seen that the mice of the present invention have a higher food intake compared to the wild type, but have less weight gain.

  FIG. 15 represents the results for energy consumption. As in the case of the normal diet, energy consumption was increased in the mouse of the present invention.

12 Clinical biochemical measurements (2)
For the same serum sample as used in “10. Clinical Biochemical Measurement”, the triiodothyronine (T3), growth hormone (GH), and glucagon concentrations in the serum were measured using the RIA kit (Endocrinetech. Co., respectively). Amersham Biosciences, Yanaihara Research Co.). Albumin concentration was measured by enzymatic assay (Wako Pure Chemical Industries).
The results are shown in Table 4.

Each measured value in Table 4 is expressed as mean ± se for 15-week-old male mice (n = 8-10) fed a normal diet.
There were no significant differences in blood GH, glucagon and albumin concentrations in KRAP + / + and KRAP − / − mice fed a normal diet. This suggests that the growth delay observed in KRAP − / − mice is not caused by abnormal GH secretion. KRAP − / − mice also showed mildly reduced T3 concentrations. This suggests that the increased energy metabolism rate seen in KRAP − / − mice is not due to hyperthyroidism.

13 Comparison of tissue gene expression Total RNA was extracted from the liver, skeletal muscle, epididymal white fat and interscapular brown adipose tissue of KRAP + / + and KRAP − / − mice according to a conventional method and subjected to Northern blotting. . As a probe, a cDNA fragment obtained by RT-PCR using mouse liver total RNA or mouse epididymal white fat total RNA as a template and the following primers was used.

  In the table, ACC1 = acetyl-CoA carboxylase 1; ACC2 = acetyl-CoA carboxylase 2); ACO = acyl-CoA oxidase; UCP2 = uncoupled protein 2; β-actin = β-actin; LPL = lipoprotein lipase; HSL = PPARγ = peroxisome proliferator-activated receptor γ; Leptin = leptin; aP2 = adipocyte fatty acid binding protein; 36B4 = acidic ribosome phosphorylated protein P0; UCP1 = uncoupling protein 1; PGC1α = PPARγ-coacty Represents beta 1α.

  The results are shown in FIG. The expression levels of acetyl-CoA carboxylase (ACC) 1 and ACC2 in the liver of KRAP − / − mice fed a normal diet were lower than that of KRAP + / +. Acyl-CoA oxidase (ACO), uncoupled protein (UCP) 2 and internal standard β-actin in the liver were not different. Genes related to lipid metabolism and energy metabolism in skeletal muscle, epididymal white fat and interscapular brown adipose tissue (lipoprotein lipase (LPL); hormone sensitive lipase (HSL); peroxisome proliferator activated receptor γ (PPARγ)) ; PPARγ-coactivator 1α (PGC1α)) showed no remarkable abnormality as far as it was examined.

14 Measurement of Spontaneous Momentum Spontaneous momentum is measured by Digital Acquisition System (trade name; Bioresearch Center Co., Ltd., device model number: NS-DAS-8-A) and a spontaneous motion sensor for experimental animals (trade name). Measurement was performed using Neuroscience, apparatus model number: NS-AS01). During the test, mice were allowed to freely feed (normal food) and water. The sensor counts 1 count (moving action) when there is a movement change of 0.5 seconds or more.

  The results are shown in FIG. Compared with KRAP + / + mice, KRAP − / − mice showed a significant increase in momentum during the night (dark period). In daytime (light period), no significant difference was observed, but the same tendency was observed. The results of energy metabolism obtained show that the energy metabolism rate is equally enhanced in KRAP − / − mice regardless of day or night, which does not completely match the momentum curve. Therefore, the energy consumption is not increased due to abnormal movement, and the obtained tendency to increase the amount of exercise is that KRAP − / − mice are exercising more rapidly than KRAP + / + mice because they are underweight, Is suggested. In addition, no behavior suggestive of marked neurobehavioral abnormalities was observed.

15. Liver tissue triglyceride content when fed a high fat diet (19 weeks old) Male KRAP + / + (n = 7) and KRAP − / − (n = 6) mice were fed a high fat diet sample (“ After giving High Fat Diet 32 "(described above); CLEA Japan) for 8 weeks, the liver was excised and the tissue triglyceride content was measured in the same manner as in" 6. Measurement of tissue triglyceride and glycogen content ".

  The results are shown in FIG. As shown in the photograph of the overview of the liver, the KRAP + / + mice showed an image of fatty liver, but the KRAP − / − mice maintained a healthy state. From the measurement results of liver triglyceride content, no triglyceride accumulation was observed in the liver of KRAP − / − mice. This result is consistent with the fact that KRAP-/-mice show an increased energy metabolism rate, the epididymal white adipose tissue weight is low, and the expression levels of ACC1 and ACC2 which are adipose synthesis-related genes in the liver are low. From this result, it is clear that the KRAP gene mutation has an effect of preventing obesity caused by diet.

  This application is based on Japanese Patent Application No. 2005-210744 filed on July 21, 2005, and the contents described in the specification and claims of Japanese Patent Application No. 2005-210744 are all Included in the application specification.

Claims (18)

  A transgenic non-human animal with reduced or absent expression of the KRAP gene.   The transgenic non-human animal according to claim 1, comprising a KRAP gene disrupted by homologous recombination.   The transgenic non-human animal according to claim 1, which has a modified lipid metabolism and / or sugar metabolism.   The transgenic non-human animal according to claim 1, wherein the transgenic non-human animal exhibits increased energy consumption, increased insulin sensitivity, and / or abnormal blood hormone levels.   An isolated mutant KRAP gene nucleic acid molecule capable of homologous recombination with a KRAP gene in the genome and capable of reducing or eliminating expression of the KRAP gene in non-human animal cells.   6. The nucleic acid molecule according to claim 5, wherein the mutation is a deletion of at least one exon.   The nucleic acid molecule according to claim 6, wherein the mutation is a deletion of a region containing at least one of exons 11 to 16.   A vector comprising the nucleic acid molecule according to any one of claims 5 to 7.   The vector according to claim 8, which is an expression vector for producing a transgenic animal or gene therapy.   A host cell into which the vector according to claim 8 or 9 has been introduced.   An isolated interfering nucleic acid molecule having a sequence that is substantially identical or substantially complementary to at least a portion of a KRAP gene or mRNA, and that can inhibit expression of the KRAP gene in a cell, tissue or individual.   12. The interfering nucleic acid molecule according to claim 11, wherein at least a part is double stranded.   A binding protein that specifically binds to a KRAP protein or a part thereof and can inhibit the function of the KRAP protein in a cell, tissue or individual.   A lipid metabolism and / or sugar metabolism-modifying agent comprising the interfering nucleic acid molecule or binding protein according to any one of claims 11 to 13.   The lipid metabolism and / or sugar metabolism-modifying agent according to claim 14, which is an energy consumption enhancer, an obesity prevention or elimination agent, or an insulin sensitivity enhancer.   A method of inducing alteration of lipid metabolism and / or sugar metabolism in a mammalian cell, tissue or non-human animal by inhibiting the expression or function of a KRAP gene or protein.   The method according to claim 16, wherein the alteration of lipid metabolism and / or sugar metabolism is increased energy consumption, prevention or elimination of obesity, increased insulin sensitivity, and / or abnormal blood hormone levels.   An interfering nucleic acid molecule having a sequence that is substantially identical or substantially complementary to at least a portion of a KRAP gene or mRNA and that can inhibit expression of the KRAP gene in a cell, tissue or individual, and / or a KRAP protein or Inhibiting the expression of the KRAP gene or the function of the KRAP protein using a binding protein that specifically binds to a part thereof and can inhibit the function of the KRAP protein in a cell, tissue or individual. A method according to ranges 16 or 17.

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