WO2007010999A1 - Animal in which lipid metabolism and sugar metabolism are modified by krap gene mutation, modification method and modifying agent - Google Patents

Abstract

It is intended to provide a novel agent which can contribute to prevention and/or treatment of obesity and/or diabetes, a transgenic animal which can be used for the purpose of elucidating pathologic conditions of such as obesity and/or diabetes or the like, a nucleic acid molecule which can be used for the production thereof and the like. One aspect of the invention is a transgenic nonhuman animal in which the expression of KRAP gene is reduced or lost.

Description

 Specification

 Lipid and sugar metabolism-modified animals by KRAP gene mutation, modification methods and modification agents

 Technical field

 [0001] The present invention relates to a nucleic acid molecule capable of inducing an increase in energy consumption, an increase in insulin sensitivity, prevention or elimination of fattening, and an abnormality in Z or blood hormone concentration in an animal, and a drug containing the same The invention relates to a transgene animal containing such a nucleic acid molecule, increased energy consumption, increased insulin sensitivity, prevention or elimination of obesity, and a method for inducing abnormalities in Z or blood hormone levels.

 Background art

[0002] To date, several genes are known to be involved in obesity in mammals! By altering these genes positively or negatively, animals exhibiting anti-obesity or obesity have been produced. The target gene such modifications, for example, leptin can function systemically as endocrine signaling factor (Leptin), FGF19, neuropeptide, Y (neu ropeptide Y), Anne ³ r Ohoeten communicating growth factor (Angiopoietin- related growth factor) and transcription factors PGC1 α (ΡΡΑ R gamma coactivator 1 α), CBP (CREB binding protein) ゝ SRC—1, TIF2, RXR (retinoid X receptor) a), intracellular lipid metabolism-related enzymes ACC2 (acetyl CoA carboxylase2), SCDKstealoyl CoA desaturasel), intracellular signal regulators SHIP2, PTP1B, S6 kinase, SOCS-6, mitochondrial function protein UCP (uncoupling protein) One that codes for the species. Many of these proteins do not function well or are multifunctional (those that differ depending on the cell in which they are expressed, or that act differently depending on the interacting protein). 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's situation. It is known to show metabolic changes. In addition, transgenic mice with enhanced expression of FGF19 and FGF19-administered mice (FGF is a humoral factor that acts remotely in blood. However, the anti-obesity effect by FGP19 is shown, and it is attracting attention from the viewpoint of obesity / diabetes treatment (Non-Patent Documents 1 to 31).

 [0003] The KRAP (Ki-ras-induced actin- interacting protein) gene has recently been identified as one of the cancer-related genes up-regulated by Ki-ras that has been activated (non-patent literature) 32). The protein encoded by this gene had a coiled coil region, but its physiological expression site and function were unknown.

 [0004] Non-Patent Document 1: Sleeman MW, Wortley KE, Lai KM, Gowen LC, Kintner J, Kline WO,

 Garcia K, Stitt TN, Yancopoulos GD, Wiegand SJ, Glass DJ. Absence of the lipid p hosphatase SHIP2 confers resistance to dietary obesity. Nat Med. 2005 Feb; 11 (2): 1 99-205.

 Non-Patent Document 2: Oh W, Abu-Elheiga L, Kordari P, Gu Z, Shaikenov T, Chirala SS, Wa kil SJ. Glucose and fat metabolism in adipose tissue of acetyl— CoA carboxylase 2 kn ockout mice. Proc Natl Acad Sci US A. 2005 Feb 1; 102 (5): 1384— 9.

 Non-Patent Document 3: Abu- Elheiga L, Oh W, Kordari P, Wakil SJ. Acetyl-CoA carboxylase

2 mutant mice are protected against obesity and diabetes induced by high-fat / high -carbohydrate diets.Proc Natl Acad Sci US A. 2003 Sep 2; 100 (18): 10207— 12. Non-Patent Document 4: Jiang G, Li Z , Liu F, Ellsworth K, Dallas-Yang Q, Wu M, Ronan J, Es au C, Murphy C, Szalkowski D, Bergeron R, Doebber T, Zhang BB. Prevention of o besity in mice by antisense oligonucleotide inhibitors of stearoyl— CoA desaturase— 1.

J Clin Invest. 2005 Apr; 115 (4): 1030— 8.

 Non-Patent Document 5: Dobrzyn A, Ntambi JM. The role of stearoyl-CoA desaturase in body weight regulation.Trends Cardiovasc Med. 2004 Feb 4 (2): 77- 81. Review.

 Non-Patent Document 6: Abu- Elheiga L, Matzuk MM, Abo- Hashema KA, Wakil SJ. Continuo us fatty acid oxidation and reduced fat storage in mice lac ing acetyl— CoA carboxyla se 2. Science. 2001 Mar 30; 291 (5513 ): 2613— 6.

Non-Patent Document 7: Yamauchi T, Oike Y, Kamon J, Waki H, Komeda K, Tsuchida A, Date Y, Li MX, Miki H, Akanuma Y, Nagai R, Kimura S, Saheki T, Nakazato M, Naitoh T, Yamamura K, Kadowa i T. Increased insulin sensitivity despite lipodystrophy in C rebbp heterozygous mice. Nat Genet. 2002 Feb; 30 (2): 221— 6

Non-Patent Document 8: Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, Fu magalli S, Allegrini PR, Kozma SC, Auwerx J, Thomas G. Absence of S6K1 protects against age— and diet-induced obesity while enhancing insulin sensitivity. Nature. 2004 Sep 9; 431 (7005): 200-5. Epub 2004 Aug 11. Erratum in: Nature. 2004 Sep 23; 4 31 (7007): 485.

Non-Patent Document 9: Fu L, John LM, Adams SH, Yu XX, Tomlinson E, Renz M, Williams PM, Soriano R, Corpuz R, Moffat B, Vandlen R, Simmons L, Foster J, Stephan JP, Ts ai SP , Stewart TA. Fibroblast growth factor 19 increases metabolic rate and reverse s dietary and leptin- deficient diabetes. Endocrinology. 2004 Jun; 145 (6): 2594-603. Non-patent literature 10: Tomlinson E, Fu L, John L, Hultgren B, Huang X, Renz M, Stephan JP, Tsai SP, Powell-Braxton L, French D, Stewart TA.Transgenic mice expressing human fibroolast growth factor— 19 display increased metabolic rate and decreased a diposity. Endocrinology. 2002 May; 143 ( 5): 1741-7.

Non-Patent Document 11: Molero JC, Jensen TE, Withers PC, Couzens M, Herzog H, Thien CB, Langdon WY, Walder K, Murphy MA, Bowtell DD, James DE, Cooney GJ.c- Cb 1— deficient mice have reduced adiposity, higher energy expenditure, and improved p eripheral insulin action.J Clin Invest. 2004 Nov; l 14 (9): 1326-33.

Non-Patent Document 12: Lin J, Wu PH, Tarr PT, Lindenberg KS, St-Pierre J, Zhang CY, Moo tha VK, Jager S, Vianna CR, Reznick RM, Cui L, Manieri M, Donovan MX, Wu Z, Cooper MP, Fan MC, Rohas LM, Zavacki AM, Cinti S, Shulman GI, Lowell BB, Krain c D, Spiegelman BM. Defects in adaptive energy metabolism with CNS— linked hyper activity in PGC— lalpha null mice. Cell. 2004 Oct 1; 119 (1): 121— 35.

Non-Patent Document 13: Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajim a Y, Nakayama O, Makishima M, Matsuda M, Shimomura I. Increased oxidative stre ss in obesity and its impact on metabolic syndrome. Clin Invest. 2004 Dec; 114 (12): 1752-61.

Non-Patent Document 14: Krebs DL, Uren RT, Metcalf D, Rakar S, Zhang JG, Starr R, De Sou za DP, Hanzinikolas K, Eyles J, Connolly LM, Simpson RJ, Nicola NA, Nicholson SE, Baca M, Hilton DJ, Alexander WS.SOCS-6 binds to insulin receptor substrate 4, and mice lacking the SOCS-6 gene exhibit mild growth retardation. Mol Cell Biol. 2 002 Jul; 22 (13): 4567-78.

Non-Patent Document 15: Leone TC, Lehman JJ, Finck BN, Schaeffer PJ, Wende AR, Boudina S, Courtois M, Wozniak DF, Sambandam N, Bernal-Mizrachi C, Chen Z, Holloszy JO, Medeiros DM, Schmidt RE, Saffitz JE, Abel ED, Semenkovich CF, Kelly DP.PG C-1 alpha denciency causes multi-system energy metabolic derangements: muscle dy sfunction, abnormal weight control and hepatic steatosis.PLoS Biol. 2005 Apr; 3 (4): elOl.

Non-Patent Document 16: Tsukiyama- Kohara K, Poulin F, Kohara M, DeMaria CT, Cheng A, Wu Z, Gingras A, Katsume A, Elchebly M, Spiegelman BM, Harper ME, Tremolay ML, Sonenberg N. Adipose tissue reduction in mice lacking the translational inhibit or 4E-BP1. Nat Med. 2001 Oct; 7 (10): 1128-32.

Non-Patent Document 17: Cohen P, Miyazaki M, Socci ND, Hagge— Greenberg A, Liedtke W, Soukas AA, bharma R, Hudgins LC, Ntambi JM, Friedman JM. Role for stearoyl-Co A desaturase- 丄 m leptin- mediated weight loss. Science. 2002 Jul 12; 297 (5579): 240 -3.

Non-Patent Document 18: Ntambi JM, Miyazaki M, Stoehr JP, Lan H, Kendziorski CM, Yandell BS, Song Y, Cohen P, Friedman JM, Attie AD. Loss of stearoyl-CoA desaturase— 1 lunction protects mice against adiposity. Proc Natl Acad Sci US A. 2002 Aug 20; 9

9 (17): 11482-6.

Non-Patent Document 19: Cline GW, Vidal-Puig AJ, Dufour S, Cadman KS, Lowell BB, Shulma n GI.In vivo effects of uncoupling protein— 3 gene disruption on mitochondrial energ y metabolism. J Biol Chem. 2001 Jun 8; 276 (23): 20240-4.

Non-Patent Document 20: Picard F, Gehin M, Annicotte J, Rocchi S, Champy MF, O'Malley BW, Chambon P, Auwerx J. SRC— 1 and TIF2 control energy balance between white a nd brown adipose tissues. Cell. 2002 Dec 27; 111 (7): 931— 41. Non-Patent Document 21: Ohki- Hamazaki H, Watase K, Yamamoto K, Ogura H, Yamano M, Y amada K, Maeno H, Imaki J, Kikuyama S, Wada E, Wada K. Mice lacking bombesin receptor subtype— 3 develop metabolic defects and obesity. Nature. 1997 Nov 13; 39 0 (6656): 165-9.

Non-Patent Document 22: Kushi A, Sasai H, Koizumi H, Takeda N, Yokoyama M, Nakamura M. Obesity and mild hyperinsulinemia found in neuropeptide Y-Yl receptor-deficient mice.Proc Natl Acad Sci US A. 1998 Dec 22; 95 (26): 15659-64.

Non-Patent Document 23: Imai T, Jiang M, Shi hambon P, Metzger D. Impaired adipogenesis and lipolysis in the mouse upon selective ablation of the retinoid X receptor alpha media ted by a tamoxifen-inducible chimeric Cre recombinase (Cre- ERT2) in adipocytes. Proc Natl Acad Sci US A. 2001 Jan 2; 98 (1): 224— 8.

Non-Patent Document 24: Dube MG, Beretta E, Dhillon H, Ueno N, Kalra PS, Kalra SP. Cent ral leptin gene therapy blocks high-fat diet-induced weight gain, hyperleptinemia, an d hyperinsulinemia: increase in serum ghrelin levels. Diabetes. 2002 Jun; 51 (6): 1729 -36.

Non-Patent Document 25: Zhou Z, Yon Toh S, Chen Z, Guo K, Ng CP, Ponniah S, Lin SC, Hong W, Li P. Cidea— deficient mice have lean phenotype and are resistant to obesity. Nat Genet. 2003 Sep; 35 (l): 49-56.

Non-Patent Document 26: Oike Y, Akao M, Yasunaga K, Yamauchi T, Morisada T, Ito Y, Uran o T, Kimura Y, Kubota Y, Maekawa H, Miyamoto T, Miyata K, Matsumoto S, Sakai J, Nakagata N , Takeya M, Koseki H, Ogawa Y, Kadowaki T, Suda T. Angiopoietin— related growth factor antagonizes obesity and insulin resistance. Nat Med. 2005 Ap r; ll (4): 400-8.

Non-Patent Document 27: Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Defective secretion of saliva in transgenic mice lacking aquaporin— 5 water channels. J Biol Chem. 1999 Jul 16; 274 (29) : 20071-4

Non-Patent Document 28: Enerback S, Jacobsson A, Simpson EM, Guerra C, Yamashita H, Harper ME, Kozak LP. Mice lacking mitochondrial uncoupling protein are cold—sensitivity ve but not obese.Nature. 1997 May l; 387 (6628): 90-4.

 Non-Patent Document 29: Tomlinson E, Fu L, John L, Hultgren B, Huang X, Renz M, Stephan JP, Tsai SP, Powell-Braxton L, French D, Stewart TA. Transgenic mice expressing human fibroblast growth factor— 19 display increased metabolic rate and decreased a diposity. Endocrinology. 2002 May; 143 (5): 1741-7.

 Non-Patent Document 30: Fu L, John LM, Adams SH, Yu XX, Tomlinson E, Renz M, Williams PM, Soriano R, Corpuz R, Moffat B, Vandlen R, Simmons L, Foster J, Stephan JP, T sai SP , Stewart TA. Fibroblast growth factor 19 increases metabolic rate and revers es dietary and leptin-deficient diabetes. Endocrinology. 2004 Jun; 145 (6): 2594-603. Non-patent literature 31: Klaman LD, Boss〇, Peroni OD, Kim JK, Martino JL, Zabolotny JM, Moghal N, Lubkin M, Kim YB, Sharpe AH, Strieker- Krongrad A, Shulman GI, Neel BG, Kahn BB. Increased energy expenditure, decreased adiposity, and tissue— sped fic insulin sensitivity in protein — Tyrosine phosphatase IB-deficient mice. Mol Cell B iol. 2000 Aug; 20 (15): 5479-89.

 Special Article 32: Inokucni J, Komiya M, Baba I, Naito N, Sasazuki T, Shirasawa S. Deregulated expression of KRAP, a novel gene encoding actin— interacting protein, in human colon cancer cells. J Hum Genet (2004 49: 46-52.

 Disclosure of the invention

 Problems to be solved by the invention

 [0005] The present invention relates to a novel drug that can contribute to the prevention and Z or treatment of obesity and Z or diabetes, a transgenic animal that can be used for the purpose of elucidating the pathology of obesity and Z or diabetes, and the production thereof. The object is to provide nucleic acid molecules that can be used.

 Means for solving the problem

[0006] The present inventors have found that KRAP is distributed in large amounts in the liver and spleen due to physiological expression, and is also present in ubiquitous in many organs except for the heart and skeletal muscle. Furthermore, the present inventors have found that an animal in which the KRAP gene has been disrupted exhibits changes in lipid metabolism and sugar metabolism, resulting in an animal with a small amount of fat, thereby completing the present invention. The present invention

 [1] Transgenic non-human animal in which expression of KRAP gene is reduced or absent; [2] Transgenic non-human animal according to [1] above, which comprises KRAP gene disrupted by homologous recombination;

 [3] The transgenic non-human animal according to [1], which has a modified lipid metabolism and Z or sugar metabolism;

 [4] Transgenic non-human animal according to [1], which exhibits increased energy consumption, increased insulin sensitivity, and abnormal Z or 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 KR AP gene in non-human animal cells;

 [6] The nucleic acid molecule according to the above [5], wherein the mutation is a deletion of at least one etason; [7] the mutation is a deletion of a region containing at least one of the exons 11 to 16 [6] The described nucleic acid molecule;

 [8] A vector comprising the nucleic acid molecule according to any one of [5] to [7] above; [9] an expression vector for producing a transgenic animal or for gene therapy,

[8] The vector according to

 [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 identical 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 the above [11], wherein at least a part is double-stranded; [13] specifically binding to the KRAP protein or a part thereof; A binding protein that can inhibit the function of the KRAP protein;

 [14] A lipid metabolism and Z or sugar metabolism modifier comprising the interfering nucleic acid molecule or the binding protein according to any one of [11] to [13] above;

[15] The lipid metabolism and Z or sugar metabolism-modifying agent according to [14] above, 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 Z or sugar metabolism in mammalian cells, tissues or non-human animals by inhibiting the expression or function of KRAP gene or protein;

 [17] The method according to [16] above, wherein the alteration of lipid metabolism and Z or sugar metabolism is an increase in energy consumption, prevention or elimination of obesity, an increase in insulin sensitivity, and an abnormality in Z or blood hormone concentration ;

 (18) An interfering nucleic acid molecule 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, and Z or Including 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.

 The invention's effect

 [0008] According to the administration of the nucleic acid molecule or the like of the present invention or the method of the present invention, the lipid metabolism and sugar metabolism are artificially altered or energy consumption is artificially inhibited in animals by inhibiting the expression or function of the KRAP gene or protein. Can also lead to prevention or elimination of obesity and prevention or treatment of Z or 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, no adverse effects have been found due to inhibition of KRAP gene or protein expression or function.

[0009] The transgenic animal of the present invention is characterized in that lipid metabolism and sugar metabolism are modified, and in particular, as a result of increased energy consumption, the physical strength is small and the amount of fat is small compared to the wild type. Have Therefore, it is useful as a model animal in studies on body weight control and obesity mechanisms, glucose metabolism, etc., for example, studies for elucidation of pathological conditions such as obesity and diabetes.

 Brief Description of Drawings

[0010] [Figure 1] shows a comparison of the amino acid sequences of the KRAP gene products of human (upper) and mouse (lower). FIG. Thick, underlined partial force S coiled-coil motif.

 [Fig. 2] is a diagram showing targeted disruption of the mouse KRAP gene. Panel (A): A region of the mouse KRAP gene (Wild-type locus) including exons 8 to 17 is schematically shown along with related restriction enzyme sites. Panel): 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 the mutant allele predicted to be generated by homologous recombination (Targeted locus). Panel (D) left: A photo of a Southern plot using tail DNA digested with EcoRV. Middle: Northern 'plot photo showing specific deletion of KRAP mRNA in mutant (one Z-) liver compared to wild-type (+ Z +). Below is the -actin. Right: Western blot photo showing specific deletion of KRAP protein in mutant (one Ζ—) liver compared to wild-type (+ Ζ +). Below is j8-tubulin.

 [Fig. 3] is a graph showing growth curves (vertical axis: body weight, horizontal axis: age) for wild type (Wt), heterozygote (Ht), and KRAP knockout mice (Ko) (vertical axis: body weight, horizontal axis: age). Average person se; η = 5-14Z group). Left: male, right: female.

 FIG. 4 shows a comparison of organ weights for wild type (Wt) and KRAP knockout mice (Ko). The average for the 20-week-old male (n = 9), the vertical axis is the percentage of the tissue weight Z body weight. WAT = white adipose tissue, liver = liver, BAT = brown adipose tissue, kidney = kidney, heart = heart.

 [Fig. 5] shows a wild-type (Wt) and KRAP knockout mouse (Ko) prepared after fasting a 22-week-old male fed a normal diet for 16 hours. It is a photograph showing an HE stained image. WAT = white adipose tissue, BAT = brown adipose tissue, Liver = liver. Ske Noreno is 50 πι.

 FIG. 6 shows the relative amounts of liver triglycerides and glycogen in wild-type (Wt) and KRAP knockout mice (Ko) in the fed state. Left: Tridari cerido, right: Glycogen.

[Figure 7] shows a comparison of food intake per mouse (left) or normalized by body weight (right) (KRAP + Z + (n = 7), KRAP-Z— (n = 9)). The value is the average person se. Asterisk Indicates a significant difference of P <0.05, and “NS” indicates no significant difference compared to wild-type mice.

 [FIG. 8] shows the daily energy consumption profile (panel (A)) and respiratory quotient (RQ) (panel (B)) for a 22-week-old male mouse under normal diet. . The value is the average person se (KRAP + Z + (n = 7), KRAP− Z− (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. 16-week-old male mice maintained on a normal diet were used. KRAP + / + (n = 10), KRAP-/-(n = 10) (average person se). The 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 injection rate 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 KR AP + Z— (hatched column) mice under feeding.

[Fig. 12] shows blood ketone body levels of KRAP + Z + (black column) and KRAP-/-(white column) mice under fed (fed) and fasted for 15 hours (15h fast). is there. The vertical axis shows 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): Weight gain in 4 weeks, Panel (C): Graph showing weight gain in 4 weeks divided by weight at start, Panel (D): WAT Weight division display of weight. An asterisk indicates a significant difference (*: P <0.001; **: P <0.01).

 [FIG. 14] is a diagram showing the amount of intake during a period when a high fat diet was given. Left: food intake per day (g), right: 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 regarding the expression of several genes in silk and weave. Tissue: Liver = liver, Skeletal muscle = skeletal muscle, WAT = white Adipose tissue, BAT = brown adipose tissue; animal: W = wild type (+ Z +), K = KRAP knockout mouse (Z); gene: ACC1 = acetylene Co A carboxylase 1, ACC2 = acetylenore CoA canoleboxylase 2, ACO = Basil-Co A oxidase, UCP2 = uncoupled protein 2, β-Actin = j8 actin, LPL = lipoprotein lipase, HSL = hormone-sensitive lipase, PPAR y = peroxisome proliferator activated receptor γ, Leptin = Leptin, aP2 = Adipocyte fatty acid binding protein, 36B4 = acid ribosome phosphorylated protein P0, UCP1 = uncoupled protein 1, PGC1 a = PPAR y coactivator. 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 movements counted, and the horizontal axis represents time and light / dark conditions. Panel): Shows the total number of counts under rdarkj and “light” 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): A graph showing tissue triglyceride content.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0011] In the present invention, the "KRAP gene" includes all homologs and alleles that encode KRAP that can be expressed as a polypeptide. Such genes are known at least for mice and humans (eg, Figure 1), and the desired cDNA library can be obtained under stringent conditions using probes designed based on such known sequences. It can be identified by screening. “Stringent conditions” means, for example, 50% formamide, 120 mM Na HPO, 7% SDS, ImM EDTA, 250 mM N

 twenty four

 Hybridization conditions at 42 ° C in aCl solution and equivalent stringency conditions.

[0012] 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 NO: 2 and 3) or Genb 60% or more in homology search using NCBI (National Center for Biotechnology Information) B LAST search against the full length of human cDNA (mRNA) registered as ank registration number AB116937 (SEQ ID NOs: 4 and 5) Nucleotide identity or 50% or more amino acid identity, preferably 80% or more nucleotide identity or 70% or more amino acid identity, more preferably 90% or more nucleotide identity. Those having 80% or more amino acid identity are included in the gene encoding the “KRAP” protein in the context of the present invention.

 [0013] "Wild-type" KRAP gene refers to the above-mentioned mouse or human KRAP gene registered in Genbank among these KRAP genes, or a natural type having expression / function substantially equivalent to them. K The KRAP gene.

 [0014] A "mutant" KRAP gene is quantitatively characterized by a partial modification of the nucleotide sequence compared to the KRAP gene (particularly a natural or wild-type gene) obtained as described above. Or any KRAP gene that results in qualitatively different KRAP gene expression.

 [0015] In the present invention, homologous recombination or identity that allows homologous recombination with the KRAP gene in the genome is achieved, and the expression of the KRAP gene is reduced or reduced in non-human animal cells. A mutant KRAP gene that can be deleted is used. Such mutant KRAP genes include, but are not limited to, for example, those having at least one etason deficiency and those having a mutation causing a frame shift. Examples of the deletion include those in which the mutation is present in the half region on the C-terminal side of the KRAP gene, and more specifically, a deletion in a region containing at least one of exons 11-16. A region containing all of ˜16 can be missing. Alternatively, the deletion can be, for example, about 5 to 95%, 20 to 80%, or 35 to 70% of the coding region. The loss of the coding region can be widespread as long as the coding region and the Z or non-coding region to the extent that homologous recombination is sufficient as long as it causes destruction of the KRAP function remain.

[0016] By inserting such a mutant KRAP gene nucleic acid molecule into an appropriate vector, a vector for gene transfer, such as 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).

[0017] 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.

 [0018] In the transgenic animal of the present invention, it is determined 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 the production. It can be specified by confirming. In addition, lipid metabolism (especially increased energy consumption), glucose metabolism (especially increased insulin sensitivity), and fluctuations in various blood hormone levels related to these are confirmed using, for example, the assembly method described later in the Examples. By doing so, the transgenic animal of the present invention can be identified.

 [0019] 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. it can. It can also be used to elucidate the function of the KRAP gene itself.

[0020] According to the present invention, by using a vector containing a mutant KRAP gene as described above, ethas vivo gene therapy is also possible. That is, in general gene therapy, a functional gene is introduced in vitro to cells derived from a treatment target containing a defective gene, and the genetically manipulated cells are returned to the treatment target. However, in the present invention, the step of introducing the mutant KRAP gene as described above in vitro into a cell derived from a treatment target containing a functional KRAP gene, and returning the genetically engineered cell to the treatment target. Process. 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 herpes viruses are used, and their production methods are well known to those skilled in the art. [0021] The present invention involves inhibiting the expression or function of the KRAP gene or protein.

A method of inducing alterations in lipid metabolism and Z or sugar metabolism in mammalian cells, tissues or non-human animals is provided. In order to inhibit the expression or function of the KRAP gene or protein, for example, an interfering nucleic acid molecule designed based on the nucleotide sequence of the KRAP gene (e.g., antisense RNA; a short-chain containing a double-stranded portion that can cause RNA interference) (For example, 18-200 bases) RNA, double-stranded nucleic acid containing DNA and RNA, siRNA, miRNA, etc., binding protein molecules that specifically bind to KRAP protein (where "protein" It is used as a term that also includes peptides; for example, polyclonal or monoclonal antibodies, various fragments of antibodies, humanized antibodies, etc.) can be used. 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.

[0022] These interfering nucleic acid molecules and Z or binding protein molecules can be used alone as lipid metabolism and Z or sugar metabolism modifiers, for example, energy consumption enhancers, obesity prevention or elimination agents, or insulin sensitivity enhancers. In combination, or in combination with an appropriate carrier that is physiologically acceptable, and administered to animals, 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, the individual's body weight, age, sex, general health condition, etc., but a person skilled in the art can appropriately determine the appropriate animal by selecting and experimenting with an appropriate animal. Can be determined.

 Example

 [0023] 1. Construction of KRAP targeting vector

The construction of the targeting vector is described with reference to FIG. From the 129ZSV mouse genomic library (Stratagene), the 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 Xhol-Sail fragment was prepared from the isolated genomic DNA and inserted into the Sail site of pBluescript SK as a 3'arm. Second, an Xbal EcoRI fragment was prepared from the isolated genomic DNA and placed as an arm into the Xbal EcoRI site. . Third, the pGKneo cassette was inserted into the EcoRI site. Finally, the diphtheria toxin A fragment cassette was inserted into the Sail site with Xhol—Sal I. In this way, as shown in the panels (A) and (B) of FIG. 2, a 7.2 kb EcoRI-Xhol fragment containing the KRAP gene exons 11-16 (55% of the coding region) (in the sequence listing). A targeting vector was constructed by replacing the base numbers 20 598 to 28867 in SEQ ID NO: 1 with a pGKneo cassette having the opposite transcription direction, and the targeting constructs 5 and 3, and arms were 2.3 kb (each) It consisted of genomic DNA of base numbers 18288 to 20597 in SEQ ID NO: 1 in the sequence table and 6.4 kb (base numbers 27876 to 34348 in SEQ ID NO: 1 in the sequence list), 3 diphtheria toxin A fragment cassette (DTA-A) 'Adjacent to the genomic arm, this target vector was linearized with Sail.

 [0024] 2. Production of KRAP knockout mice

 The above-described linear 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 C57BLZ6 mouse, and the resulting male chimera was crossed with a C57BLZ6 mouse. Heterozygous (KRAP + Z −; sometimes abbreviated as “Ht”) mice were mated to obtain KRAP knockout (KRAP—Z—; abbreviated as “Ko”) mice.

 [0025] The observed genotype ratio of the offspring was consistent with the expected Mendelian ratio. Therefore, it was shown that embryogenesis was unaffected by the disruption of RAP gene. KR AP (—Z—) mice were addictive.

 [0026] Genomic DNA extracted from the tail of the animal was digested with EcoRV, and an external probe (350 bp Xbal fragment, base numbers 15786 to 16140 in SEQ ID NO: 1 in the sequence listing; Fig. 2, Panel (C) rprobe A) Hybridized). Wild type (KRAP + Z +; may be abbreviated as “Wt”) 21. Okb restriction fragment, mutant (—Z—) 8. Okb fragment, heterozygous (one Z +) both Fragments were detected (Figure 2, left panel of panel (D)).

[0027] Furthermore, homologous recombinants were confirmed using the neo probe, and it was confirmed that there was only one integration site. [0028] Total RNA was extracted from wild-type (+ Z +) and mutant (one Z) livers in accordance with a conventional method, and the base numbers 2962 to 3759 in SEQ ID NO: 1 in the sequence listing were used as probes. Blotting was performed (Fig. 2 (D) center). In KRAP (Z—), KRAP mRNA was specifically deleted (j8-actin used as an internal control showed equivalent expression).

 [0029] Wild type (+ Z +) and mutant (-Z-) liver lysates were prepared and Western 'blotted. Mouse tissues were sonicated in RIPA buffer [20 mM Tris—HC1, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, protease “inhibitor 1 cocktail” (Roche)]. This solution was centrifuged at 15, OOOrpm 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. This plot was blocked in blocking buffer (0.1% Tween 20, 5% non-fat 'skim milk, 50 mM Tris—HC1, 150 mM NaCl, 0.1% NaN, pH 7.5)

Three

 This was incubated overnight at 4 ° C with a purified anti-KRAP antibody (dilution ratio 1: 200,000). This was developed with HRP conjugated anti-rabbit IgG goat secondary antibody and ECL (Amersh am Pharmacia).

 [0030] 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 pGEX6P-1 vector 1 (Pharmacia) according to the instructions. This fusion protein was soluble in non-denaturing buffer and purified using dartathione-sepharose 4B (Amersham). This recombinant KRAP protein was injected into a rabbit and booster immunized to obtain antiserum. The antiserum was purified using a 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 (Z—), the KRAP protein was specifically deleted (β-tubulin used as an internal control showed equivalent expression).

 [0031] 3. Measurement of body weight change

Animals were maintained in a temperature controlled facility (23 ° C) with a 12 hour light / dark cycle. The mouse is a standard rodent feed (CE-2 (Japan Claire); And voluntary access to water. KRAP + obtained by mating heterozygous mice

Body weights were recorded weekly for Z +, + Z—, and Z— animals.

 [0032] The results are shown in FIG. The body weight of KRAP Z pups was significantly different from litter KRAP + Z + and + Z mice at least 5 days after birth. When fed with a normal diet, KRAP Z—mice gained less weight than both + Z + and + Z—mice. This difference occurred at 3 weeks of age for both males (Figure 3, left) and females (Figure 3, right).

 [0033] 4. Organ ¾ quantity 沏 constant

 Male KRAP + Z + (n = 9) and KRAP Z— (n =

9) The mice were euthanized and the wet weight of epididymal white adipose tissue (WAT), liver, scapular brown adipose tissue (BAT), kidney and heart were measured.

[0034] The results are shown in FIG. The relative weight of WAT and liver (g / g body) was lower in KRAP Z— mice than in KRAP + Z + mice. On the other hand, no significant differences were observed in BAT, kidney and heart.

 [0035] 5. mm rn

 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 norafine. Sections were deparaffinized, rehydrated, stained with hematoxylin 'eosin (H E) and observed with a light microscope.

 The results are shown in FIG. KRAP Z adipocytes (shown white) in HE-stained WAT and BAT were clearly reduced in size compared to KRAP + Z + adipocytes. KRAP Z—Lipid vacuoles were hardly observed in the liver staining of mice.

 [0037] 6. Measurement of tissue triglyceride and glycogen content

Folch et al. (Folch J, Lees M, Sloane Stanley GH "A simple method for the isolation and purification of total lipides from animal tissues, J Biol Chem. 1957 May; 226 (l): 497 -509) using the extraction protocol and enzymatic triglyceride test (Wako Pure Chemical Industries) It ’s hot. For measurement of spleen glycogen, spleen from the same mouse was homogenized in 3% (w / w) perchloric acid, and the homogenate was incubated with amyloglucosidase at 40 ° C for 2 hours to hydrolyze glycogen. The resulting glucose residues were then measured using an enzymatic glucose CII test (Wako Pure Chemical Industries).

 [0038] The results are shown in FIG. Morphological observations suggested that the triglyceride content was low in the liver of KRAP—Z—, but in fact, according to the extraction and measurement of liver tissue triglycerides, the liver of KRAP — / — It contained 40% less! / And triglyceride than the + liver. On the other hand, there was no significant difference between the two genotypes in liver glycogen content.

 [0039] 7. 沏 I constant

 22 week old male mice 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. At the end of the 24-hour period, the remaining food including food waste was weighed and the amount of food consumed per day (g / day / kg-body w eight) was calculated.

 The results are shown in Table 1 and Fig. 7.

[0040] [Table 1]

Size

 Q. CO

 o

0001 <096 .. 2 5902 50174 Soil ....

 (d / k / bt) aggyw.

 19 0 24410930175422525 Soil .....

 1 110150081 Soil ...

 Fecal consumption food amount 02 02002003 Soil ...

掣 WS set _≥3Ik Ή

ιΙπιΙ

 Ipni 瞷 瞓

+ Mouse body weight was significantly heavier than KRAP—Z—mouse (P <0. 001). Food intake was significantly higher in KRAP- Z- mice than in KRAP + Z + mice when food consumption was adjusted for body weight. These results indicate that the delay in weight gain in KRAP-Z-mouses is not due to food intake.

[0042] 8. Study of the first generation of Ene Noregi

 For simultaneous measurement of energy metabolism, male wild-type mice (n = 7) and KRAP—Z— (n = 9) mice matched in age (22 weeks of age) are individually placed in a metabolic cage for 7 days. This (Ichikawa M, Fujita Y, tiifects of nitrogen and energy metabolism on body weight ι n later life of male Wistar rats consuming a constant amount of food, J Nutr. 1987 0 ct; l 17 (10): 1751-8; Ichikawa M, Kanai S, Ichimaru Y, Funakoshi A, Miyasaka K., The diurnal rhythm of energy expenditure differs between obese and glucose-intolerant rats and streptozotocin- induced diabetic rats, J Nutr. 2000 Oct; 130 (10): 2562- 7). Using an automatic O-CO analyzer (NEC Medical System, model IH26, Tokyo, Japan)

twenty two

 Oxygen consumption and carbon dioxide production during expiration were measured continuously. The energy consumption per hour and per day was calculated.

 The results are shown in FIG. The daily energy consumption is higher in KRAP- Z—mice than in K RAP + Z + mice (P 0.01), and in KRAP—Z—mice 2929. 3 ± 16 3.1 kj / kg body weight, In KRAP + Z + mice, it was 225.7.2 ± 152.2 kj / kg body w eight. The respiratory quotient (RQ) during metabolic measurements was not significantly different between the two genotypes (P = 0.37), 0. 90 ± 0.03 for KRAP + Z + mice, 0 for KRAP-Z- mice. 88 ± 0. 02. These results indicate that KRAP deficiency resulted in increased energy consumption. In KRAP-Z- mice, the source of energy used for combustion was normal, at least on the normal diet. Measurements of food intake and energy expenditure showed that metabolic turnover was clearly enhanced by KRAP deficiency in mice.

 [0044] 9. Intraperitoneal glucose tolerance test and insulin sensitivity test

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

[0045] The results are shown in FIGS. 9 and 10. At 60, 90 and 120 minutes after glucose administration, glucose levels in KRAP− / one mice were significantly lower compared to KRAP + / + mice (FIG. 9). In the insulin sensitivity test, insulin injection resulted in a significantly lower level of dalcose in KRAP- / mice compared to KRAP + / + mice (Fig. 10).

 [0046] These results show that KRAP- Z compared to KRAP + Z + mice maintained on a normal diet

 -Mice show more glucose tolerance and insulin sensitivity.

 [0047] to. ^ ^

 Blood was collected from the retroorbital sinus vein in the normal fed state. The blood was kept on ice until subjected to centrifuge separation (3,000 g, 15 minutes, 4 ° C), serum was collected and stored at -70 ° C until used for prayer. Serum insulin, lebutin, adiponectin and thyroxine (T4) concentrations were obtained using ELISA kits (SWbayagi Co., R & D systems, Otsuka pharmaceutical Co., Endocnnetech. 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.

[0048] [Table 2]

f fNFA) ldidid (Ehli Slfidtttt lil tnassosero a noneserey acrere cemee ogycrvseu,

 fdhene w o 卜

 o Dimensions 卜

 CM CN

 o τ- ο

 CL o Ο

06 325 158 0 0 1 1440 138

 o O 56969 7554 7 (/ dl) ±± Tellures loco mg ... 0 σ

 04700860 0405E) (/ lFA ±± NEmq ....

 Value average is Shihei se

 15th week

o 00 8 0054 794 254 925 (/ dl) Risely ±±± gm .....

 2 6721 67834 / dl) ± (± Letesuloko mg ...

 (| / Yo) ibu

 9 040032003 04FA ±± NE ....

 Value level is flat es

3 female regular meals 4 weeks old,

c c

15 weeks old male mice (n = 10) of the indicated genotype and 34 weeks old The average person se for female mice (n = 8-9),

 [Table 3]

 (1 £ for ρ! 1) l! l-_s} 7e uodu uatMUIXOJ> uodl eud bi st w00a! o> EJ3nn-

990ε (i 9 inch) ρou =

 ozz

[0051] V is the average of the average genotype of a 15-week-old male mouse (n = 10) and 34-week-old female mouse (n = 11) of the indicated genotype that were fed a normal diet.

[0052] Serum cholesterol in KRAP + / + and KRAP- Z— mice fed a normal diet Terol and free fatty acid levels were not significantly different. Serum triglyceride levels tended to be lower in KRAP Z mice, although there was no statistically significant difference. KRAP Z—Mice showed mild hypoglycemia and reduced circulating insulin levels.

 [0053] A ketone body is produced by decomposing fat as an energy source in a starved state. In KRA P Z—mice, a low value that is similar to the wild type under non-fasting indicates that there is no ketoacidosis (no abnormalities like starvation). In addition, since the ketone body concentration increased under fasting, it was suggested that lipolysis and conversion to NEFA strong ketone bodies were abnormal even in KRAP-/-mice.

 [0054] Since lebutin 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 Z—mice.

 [0055] 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. Their serum thyroxine (T4) levels were lower than KRAP + Z + mice. Moreover, the fact that ketone body formation in the fed state was comparable to the wild type supported K RAP Z—mice that there were no abnormalities, such as starvation, when KRAP was normalized to body weight. -/-Consistent with mice eating more than KRAP + / +.

 [0056] 11. Adjustment of body weight when fed a high fat diet

Male fat KRAP + Z + (n = 7) and KRAP Z— (n = 6) mice matched in age (23 weeks old) high-fat diet (“High Fat Diet 32”, crude fat) The content was about 32%, and the ratio of fat-derived calories to total calories was about 60%; These mice were raised on a normal diet until age 23 weeks. Body weight was measured every two days, and the weight of the remaining food including food waste was measured every two days, and the amount of food consumed per day (g / day / kg-body weight) was estimated. After the 4-week period on the high fat diet, these mice were subjected to the same energy metabolism measurements as described above. [0057] The results are shown in FIGS. Figure 13, Panel (A) is the mouse weight curve, Panel is the weight gain at 4 weeks, Panel (C) is a graph and panel with the weight gain at the start of 4 weeks divided by the 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 due to diet. It is clear that it has a preventive effect.

 [0058] 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 mouse of the present invention has a higher food intake compared to the wild type, but has less weight gain.

 [0059] 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.

 [0060] 12. ^ ^ (2)

 For the same serum sample used in “10. Clinical Biochemical Measurements” above, the serum levels of triodothyronine (T3), growth hormone (GH), and glucagon were determined using the RIA kit (Endocrinetech). O., Amersham Biosciences, anaihara researcn o .. Albumin concentration was measured by enzymatic assay (Wako Pure Chemical Industries).

 The results are shown in Table 4.

[0061] [Table 4]

Each measured value in Table 4 is expressed as mean average 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 + Z + and KRAP- Z— mice fed a normal diet. From this, KR The growth delay seen in AP-Z-mice may not be due to abnormal GH secretion. KRAP-Z-mice also showed mildly reduced T3 concentrations. This suggests that the increased energy metabolism rate observed in KRAP-Z-mice is not due to hyperthyroidism.

 [0063] 13. Comparison of tissue gene expression

 Total RNA was extracted from KRAP + Z + and KRAP- Z—mouse liver, skeletal muscle, epididymal white fat and interscapular brown adipose tissue according to a conventional method, and subjected to Northern 'plot. As a probe, mouse liver total RNA or mouse epididymal white fat total RNA was used as a cage, and a cDNA fragment obtained by RT-PCR using the following primers was used.

[0064] [Table 5]

In the table, ACC 1 = acetyl-CoA carboxylase 1; ACC2 = acetyl-CoA ruboxylase 2); ACO = facyl-Co A oxidase; UCP2 = anchoring protein 2; β-Actin = j8 actin; LPL = lipoprotein Lipase; HSL = Hormone Sensitive lipase; PPAR y = peroxisome proliferator-activated receptor γ; Leptin = lebutin; aP2 = adipocyte fatty acid binding protein; 36B4 = acidic ribosomal phosphate protein PO; UCPl = uncoupling protein 1; PGC1 a = PPAR y—coactivator represents 1 α.

 [0066] The results are shown in FIG. The expression levels of acetyl-CoA carboxylase (ACC) 1 and ACC2 in the liver of KRAP Z mice fed a normal diet were lower than those of KRAP + / +. In liver, acyl-CoA oxidase (ACO), anchoring protein (UCP) 2 and the internal standard | 8-actin did not differ significantly. Lipid metabolism-related genes, energy metabolism-related genes (lipoprotein lipase (LPL); hormone sensitive lipase (HSL); peroxisome proliferator-activated receptor γ (PPAR) in skeletal muscle, epididymal white fat and interscapular brown adipose tissue γ); PPAR γ coactivator 1 a (PGC1)) showed no noticeable abnormality as far as it was examined.

 [0067] 14. Job title

 Spontaneous momentum is measured individually after 24 hours in the digital acquisition system (trade name; Bioresearch Center Co., Ltd., model number: NS-DAS-8-A) and experimental animal spontaneous motion sensor (brand name: neuroscience) The measurement was performed using a device model number: NS-AS0 1). During the test, the mice were allowed to freely feed (ordinary food) and water. The sensor counts 1 (moving action) when there is a movement change of 0.5 seconds or more.

 [0068] The results are shown in FIG. Compared with KRAP + Z + mice, KRAP Z—mice are at night

 Significant increase in momentum was observed during the dark period. During the daytime (light period), no significant difference was observed, but the same trend was observed. The results of energy metabolism obtained show that KRAP-/-mice have an equally enhanced energy metabolism rate both day and night, which is not completely consistent with the momentum curve. Therefore, because of abnormal movements, energy consumption is increased! /, It should not be! /, And the resulting increase in the amount of exercise is simply more agile than KRAP + / + mice because KRAP Z mice are underweight. It is suggested that. In addition, no behavior suggestive of marked neurobehavioral abnormalities was observed.

[0069] 15. Liver and weave triglyceride content when fed a succulent diet Male fat KRAP + Z + (n = 7) and KRAP—Z— (n = 6) mice matched to age (19 weeks old) high fat diet sample (“High Fat Diet 32” (mentioned above); Claire Japan) After 8 weeks, the liver was removed and the tissue triglyceride content was measured in the same manner as in “6. Measurement of tissue triglyceride and glycogen content”.

[0070] The results are shown in FIG. As shown in the photograph of the liver overview, KRAP + Z + mice showed fat liver images, but KRAP- Z- mice maintained a healthy state. Liver Triglyceride content was also measured, and KRAP-Z-mouse liver showed no triglyceride accumulation. This result is consistent with the fact that KRAP-/-mice show an increased energy metabolism rate, the weight of epididymal white adipose tissue is small, and the expression level of ACC1 and ACC2 which are genes related to fat synthesis in the liver is low. From this result, it is clear that mutations in the K RAP gene have the effect of preventing obesity caused by diet.

 [0071] 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 encompassed in this application.

Claims

The scope of the claims

 [I] Transgenic non-human animals with reduced or absent KRAP gene expression.

 [2] The transgenic non-human animal according to claim 1, comprising a KRAP gene disrupted by homologous recombination.

 [3] The transgenic non-human animal according to claim 1, which has a modified lipid metabolism and Z or sugar metabolism.

 [4] The transgenic non-human animal according to claim 1, which exhibits increased energy consumption, increased insulin sensitivity, and abnormal Z or blood hormone levels.

 [5] An isolated mutant KRAP gene nucleic acid molecule that is capable of homologous recombination with the KRAP gene in the genome and that can reduce or eliminate expression of the KRAP gene in non-human animal cells.

 [6] The nucleic acid molecule according to claim 5, wherein the mutation is deficiency of at least one etason.

[7] 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.

 [8] A vector comprising the nucleic acid molecule according to any one of claims 5 to 7.

 [9] The vector according to claim 8, which is an expression vector for producing a transgenic animal or for gene therapy.

[10] A host cell into which the vector according to claim 8 or 9 has been introduced.

 [I I] It has a sequence that is substantially identical or substantially complementary to at least part of the KRAP gene or mRNA, and can inhibit the expression of the KRAP gene in cells, tissues, or individuals

An isolated interfering nucleic acid molecule.

[12] The interfering nucleic acid molecule according to claim 11, which is at least partially double-stranded.

[13] A binding protein that specifically binds to KRAP protein or a part thereof and can inhibit the function of KRAP protein in cells, tissues, or individuals.

[14] A lipid metabolism and Z or sugar metabolism-modifying agent comprising the interfering nucleic acid molecule or binding protein according to any one of claims 11 to 13.

[15] The lipid metabolism and Z 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.

[16] A method of inducing alterations in lipid metabolism and Z or sugar metabolism in mammalian cells, tissues or non-human animals by inhibiting the expression or function of the KRAP gene or protein.

 [17] The claim 16, wherein the alteration of lipid metabolism and Z or sugar metabolism is increased energy consumption, prevention or elimination of obesity, increased insulin sensitivity, and abnormal Z or blood hormone levels the method of.

 [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 expression of the KRAP gene in a cell, tissue or individual, and Z or A process of inhibiting the expression of the KRAP gene or the function of the KRAP protein 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 cells, tissues or individuals. 18. A method according to claim 16 or 17, comprising.