WO2002033063A1 - Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis - Google Patents
Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis Download PDFInfo
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- WO2002033063A1 WO2002033063A1 PCT/KR2001/001271 KR0101271W WO0233063A1 WO 2002033063 A1 WO2002033063 A1 WO 2002033063A1 KR 0101271 W KR0101271 W KR 0101271W WO 0233063 A1 WO0233063 A1 WO 0233063A1
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P3/06—Antihyperlipidemics
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- C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01041—Isocitrate dehydrogenase (NAD+) (1.1.1.41)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
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Definitions
- the present invention relates to an isocitrate dehydrogenase which catalyze the production of NADPH necessary for the biosynthesis of lipids, including fatty acids, squalene and cholesterol, and its use in the treatment of metabolic diseases, including obesity, hyperlipidemia and fatty liver. Also, the present invention relates to an isocitrate dehydrogenase gene, fused gene constructs containing the gene, transfectant cells harboring the genes in their genome, and transgenic animals capable of expressing isocitrate dehydrogenase continuously throughout their lifespan.
- isocitrate dehydrogenase catalyses the oxidative decarboxylation of citric acid into ⁇ -ketoglutarate with concurrent production of NADH or NADPH.
- isocitrate dehydrogenase isozymes can be separated into three classes according to their cofactors and locations in the cell: mitochondrial NAD + -dependent isocitrate dehydrogenase (hereinafter referred to as "IDH”) , mitochondrial NADP + -dependent isocitrate dehydrogenase (hereinafter referred to as "IDPm”) , and cytoplasmic NADP + -dependent isocitrate dehydrogenase (hereinafter referred to as "IDPc”) .
- IDH mitochondrial NAD + -dependent isocitrate dehydrogenase
- IDDPm mitochondrial NADP + -dependent isocitrate dehydrogenase
- IDPc cytoplasmic NADP + -dependent isocitrate dehydrogenase
- IDH has been assumed to play a major role in the oxidative decarboxylation of isocitrate in the tricarboxylic acid cycle (TCA) with concurrent production of ⁇ - ketoglutarate and NADH.
- TCA tricarboxylic acid cycle
- NADH is used for energy generation through the electron transfer system
- ⁇ - ketoglutarate is a metabolite used in the synthesis of amino acids such as glutamic acid, gluta ine, arginine, and proline, and other biological products.
- IDH activity is regulated as a control point of the TCA cycle. Therefore, IDH is a key enzyme to regulate not only the TCA cycle, but also energy metabolism, protein biosynthesis and nitrogen metabolism because metabolites of the TCA cycle take part in such metabolisms .
- IDH Since its isolation from yeast and pig, IDH has been under study.
- Yeast IDH is an allosterically regulated enzyme that exists as an octamer composed of two nonidentical subunits IDH1 and IDH2 sharing high homology with each other.
- IDH1 plays a role in the regulation of the enzyme activity while IDH2 is responsible for the catalytic activity (Keys, D. A. & McAlister-Henn, L., J. Bacteriol . , 172, 4280-4287, 1990) .
- swine IDH also exists as an octamer (2( ⁇ 2 ⁇ ⁇ ) ) in active form.
- IDPm and IDPc are, however, not known as to their functions. Although both having molecular weight of about 45 kDa with high homology, the two enzymes were identified as different, independent proteins, as analyzed by immunological reaction experiments using polyclonal antibodies (Plaut, G. W. E. et al . , Biochem. Biophys. Acta., 760, 300-308, 1983; Fantania, H. R. et al . , FEBS, 322, 245-248, 1993). Particularly, IDPm and IDPc are highly tissue-specific.
- isocitrate dehydrogenase isozymes have been characterized concerning some of their structural characteristics, but not concerning functions. Particularly, nowhere had been found studies on precise mechanisms of IDPm and IDPc until the publication of recent reports which merely made the assumption that IDPm catalyzes a reverse reaction in the TCA cycle to convert ⁇ -ketoglutarate through isocitrate to citrate, which is associated with a tricarboxylate carrier to supply acetyl-CoA, a precursor for the biosynthesis of fatty acids and cholesterol, with concurrent conversion of the citrate to oxaloacetate to raise cytoplasmic phosphoenolpyruvate levels, thereby promoting gluconeogenesis (Des Rosiers, C.
- IDPc is known to be expressed in large quantities in the ovary and the mammary gland.
- IDPc has been quantitatively analyzed to produce NADPH in greater quantities than do important enzymes of the pentose phosphate pathway; i.e., glucose-6-phosphate dehydrogenase for the conversion of glucose-6- phosphate to 6-phosphoglucono- ⁇ -lactone and NADPH, 6- phosphogluconate dehydrogenase for the conversion of 6-phosphogluconate to ribulose-5-phosphate and NADPH, and cytoplasmic malic enzyme for the conversion of malate to pyruvate and NADPH; by factors of 16, 8 and 18, respectively (Veech, R. L. et al . , Biochem. J. , 115, 609-619, 1969) .
- ⁇ - ketoacyl-ACP reductase and enoyl-ACP reductase require NADPH as a cofactor for their catalysis.
- NADPH NADPH
- a large quantity of NADPH is required for the reactions catalyzed by HMG-CoA reductase and squalene synthetase and for the final 19-step reaction from lanosterol to cholesterol.
- control of the activity of IDPc which functions to supply most of the NADPH required in the cell, is very important to regulate the biosynthesis of fatty acids and their derivatives, lipids, squalene, and cholesterol and its derivatives.
- lipid deposition follows the following procedure.
- excess energy sources are available, the differentiation of adipose cells is accelerated, resulting in an increase in the number and size of white adipose tissues with concomitant deposition of lipids .
- the white adipose tissue allows the ob gene to be actively expressed, which leads to an increase in body leptin level.
- the hormonal action in the brain is changed toward the decreasing of appetite. Meanwhile, excess calories are consumed to maintain the body temperature, using uncoupler proteins (UCP) .
- UCP uncoupler proteins
- adipose cell differentiation and lipid deposition are promoted and excess body energy is stored in lipid form, so that body energy is balanced (Hu, E. et al., Proc. Natl. Acad. Sci. USA, 92, 9856-9860, 1995; Keller, H. et al., Proc. Natl. Acad. Sci. USA, 20, 9856-9860, 1993; Freytag S.
- PPAR ⁇ peroxysome proliferator-activated receptor ⁇
- ligands necessary for the activation of PPAR ⁇ include polyunsaturated fatty acids such as linoleic acid, docosahexanoic acid (DHA) , and arachidonic acid (Krey, G. et al., Mol. Endocrinol . , 11, 779-791, 1997; Yu et al., J. Biol.
- prostaglandin J2 is known to serve as a ligand of the master transcription factor (Forman B. M. et al., Cell, 83, 803-812, 1995; Kliewer S. A. et al . , Cell, 83, 813-819, 1995).
- IDPc might be directly involved in controlling the biosynthesis of various fatty acids, cholesterol and hormones owing to its ability to produce NADPH.
- IDPc can be assumed to play a key role in obesity and fatty liver by encouraging the production of activating ligands for PPAR ⁇ , such as polyunsaturated fatty acids and arachidonic acid to trigger the cascade expression of various genes related to the differentiation of adipose cells.
- activating ligands for PPAR ⁇ such as polyunsaturated fatty acids and arachidonic acid to trigger the cascade expression of various genes related to the differentiation of adipose cells.
- NADPH for cholesterol biosynthesis offers the possibility that artificial control of intracellular levels of IDPc and its reaction product NADPH might provide a means of controlling cholesterol biosynthesis.
- Fig. 1 provides schematic diagrams showing structures of a basic LNCX-vector (top) , a recombinant vector into which an IDPc gene is inserted in the sense orientation to increase the expression of the IDPc gene in NIH3T3 F442A adipocytes (middle), and a recombinant vector into which an IDPc gene is inserted in the antisense orientation to decrease the expression of the IDPc gene in NIH3T3 F442A adipocytes (bottom) .
- Fig. 2a provides optical photographs showing Oil- Red-0-dyed adipocytes differentiated from normal NIH3T3 F442A (left), the transfectant FS1 cells with improved IDPc gene expression (middle) , and the transfectant FAS1 cells with decreased IDPc gene expression (right) on plates (upper panel) and in part, magnified at 200 power (lower panel) .
- Fig. 2b provides optical photographs showing the lipid deposition in adipocytes, which is in a NADPH dose-dependent pattern.
- Fig. 3 is a diagram illustrating the construction of a recombinant expression vector for use in generating a transgenic animal, in which an IDPc cDNA is inserted downstream of a rat-derived PEPCK (phosphoenolpyruvate carboxykinase) gene promoter.
- Fig. 4 provides photographs showing a comparison in body size and epididymal fat pad deposit between F x progeny from the transgenic mice of the present invention and normal mice .
- Fig. 5 provides autoradiographs showing an increase in the expression level of obesity-indicative genes in the adipose tissue of the transgenic mice of the present invention, compared to normal mice.
- Fig. 6a is a histogram comparing the body weight of the transgenic mice F x to that of normal mice.
- Fig. 6b is a histogram comparing the liver weight of the transgenic mice F ⁇ to that of normal mice.
- Fig. 6c is a histogram comparing the IDPc activity and blood IDPc level of the transgenic mice F x to those of normal mice.
- Fig. 6d is a histogram comparing the [NADPH] / [NADPH+NADP + ] of the transgenic mice F x to that of normal mice .
- Fig. 6e is a histogram comparing the epididymal fat pad weight of the transgenic mice F x to that of normal mice.
- Fig. 6f is a histogram comparing the blood triglyceride and cholesterol levels of the transgenic mice F x to those of normal mice.
- Fig. 6g is a histogram comparing the triglyceride and cholesterol levels in the liver of the transgenic mice F 1 to those of normal mice.
- Fig. 6h is a histogram comparing the blood leptin level of the transgenic mice F ⁇ to that of normal mice.
- Fig. 7a provides photographs showing liver tissues of the transgenic mice of the present invention and the control mice.
- Fig. 7b provides photographs showing adipocyte of the transgenic mice of the present invention and the control mice.
- Fig. 8a is a graph illustrating the inhibitory activity of oxalomalic acid against isocitrate dehydrogenase activity.
- Fig. 8b is a graph illustrating the inhibitory activity of methyl isocitrate against isocitrate dehydrogenase activity.
- Fig. 9 provides optical photographs showing Oil- Red-O-dyed adipocytes differentiated from NIH3T3 F442A cell treated with no isocitrate dehydrogenase inhibitors (left), oxalo alate (middle), and methyl isocitrate (right) , magnified at 100 power (upper panel) and 200 power (lower panel) .
- Fig. 10a is a histogram illustrating comparing the weights of the liver and epididymal fat pad of the rats in which the isocitrate dehydrogenase inhibitor of the present invention is administered, to those of rats administered with no inhibitors.
- Fig. 10b is a histogram illustrating comparing the blood triglyceride and cholesterol levels of the rats into which the isocitrate dehydrogenase inhibitor of the present invention is administered, to those of non-administered rats.
- Fig. 10c is a histogram illustrating comparing the blood HDL level of the rats into which the isocitrate dehydrogenase inhibitor of the present invention is administered, to that of non-administered rats.
- the present invention pertains to an isocitrate dehydrogenase enzyme which catalyzes the production of NADPH necessary for the biosynthesis of fatty acids and cholesterol and the deposition of lipids, and to a gene encoding the isocitrate dehydrogenase .
- the mouse-derived IDPc gene of the present invention has an open reading frame (ORF) 1,245 bp in size, with a 3'- untranslated region (UTR) in which a base sequence AATAAA, a putative poly-A signal, exists.
- the IDPc protein for which the IDPc gene codes consists of 414 amino acids, listed in Sequence No. 4, with a molecular weight of 46,575 Da.
- Alignment of the IDPc amino acid sequences from various species indicates that the mouse IDPc of the present invention shares a homology of 97.8 % with rat IDPc, 68.5 % with bovine IDPm, and 64.4 % with yeast IDPc.
- an amino acid sequence from 412 to 414 of the mouse IDPc is identical to the target sequence of peroxisome, which is known to be involved in the biosynthesis and degradation of fatty acids and cholesterol. Therefore, this suggests the high possibility that IDPc moves tq_ peroxisomes and takes part in the synthesis of fatty acids and cholesterol thereat.
- IDPm a gene having a base sequence similar to that of the IDPc gene, is also used for producing NADPH required for the biosynthesis of fatty acids and cholesterol and the deposition of lipids in accordance with the present invention.
- the present invention pertains to a fused gene construct containing the gene, a novel cell strain which anchors the gene, and a transgenic animal which expresses the gene continuously throughout its lifespan.
- the gene of interest is inserted into a mammalian expression vector in such a way as to transcribe the gene in the sense direction or in the antisense direction.
- retroviral expression vectors are preferably used as the gene carrier, with highest preference for pLNCX retroviral vector.
- pLNCX which is derived from MMLV (Moloney murine leukemia virus), has a CMV (cytomegalovirus) promoter for expressing exogenous genes in mammalian cells, and a neomycin gene as a selection marker, along with an LTR (long terminal repeat) sequence, an identification factor of retroviral vectors .
- the enzyme activity was measured to be higher by about 2 fold in the mouse NIH3T3 Ll transfectant cell in which the IDPc gene was inserted in the sense direction (FSl), but lower by about 0.4 fold in the mouse NIH3T3 Ll transfectant cells in which the IDPc gene was inserted in the antisense direction (FASl) .
- the effect of IDPc on the biosynthesis of fatty acids can be quantitatively measured with Oil-Red-O, a dye specific for lipids, which is applied to the adipocytes which have been differentiated from the transfectant cells after treatment with insulin.
- the fused gene construct is used to prepare a transgenic animal which harbors the IDPc gene within its genome.
- fused gene construct means a functional assembly of genes for use in transformation of certain organisms, which is comprised essentially of at least one structural gene, and at least one cis- acting regulatory element for controlling the expression of the structural gene.
- a cis-acting regulatory element may be in the form of a promoter, an enhancer, an intron, a 5'-UTR (untranslated region), and a 3'-UTR.
- the cis-acting regulatory element may be located at any site of 10 kb or less distant from the 5' -flanking region, 3' -flanking region, 5' -end or 3' -end of the structural gene or inside the structural gene (in the case of an intron) .
- the fused gene construct further comprises various components, including a polyadenylation signal for improving transcription or translation rates, a ribosome-binding sequence, an intron, etc. Further to these, a base sequence for improving the efficiency of the insertion of a gene of interest into the genome or certain sites, and a marker gene for identifying the insertion may be provided for the fused gene construct.
- a promoter for the fused gene construct to be used in making a transgenic animal include the CMV promoter, or expression regulatory regions for genes expressible in white adipose tissues, such as genes coding for lipoprotein lipase (LPL) , adipsin, adipocyte protein 2 (aP2) and IDPc.
- LPL lipoprotein lipase
- adipsin adipsin
- adipocyte protein 2 adipocyte protein 2
- IDPc IDPc.
- a rat-derived promoter for a cytosolic phosphoenolpyruvate carboxykinase (PEPCK) gene which is expressed in both the liver and the white adipose tissues .
- PEPCK cytosolic phosphoenolpyruvate carboxykinase
- the preparation of a transgenic animal in which the permanent expression of the IDPc gene is conducted starts with the cytosolic PEPCK gene of rats. From this gene, a 2.2 kb 5' -upstream sequence containing a promoter was obtained. Downstream of this sequence, a mouse IDPc cDNA was inserted in the sense orientation to prepare a fused gene construct, which was named pPEPCKIDPc.
- PEPCK-C a 5' -upstream sequence of the gene encoding the cytosolic PEPCK
- the tissues, where the PEPCK-C gene is expressed under the regulation of the promoter are determined depending on the regulatory regions existing in the 5'-upstream sequence.
- the 2.2 kb 5'- upstream sequence of the PEPCK-C gene used in the present invention contains a gene sequence near nt 987, which is known as a regulatory region necessary for efficient expression in white adipose tissue (Hanson, R. W. Annu. Tev. Biochem., 66, 581-611, 1997).
- mice are useful for making transgenic animals, but any animal, if it can be made transgenic, is available in the present invention because IDPc is an enzyme expressed in all higher animals.
- Embryo One of the most important steps in making of a transgenic animal is to introduce the fused gene construct into an embryo. The introduction is conducted with the aid of a microinjection system. When microinjecting the fused gene construct to an embryo, an automatic microinjection system which is able to automatically control amounts of DNA to the limit of 4 pi is preferably used because of it being superior in success rate to conventional manual microinjection systems.
- the mouse embryo which contains the IDPc fused gene construct was deposited with the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) under the deposition No. KCTC 0874 BP, on Nov. 4, 2000.
- the embryo containing the fused gene construct is implanted into a surrogate mother to afford a transgenic animal .
- the implantation of the embryo into a surrogate mother is conducted at the one-cell stage of the embryo rather than the two-cell stage, for convenience.
- the embryo of the one-cell stage is implanted to the oviduct of a surrogate mother, so as to reduce various processes necessary to culture the embryo to the two-cell stage.
- an embryo is required to be cultured for one additional day in an incubator.
- the embryo In order to implant a two-cell stage embryo to the oviduct funnel, the embryo must be inserted deep into the oviduct, or it is necessary to perforate the oviduct by use of a needle. However, the implantation of the one-cell stage embryo to a surrogate mother may be conducted under conditions similar to those for general mouse embryos, although the implantation site is the oviduct funnel .
- F ⁇ heterozygous transgenic mice had grown bigger than control mice.
- F x heterozygous transgenic mice were measured to be significantly increased in the size of the epididymal fat pad with a body weight 14 times as heavy as that of the control mice.
- the transgenic mice were observed to have large and many dermal mast cells on their backs, compared to control mice .
- a significant increase in epididymal fat pad was observed in the transgenic mice (see Fig. 4) .
- obesity- indicative genes such as genes coding for adipocyte protein 2 (aP2), adipsin, lipoprotein lipase (LPL) , leptin, tumor necrosis factor ⁇ (TNF- ⁇ ), and peroxysome proliferator-activated receptor ⁇ (PPAR ⁇ ), which are all known to show increased expression with advance in the differentiation of mast cells (Hwang, C. S. et al., Ann. Rev. Cell Eev. Biol., 13, 231-259, 1997; Lemberger, T. et al., Annu. Rev. Cell Dev. Biol., 12, 225-362, 1996; Spiegel an, B. M. et al .
- aP2 adipocyte protein 2
- LPL lipoprotein lipase
- PPAR ⁇ peroxysome proliferator-activated receptor ⁇
- the transgenic mice were found to show IDPc activity 2.7 and 1.4 fold greater in the liver and the epididymal fat pad than in those of control mice (see Fig. 6c) , respectively. Accordingly, the ratio of NADPH to total NADP pool ( [NADPH] / [NADP + ] + [NADPH] ) increased with increasing the enzymatic activity of IDPc (see Fig. 6d) .
- the weight of the transgenic mice was increased by 35 % or larger compared to that of the normal mice (see Fig. 6a) with a more significant increase in the epididymal fat pad of the transgenic mice than in that of control mice (see Fig. 6e) . However, no changes were found in the weight of the liver (see Fig. 6b) .
- triglyceride and total cholesterol levels in blood of the transgenic mice were measured to be 1.8 and 2.4 fold greater than those of the control mice (see Fig. 6f) .
- the liver of the transgenic mice was increased in both triglyceride and cholesterol levels (see Fig. 6g) .
- Leptin a protein produced mainly from mast cells, was detected to be twice as high in the blood level of the transgenic mice as in that of the control mice (see Fig. 6h) .
- transgenic mice were observed to " have livers in which a greater quantity of fats were deposited compared to those of the control mice.
- Another significant increase in the transgenic mice over the control mice was found to be the size of adipocytes in the epididymal fat pad (see Figs. 7a and 7b) .
- the weight gain of the transgenic mice that show more active expression of the IDPc gene is attributed to an increase in the quantity of body fat, and various obesity-indicative genes are more actively expressed in adipose tissues of the transgenic mice than in those of the control mice.
- various obesity-indicative genes are more actively expressed in adipose tissues of the transgenic mice than in those of the control mice.
- there was a significant increase in the level of PPAR ⁇ a transcription factor activating the transcription of genes coding for the enzymes which are responsible for the biosynthesis of lipids.
- an increase in the expression of the IDPc gene primarily results in the production of greater quantities of NADPH which is necessary for the biosynthesis of fatty acids and allows thus abundant lipid derivatives to induce the activation and gene expression of PPAR ⁇ , which, in turn, activates the expression of obesity-indicative genes and finally cause obesity in the IDPc-transgenic mice.
- an increase in the gene expression and activity of IDPc and in the cellular level of NADPH increases the activity of the enzymes that are involved in the biosynthesis of cholesterol, as well as activating the biosynthesis of lipoproteins through the increasing of lipid levels to produce more quantities of cholesterol composites in which cholesterol is associated with lipoproteins .
- the present invention pertains to a method for screening materials inhibitory of the enzymatic activity and gene expression of isocitrate dehydrogenase and thus effective for the treatment of metabolic diseases such as obesity, hyperlipidemia, and fatty liver.
- the present invention suggests therapeutics for the treatment of metabolic diseases caused by an increase in fat levels in vivo, such as obesity, hyperlipidemia and fatty liver, by taking advantage of the fact that an increase in the enzymatic activity and gene expression of isocitrate dehydrogenase promotes the biosynthesis of NADPH, in turn, activating PPAR ⁇ and raising in vivo levels of fatty acids, squalene and cholesterol .
- spectrophoto etry is very useful.
- a spectrophotometer is adjusted to zero absorbance at 340 nm using a mixture of a ten-fold concentrated reaction buffer plus 3rd distilled water.
- a test sample and 3rd distilled water is installed in the spectrophotometer, isocitrate dehydrogenase is added to the cuvette and a measurement is made of the change in absorbance at 340 nm with time.
- the present invention pertains to the use of NADPH in promoting the biosynthesis of lipids, cholesterol and squalene and activating PPAR ⁇ on the basis of the first finding of the present invention that an artificial increase in the cellular level of NADPH gives great rise to obesity and hyperlipidemia and raises the cellular level of triglyceride.
- a PCR started with 94 °C pre-denaturation for 4 min and carried out with 25 cycles of denaturing at 94 °C for 1 min, annealing at 50 °C for 1 min and extending at 72 °C for 2 min, finally followed by 72 °C extension for an additional 10 min, while 100 ng of the cDNA library was used as a template.
- a 0.8 kb DNA sequence was amplified.
- This PCR product was cloned into pCR II (Invitrogen Co.). The inserts of clones were sequenced to identify a rat IDPc gene.
- cDNA library phage with 5xl0 4 PFU was mixed with 3xl0 8 cells of E. coli XLl-blue at 37 °C for 15 min.
- the mixture of phage and host was poured in a 150 mm TYM-Ap agarose plate (1 % tryptone, 0.5 % yeast extract, 1 % NaCl, 1.5 % agar, 10 mM MgS0 4 , ampicillin 50 ⁇ g/ml) , solidified, and incubated at 37 °C for 12 hours. Following the formation of phage plaques, the plate was stored at 4 °C for 1 hour and then, the phages were transferred onto a nitrocellulose membrane.
- the nitrocellulose membrane had been soaked in distilled water and 1 M NaCl, in sequence, and dried on a 3 MM filter paper.
- the phage-coated soft agar plate was covered with the nitrocellulose membrane and then with another one for duplication.
- These duplicate membranes were immersed in a denaturation buffer (0.5 M NaOH, 0.5 M NaCl) for 5 min to denature the phages from host cell lysis, and phage DNA, and allowed to stand in a neutralization buffer (0.5 M Tris-Cl, pH 8.0, 0.5 M NaCl) for 5 min, finally followed by drying on a 3 MM filter paper.
- the mouse IDPc cDNA gene isolated in Example 1-1 was analyzed for base sequence with the aid of Sequenase version 2.0 kit (United States Biochemicals) . From the obtained base sequence, the amino acid sequence was determined. The GeneBank database was used to search for similar amino acid sequences and compared for homology.
- mouse IDPc cDNA has a 1,245 bp ORF listed in Sequence No. 3 with the sequence AATAAA, which is regarded as a poly (A) + signal, existing in the 3'-UTR.
- the amino acid sequence deduced from the base sequence of the IDPc gene is described to consist of 414 residues with a molecular weight of 46,575 Da, as shown in Sequence No. 4.
- mouse IDPc Alignment of the mouse IDPc and other species- derived isocitrate dehydrogenase proteins indicated that the mouse IDPc of the present invention shares a homology of 97.8 % with rat IDPc, 68.5 % with bovine IDPm, and 64.4 % with yeast IDPc.
- the sequence nt 412-414 is found to be identical to a peroxisome targeting sequence.
- Peroxisome is known to be involved in the biosynthesis and degradation of fatty acids and cholesterol.
- IDPc cDNA obtained in Example 1 was subcloned into the retroviral vector pLNCX (Miller, A. D. and Rosman, G.
- a retroviral vector pLNCX that did not anchor the gene.
- the recombinant vectors thus obtained were introduced into mouse NIH3T3, a fibroblast cell line.
- a pLNCX vector containing a GFP (green fluorescence protein) cDNA was also introduced into the cell, simultaneously.
- NIH3T3 cells were inoculated at a density of 5xl0 5 cells/ml and cultured in a 10 % FBS-supplemented DMEM. When the number of the cells increased by 50 %, the medium was removed and the retrovirus particles separated from the packaging cells were added to the NIH3T3. After 5 hours of incubation, the cells were provided with fresh medium and cultured for 2 days .
- Counts of the NIH3T3 infected by the recombinant retrovirions were measured, followed by aliquoting the cells at a density of 50 cells per well into 96-well plates in which DMEM added with G-418 (Gibco BRL) 400 ⁇ g/ml was contained. While the G-418 medium was changed every other day, the NIH3T3 cells in each well were re-separated and cultured to select first NIH3T3 transformants. For secondary screening, PCR with genomic DNA was performed to verify the integration of IDPc cDNA into the genome.
- the cell line FSl which harbors the IDPc gene in the sense direction in its genome, was deposited with the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) under the deposition No. KCTC 0861BP on Sep. 6, 2000.
- the cytoplasm was separated from the cells, and concentration of protein was determined using the Bradford assay.
- 3xl0 7 cells/ml were washed twice with lx PBS and lysed with a sucrose buffer (0.32 M sucrose, 0.01 M Tris-Cl, pH 7.4).
- the cell lysate was centrifuged at l,000x g to remove cell debris and then at 15,000x g to pellet mitochondria.
- PBS containing 0.1 % Triton X-100 was added at 1/10 the total solution volume, followed by quantification by use of the Bradford assay.
- the enzyme activity of IDPc was determined by measuring the change in the production amount of NADPH in a buffer (50 mM MOPS, pH 7.2, 35.5 mM triethanolamine, pH 7.2, 2 mM NADP + , 2 mM MgCl 2 , 5 mM isocitrate, and rotenone 1 ⁇ g/ml) maintained at 25 °C. Using a spectrophotometer, absorbance at 340 nm was measured for 2 min to quantify the amount of NADPH produced by the IDPc contained in the cytoplasmic protein, thereby determining the enzyme activity of IDPc. For the quantification of the enzyme, the amount which could produce 1 ⁇ M of NADPH in 1 min was defined as 1 unit.
- the enzyme activity of IDPc was increased by a factor of about 2 in the transfectant FSl cells into which the IDPc gene was introduced in the sense direction while being decreased by a factor of about 0.4 in the transfectant FASl cells into which the IDPc gene was introduced in the antisense direction.
- the transfectant cell lines were cultured in 10% FBS-supplemented DMEM containing insulin 5 ⁇ g/ml, 0.5 mM 3-isobutyl-l-methylxanthine (IBMX, Sigma) , 1 ⁇ M dexamethasone (DEX) , and penicillin-streptomycin (Gibco BRL), each 50,000 units, for two days to increase the counts of the cells to a density of approximately 3xl0 4 cells/cm 2 . Thereafter, the cells were further cultured for 12 days in DMEM free of IBMX and DEX while the medium was refreshed every other day.
- IBMX 3-isobutyl-l-methylxanthine
- DEX dexamethasone
- Gibco BRL penicillin-streptomycin
- Cell culturing was carried out in a C0 2 incubator at 37 °C under a wet, 5% C0 2 atmosphere. After culturing, the cells were treated with Oil- Red-0, which specifically dyes oil, to observe oil deposits formed in adipocytes.
- the medium was depleted, after which 10 ml of a cacodylate buffer (90 mM cacodylate, pH 7.2, 2 % formaldehyde, 2.5 % glutaraldehyde, 0.025 % CaCl 2 , 5 % sucrose) was added to the cells, which was then allowed to stand at 4 °C for 1 hour. After removal of the buffer, 5 ml of Oil-Red-O in 40 % isopropanol was added to the cells and slowly mixed over 1 hour, followed by washing with 40 % isopropanol.
- a cacodylate buffer 90 mM cacodylate, pH 7.2, 2 % formaldehyde, 2.5 % glutaralde
- Fig. 2a there are observations of the adipocytes dyed with Oil-Red-O.
- the transfectant FSl cells in which the gene expression of the IDPc is increased have produced oils at a greater amount than did the control cells.
- the transfectant FASl cells with decreased expression of the IDPc gene show significantly reduced oil deposits relative to the control cells.
- Photographs magnified at 200x power further show the difference in adipocyte size among the cell groups.
- EXAMPLE 5 Change in Di ferentiation of Adipocytes and Oil Deposition within Cells According to Concentration of NADPH
- NADPH which can be obtained as a reaction product of not only isocitrate isoenzymes IDPc and IDPm, but also glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and malate dehydrogenase, was identified to have direct positive influence on the differentiation of adipocytes and the concomitant deposition of oils even when it was artificially added.
- TCVAMPT.F. 6 Identification of In Vivo Activity of IDPc Using Transgenic Animal Containing IDPc Gene in Its Genome
- a 2.2 kb 5' -upstream sequence containing a promoter was amplified from the cytosolic PEPCK gene of rats with the aid of PfuTurbo DNA polymerase (Stratagene) using a set of primers listed in Sequence Nos. 5 and 6.
- the PCR product was digested with Bglll and Smal and then with I-Pop-I, and treated with Mung Bean nuclease to produce blunt-ends, one of which was cut with Bglll.
- This DNA digest was inserted into the mammalian expression vector pCI-neo containing a CMV promoter.
- the mouse IDPc fused gene construct (ca. 10 ⁇ g) obtained in Example 6-1-1 was subjected to double digestion with restriction enzymes Bglll and Nsil , after which the digestion solution was resolved on 0.7 % agarose gel to separate a 4.9 kb DNA fragment containing the gene of interest.
- the upper part was centrifuged at 12,000 rpm for 3 min. To the supernatant recovered, an equal volume of ether was added, followed by centrifugation at 10,000 rpm for 5 sec.
- the lower DNA part was added with two volumes of absolute ethanol to precipitate DNA.
- the DNA pellet was well dissolved in a microinjection solution (10 mM Tris pH 7.4, 0.1 mM EDTA) at a concentration of 2-10 ⁇ g/2.4 ml.
- the resulting solution was dialyzed against a microinjection solution at 4 °C for 24 hours.
- the DNA was controlled to have a concentration of 2-4 ng/ ⁇ l in total and stored at -20 °C until use.
- hCG human oviduct ampulla
- hyaluronidase 300 ⁇ g/ml
- DIC Normarski differential interference contrast
- mice After completion of microinjection, survivors were cultured in Ml6 medium at 37 °C in a 5 % C0 2 atmosphere in a C0 2 incubator.
- the resulting mouse embryo containing the IDPc fused gene construct was deposited with the Korean Collection for Type Culture of Korea Research Institute of Bioscience and Biotechnology (KRIBB) under the deposition No. KCTC 0874 BP, on Nov. 5, 2000.
- FVB/N lineage mice were used as recipients.
- Vasectomy was performed on male mice in the estrus stage, which were allowed to mate with females. The next morning, the female mice which showed vaginal plugs were selected as final recipients.
- dissection was performed at the subcutis of the sperm duct of the recipients to the length of 1 cm and subsequently at the muscle layer, followed by implanting the embryo of Example 6- 1-3 into the opposite oviduct thus exposed.
- Offspring from the recipients were examined by PCR for the insertion of the microinjected mouse IDPc fused gene construct DNA, that is, pPEPCKIDPc, into their genome.
- a tail part 2-3 cm long was cut from 2 or 3-week-old mice bred by the recipient, and immersed in 700 ⁇ l of a lysis buffer (50 mM Tris- Cl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1 % SDS) for 15- 18 hours at 55 °C in the presence of 35 ⁇ l of proteinase K (10 mg/ml) with agitation.
- a lysis buffer 50 mM Tris- Cl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1 % SDS
- mice genomic DNA (1 ⁇ g) thus obtained was partially amplified by PCR using a 3' -flaking region of the CMV promoter as a sense primer PI
- mice Offspring from the crossing of selected transgenic male mice with wild-type female FVB/N mice had their tails cut and examined for transgenicity in the same manner to select the transgenic mice, which inherited the recombinant IDPc gene in a germ line. After being identified by PCR as having the recombinant gene, the transgenic mouse offspring were maintained in a heterozygous F 2 line. Like the F ⁇ heterozygous transgenic mice, these F 2 heterozygous transgenic mice were found to show obesity, hyperlipidemia, and fatty liver. For managing the transgenic mouse species, 2- week-old mice had their tails cut partially and genomic DNA was prepared from the tail segments and analyzed for the insertion of exogenous gene of interest. Once being identified as transgenic, mice were separated according to sex and marked in their ears.
- 6-2-1 Enlargement of Epididymal Fat Pad in Transgenic Animal Transgenic mice and normal mice, both being 28 weeks old and bred from the same parents, were sacrificed by separation of their spines, after which their exodermis was partially dissected with scissors while being hold by a pincette. After the complete peeling of the exodermis, the endodermis was dissected to expose epididymal fat pads which were then compared in size to those of wild-type FVB/N mice. Afterwards, total adipose tissues were taken from both the transgenic mice and wild-type mice in order to compare the total weights therebetween. Soon after being measured for weight, the separated total adipocytes were fixed in formalin and rapidly cooled. Using a microtome, the frozen adipose tissues were sliced at - 20 °C to pieces 10 ⁇ m thick, which were dyed with hematoxylin and eosin for visualization under a microscope.
- Fig. 4a Microscopic observations are given in Fig. 4. 26 weeks after birth, as seen in Fig. 4a, F 1 heterozygotic transgenic mice had grown bigger than control mice. When being frayed, the transgenic mice were observed to have large and many dermal mast cells on their backs, compared to control mice, as shown in Fig. 4b. In addition, the transgenic mice were identified to have significantly larger epididymal fat pads. When being further anatomized, F 1 heterozygotic transgenic mice were measured to have significantly larger epididymal fat pads compared to control mice, as shown in Fig. 4c. After complete removal of the abdominal skin, a significant increase in the weight of white adipose tissue was observed in the transgenic mice relative to control mice, as seen in Fig. 4d. No difference in the size and color of the liver between the transgenic mice and the normal mice was seen with the naked eye.
- each obesity-indicative gene was quantitatively measured as to its expression level as follows.
- 1 g of the epididymal fat pad taken from each of the IDPc gene-transgenic and the normal mice was added in 9 ml of a lysogenic solution (4M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5 % sarkosyl, 0.72 % ⁇ -mercaptoethanol) and homogenized by use of a homogenizer. After being cooled for 2-3 min in ice, the homogenate was added with a mixture of 1 ml of an extraction solution (2M sodium acetate, pH 4.0) and 10 ml of DEPC-water saturated phenol, 2 ml of chloroform-isoamyl alcohol (24:1) and let to stand in ice for 15 min.
- a lysogenic solution 4M guanidium thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5 % sarkosyl, 0.72 % ⁇ -mercaptoethanol
- RNA pellet After being dissolved in an aqueous 36 % formaldehyde solution, the RNA was resolved on 1 % agarose gel containing formaldehyde at an amount of 6.7 % under an electric field. Following the electrophoresis, separated RNA bands were transferred onto a nylon membrane in 20x SSC solution, dried and fixed by UV cross-linking.
- the nylon membrane was washed for 5 min in 6x SSC solution and then subjected to pre-hybridization at 42 °C for 2 hours in an appropriate amount of a hybridization solution (50 % formamide, 6x SSC, 5x Denhardt's solution, 1.2 % SDS, 10 ⁇ g/ml salmon sperm DNA).
- a hybridization solution 50 % formamide, 6x SSC, 5x Denhardt's solution, 1.2 % SDS, 10 ⁇ g/ml salmon sperm DNA.
- various obesity- indicative cDNAs dP2, adipsin, LPL (lipo protein lipase), leptin, tumor necrosis factor ⁇ [TNF ⁇ ], and PPAR ⁇
- dP2 adipsin, LPL (lipo protein lipase), leptin, tumor necrosis factor ⁇ [TNF ⁇ ], and PPAR ⁇
- the nylon membrane was washed at 65 °C with 6x SSC solution containing 0.1 % SDS for 30 min and then with 2x SSC containing 0.1 % SDS for 20 min and additionally washed in the same manner as above at least one more. Finally, the nylon membrane was washed twice with 0.2x SSC solution at room temperature.
- the autoradiogram obtained by exposing the membrane to X-ray film at -70 °C allowed the identification of mRNAs transcribed from obesity-indicative genes in the epididymal fat pad.
- the hybridization results are shown in autoradiographs .
- the expression of the recombinant IDPc gene introduced to the transgenic mice was found, along with the expression of the endogenous IDPc gene, demonstrating that the total IDPc activity was increased.
- an increase was found in the expression of obesity-indicative genes, such as genes coding for aP2, adipsin, LPL, leptin, TNF- ⁇ , and peroxisome proliferator-activated receptor ⁇ (PPAR ⁇ ), which are all known to show increased expression with the advance in the differentiation of mast cells.
- PPAR ⁇ peroxisome proliferator-activated receptor ⁇
- obesity transgenic mice F- L and normal mice were compared for intracellular lipid deposition.
- a container suitable for receiving a mouse was placed on a scale which was then subjected to null adjustment, after which a mouse was carefully put in the container. Because the numeral read on the scale was changed whenever the mouse moved, the value detected when the mouse did not move was set forth as the body weight for the mouse.
- livers and adipose tissues taken from the transgenic mice and normal mice were homogenized in a buffer (0.32 M sucrose, 0.01 M Tris-Cl pH 7.4) and centrifuged at 3,000x g for 15 min. Then, the supernatant was recentrifuged at 10,000xg for 15 min. A pure cytosolic fraction was obtained as the supernatant.
- the enzyme activity of IDPc was determined by measuring the change in the production amount of NADPH in a buffer (50 mM MOPS, pH 7.2, 35.5 mM triethanolamine, pH 7.2, 2 mM NADP + , 2 mM MgCl 2 , 5 mM isocitrate, and rotenone 1 ⁇ g/ml) maintained at 25 °C. Using a spectrophotometer, absorbance at 340 nm was measured for 2 min to quantify the amount of NADPH produced by the IDPc contained in the cytoplasmic protein, thereby determining the enzyme activity of IDPc. For the quantification of the enzyme, the amount which produced 1 ⁇ M of NADPH for 1 min was defined as 1 unit .
- Two cytosolic extracts containing 100 ⁇ g of proteins were prepared: one was pre-treated by reaction at 60 °C for 30 min and cooling to 0 °C, so as to degrade all NADP + to measure the amount of the preexisting NADPH [NADPH] (sample 1) ; and the other was stored at 0 °C without pretreatment and used to measure the total NADP pool [NADPH+NADP + ] (sample 2) .
- reaction solution 0.1 M Tris-HCl buffer, pH 8.0, 5 mM EDTA, 2 mM phenazine ethosulfate, 0.5 mM MTT
- glucose-6-phosphate dehydrogenase was added at an amount of 1 mM to each sample (samples 1 and 2), but not to a control.
- a triglyceride-assay kit [a solution of lipoproteinase 10800 U, glycerol kinase 5.4 U, peroxidase 135,000 U, and L- ⁇ -glycerophosphate oxidase 160 U in 72 ml of N,N-bis (2-hydroxyethyl) -2- amino ethanesulfonic acid buffer] or 1.5 ml of a cholesterol enzyme-assay kit (a mixture of an enzyme solution (cholesterol esterase 20.5 KU/1, cholesterol oxidase 10.7 KU/1, and sodium hydroxide 1.81 g/1) and a buffer (potassium monophosphate 13.6 g/1, phenol 1.88 g/1) in the proportions of 1:1) and incubated at 37 °C for 5 min for reaction.
- a triglyceride-assay kit a solution of lipoproteinase 10800 U, glycerol kinase 5.4 U, peroxidase
- 100 ⁇ l of the homogenate was dissolved in 200 ⁇ l of pure ethyl alcohol and mixed with 500 ⁇ l of each of the assay kits for triglyceride and cholesterol, manufactured by Asan Pharmaceutics, Co. Ltd., containing 0.5 % Triton X-100 and 3 mM sodium cholate, followed by incubation at 37 °C for 10 min. After being added with 800 ⁇ l .
- each sample was measured for liver triglyceride and total cholesterol levels with the aid of a microplate reader in the same manner as in above.
- the above procedure was applied to various known concentrations of a standard solution to acquire a standard curve which was used to quantify triglyceride and cholesterol levels in the liver.
- the resulting sample mixture was diluted 1/20 with a calibrator diluent (RD5-3) .
- 50 ⁇ l of an assay diluent (RDlW) was added to each well of 96-well plates, followed by the addition of 50 ⁇ l of the serum sample or 50 ⁇ l of a reference material.
- the resulting solution in each well was completely mixed for 1 min and incubated at room temperature for 2 hours to perform the reaction. After complete removal of liquid, each well was washed 4-5 times with a washing buffer. The residue in each well was reacted with 100 ⁇ l of a mouse leptin conjugate at room temperature for 2 hours and washed 4-5 times again. Within 30 min, blood leptin levels were quantified by measuring the absorbance at 450 nm in a microplate reader.
- Example 6-2-3 Measurements obtained in Example 6-2-3 were analyzed and graphed in Fig. 6.
- the transgenic mice (Tg) were measured to have weights 23-35 % higher than the normal mice (Non-Tg) .
- the transgenic mice were found to show 1.4- and 2.7-fold greater activity of IDPc in the liver and adipose tissues, respectively, than the normal mice, as shown in Fig. 6c.
- Fig. 6d compares concentration ratios of NADPH to total NADP pool ( [NADPH] / [NADP + ] + [NADPH] ) in the liver and the adipose tissue between the transgenic mice and the normal mice.
- the concentration ratios in the liver and adipose tissue of the transgenic mice was increased by factors of 1.2 and 1.3, respectively, relative to those of the normal mice. A great increase was detected in the weight of epididymal fat pad.
- the transgenic mice were measured to have epididymal fat pad about 13.6-fold heavier than that of the normal mice as shown in Fig. 6e. However, no significant difference was found in the weight of the liver of the normal mice and transgenic mice, as illustrated in Fig. 6b.
- the blood levels were higher in the transgenic mice by factors of 1.8 and 2.4, respectively, compared to those in the normal mice, as shown in Fig. 6f.
- triglyceride and total cholesterol levels in the liver of the transgenic mice were 1.8- and 2.4- fold higher respectively, compared to normal mice, as shown in Fig. 6g.
- Fig. 6h show blood levels of leptin, a protein produced mainly from mast cells are high by a factor of about 2 in the transgenic mice relative to normal mice.
- liver and adipose tissues taken from obesity gene-transgenic mice F x and normal mice were observed under a microscope. For observation convenience, the tissues were sliced into sections. The liver of the transgenic mice was identified to be a fatty liver which had accumulated more fat than that of the normal mice, as seen in Fig. 7a. Adipocytes of the transgenic mice were also observed to be five-fold larger in size compared to those of normal mice (Fig. 7b) .
- the weight gain of the transgenic mice in which IDPc gene is actively expressed results from body fat accumulation.
- cellular levels of various obesity- indicative proteins, including PPAR ⁇ which is a transcriptional factor promoting the expression of genes encoding enzymes involved in the metabolism of lipids and cholesterol, are significantly elevated in the adipose tissue of the transgenic mice, compared to normal mice. Therefore, an increase in the expression of the IDPc gene primarily results in the production of greater quantities of NADPH, which is necessary for the biosynthesis of fatty acids, and allows the resulting abundant lipid derivatives to induce the activation and gene expression of PPAR ⁇ , which, in turn, activates the expression of obesity-indicative genes and finally causes obesity in the IDPc-transgenic mice.
- An assay buffer 50 mM MOPS, pH 7.2, 35.5 mM triethanola ine, pH 7.2, 2 mM NADP + , 2 mM MgCl 2 , 5 mM isocitrate, rotenone 1 mg/ml
- test samples were all prepared at lOx concentration. Enzymatic reactions necessary for the selection were conducted in a final volume of 1 ml at 25 °C in crystal cuvette. For use, the concentrated test samples were diluted with 3rd distilled water.
- the assay buffer, enzyme inhibitors, and isocitrate dehydrogenase, which all at lOx concentration, were maintained at low temperature, i.e., in ice.
- a spectrophotometer was first subjected to null adjustment at 340 nm using the lOx assay buffer and 3rd distilled water. 100 ⁇ l of the sample was added, along with 100 ⁇ l of the lOx assay buffer, in a crystal cuvette, and mixed with 600 ⁇ l of 3rd distilled water. After the completion of the null adjustment, the sample cuvette was installed in the spectrophotometer, followed by adding 200 ⁇ l of isocitrate dehydrogenase and mixing the solution with the aid of a pipette. Changes in absorbance at 340 nm were monitored with time. In principle, an absorbance decreases faster in the cuvette containing a greater concentration of a sample inhibitory against the activity of the enzyme. Based on this principle, the quantification of the spectrophotometric data enabled the screening of inhibitors of isocitrate dehydrogenase.
- IDPc inhibitors play an important role in the lipid metabolism in vivo, reducing cellular oil deposits.
- rats were compared for cellular lipid deposition when they were treated with no isocitrate dehydrogenase inhibitors and methyl isocitric acid.
- a container suitable for receiving a rat was placed on a scale which was then subjected to null adjustment, after which a rat was carefully put in the container. Because the numerals read on the scale changed whenever the rat moved, the value detected when the rat did not move was set forth as the body weight for the rat .
- livers and adipose tissues taken from inhibitor-administered rats and non-administered rats were homogenized in a buffer (0.32 M sucrose, 0.01 M
- the enzyme activity of IDPc was determined by measuring the change in the production amount of NADPH in a buffer (50 mM MOPS, pH 7.2, 35.5 mM triethanolamine, pH 7.2, 2 mM NADP + , 2 mM MgCl 2 , 5 mM isocitrate, and rotenone 1 ⁇ g/ml) maintained at 25 °C.
- a triglyceride-assay kit [a solution of lipoproteinase 10800 U, glycerol kinase 5.4 U, peroxidase 135000 U, and L- ⁇ -glycerophosphate oxidase 160 U in 72 ml of N,N-bis (2-hydroxyethyl) -2- aminomethanesulfonic acid buffer] or 1.5 ml of a cholesterol enzyme-assay kit (a mixture of an enzyme solution (cholesterol esterase 20.5 KU/1, cholesterol oxidase 10.7 KU/1, and sodium hydroxide 1.81 g/1) and a buffer (potassium monophosphate 13.6 g/1, phenol 1.88 g/1) in the proportions of 1:1) and incubated at 37 °C for 5 min for reaction.
- a triglyceride-assay kit a solution of lipoproteinase 10800 U, glycerol kinase 5.4 U, peroxidase 13
- a measurement was done by reading absorbance at 500 nm for cholesterol and at 540 nm for triglyceride, so as to quantify blood levels of triglyceride and total cholesterol.
- a standard curve which was obtained by applying the above procedure to various known concentrations of a standard solution.
- 100 ⁇ l of the homogenate was dissolved in 200 ⁇ l of pure ethyl alcohol and mixed with 500 ⁇ l of each of the assay kits for triglyceride and cholesterol, manufactured by Asan Pharmaceutics, Co. Ltd., containing 0.5 % Triton X-100 and 3 mM sodium cholate, followed by incubation at 37 °C for 10 min.
- each sample was measured for liver triglyceride and total cholesterol levels with the aid of a microplate reader in the same manner as in above.
- the above procedure was applied to various known concentrations of a standard solution to acquire a standard curve which was used to quantify triglyceride and cholesterol levels in the liver.
- Example 8-2 Measurements obtained in Example 8-2 were analyzed and graphed in Fig. 10. No significant difference was found in body weight between the inhibitor-administered and non-administered rats, both being 10-weeks old. As seen in Fig. 10a, the inhibitor-administered rats were measured to be lower in the weight of epididymal fat pad by about 12 %, compared to the non-administered rats, with no difference in the weight of the liver of the normal and inhibitor administered rats. As for triglyceride and total cholesterol, their blood levels were lower in the inhibitor-administered rats by 20 % and 11 %, respectively, compared to those in the non- administered rats, as shown in Fig. 10b. Also, the arteriosclerosis index was 10 lower.
- HDL high-density lipoproteins
- the weight gain of the transgenic mice in which IDPc gene is actively expressed results from body fats accumulation and that cellular levels of various obesity-indicative proteins, including PPAR ⁇ , which is a transcriptional factor promoting the expression of genes encoding enzymes involved in the metabolism of lipids and cholesterol, are significantly elevated in the adipose tissue of the transgenic mice, compared to in that of the normal mice.
- PPAR ⁇ a transcriptional factor promoting the expression of genes encoding enzymes involved in the metabolism of lipids and cholesterol
- an increase in the expression of the IDPc gene primarily results in the production of greater quantities of NADPH, which is necessary for the biosynthesis of fatty acids, and allows the resulting abundant lipid derivatives to induce the activation and gene expression of PPAR ⁇ , which, in turn, activates the expression of obesity- indicative genes and finally causes obesity in the IDPc-transgenic mice.
- IDPc inhibitors were demonstrated to be useful in treating metabolic diseases. That is, when rats are administered with isocitrate dehydrogenase inhibitors, they are restricted from being obese in addition to showing small epididymal fat pads, low levels of triglyceride and total cholesterol, and low arteriosclerosis indexes. Therefore, isocitrate dehydrogenase inhibitors can suppress obesity-induced arteriosclerosis. Also, the increase of HDL level attributed to IDPc inhibitors demonstrates that the decrease in the level of total cholesterol upon the administration of IDPc inhibitors is thanks to the increase of HDL level.
- the expression of the IDPc gene and the concomitant increase in IDPc level is identified to bring about an increase in the cellular level of NADPH, which, in turn, causes the lipid deposition in adipocytes, leading to obesity and fatty liver.
- NADPH cellular level of NADPH
- blood triglyceride levels and total cholesterol are increased as a result of the expression of the IDPc gene.
- a decrease in the cellular level of NADPH resulting from the suppression of the gene expression of IDPc and concurrent intracellular decrease of IDPc levels, has the effect of inhibiting the lipid deposition in adipocytes.
- IDPc gene, IDPc and NADPH can be used for the synthesis of lipids, including fatty acids, squalene, DHA, etc., and for the activation of PPAR ⁇ .
- IDPc-gene transgenic mice of the present invention clearly exhibit symptoms of obesity, hyperlipidemia and fatty liver, NADPH- producing isocitrate dehydrogenase, including IDPc, and their genes can be directly used to identify materials suppressive of obesity and fatty liver as well as materials inhibitory of the biosynthesis of triglyceride and cholesterol.
- microorganism identified under I above was ⁇ ecornpw ⁇ ied by'
- microorganism identified under I above was received by this Intern-iticirml Depositary Authority on and a request to convert the orurinal deposit to a deposit under the Budapest Treaty was received by it on
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US10/239,800 US20040214270A1 (en) | 2000-10-20 | 2001-07-26 | Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fattly liver in lipid biosynthesis |
EP01952036A EP1334180A4 (en) | 2000-10-20 | 2001-07-26 | Isocitrate dehydrogenase, gene thereof, and use of the same in the treatment of obesity, hyperlipidemia, and fatty liver in lipid biosynthesis |
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JP2002536433A JP2004511256A (en) | 2000-10-20 | 2001-07-26 | Isocitrate dehydrogenase, its gene, and use of the gene for the treatment of metabolic diseases including obesity, hyperlipidemia, fatty liver and the like |
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KR100821457B1 (en) * | 2006-11-24 | 2008-04-11 | (주)아모레퍼시픽 | Cell line which is transfected with vectors containing IDH3 alpha promoter and luciferase and searching method of obesity controlling material using the same |
KR101327028B1 (en) * | 2007-01-17 | 2013-11-13 | (주)아모레퍼시픽 | Transgenic mouse overexpressing isocitrate dehydrogenase 3 alpha and manufacturing method of the same |
CN101157955B (en) * | 2007-10-09 | 2011-03-16 | 华中农业大学 | Pig isocitric acid dehydrogenase gene IDH3-gamma as genetic marker of production trait in pigs |
PT3434671T (en) * | 2016-03-22 | 2020-12-24 | Lianyungang Runzhong Pharmaceutical Co Ltd | Sultam compound and application method thereof |
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US5258286A (en) * | 1985-07-02 | 1993-11-02 | Oriental Yeast Co., Ltd. | Method of terminating isocitrate dehydrogenase reaction |
US5952177A (en) * | 1997-12-03 | 1999-09-14 | Incyte Pharmaceuticals, Inc. | Human cytosolic isocitrate dehydrogenase |
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- 2001-07-26 RU RU2003113553/14A patent/RU2266131C2/en not_active IP Right Cessation
- 2001-07-26 WO PCT/KR2001/001271 patent/WO2002033063A1/en not_active Application Discontinuation
- 2001-07-26 AU AU7283101A patent/AU7283101A/en active Pending
- 2001-07-26 CN CNA018174922A patent/CN1524122A/en active Pending
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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US5258286A (en) * | 1985-07-02 | 1993-11-02 | Oriental Yeast Co., Ltd. | Method of terminating isocitrate dehydrogenase reaction |
US5952177A (en) * | 1997-12-03 | 1999-09-14 | Incyte Pharmaceuticals, Inc. | Human cytosolic isocitrate dehydrogenase |
Non-Patent Citations (3)
Title |
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DATABASE GENBANK [online] NEKRUTENKO A. ET AL.: "Cytosolic isocitrate dehydrogenase in humans, mice and voles and phylogenetic analysis of the enzyme family", XP002907139, Database accession no. (AF020039) * |
MOL. BIOL. EVOL., vol. 15, no. 12, 1998, pages 1674 - 1684 * |
See also references of EP1334180A4 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011050210A1 (en) | 2009-10-21 | 2011-04-28 | Agios Pharmaceuticals, Inc. | Methods and compositions for cell-proliferation-related disorders |
WO2011050211A2 (en) | 2009-10-21 | 2011-04-28 | Agios Pharmaceuticals, Inc. | Methods and compositions for cell-proliferation-related disorders |
EP3064595A1 (en) | 2009-10-21 | 2016-09-07 | Agios Pharmaceuticals, Inc. | Methods and compositions for cell-proliferation-related disorders |
EP3561077A1 (en) | 2009-10-21 | 2019-10-30 | Agios Pharmaceuticals, Inc. | Methods and compositions for cell-proliferation-related disorders |
Also Published As
Publication number | Publication date |
---|---|
US20040214270A1 (en) | 2004-10-28 |
EP1334180A4 (en) | 2005-04-06 |
CN1524122A (en) | 2004-08-25 |
CA2426403A1 (en) | 2002-04-25 |
RU2003113553A (en) | 2005-01-10 |
AU2001272831B2 (en) | 2005-09-15 |
EP1334180A1 (en) | 2003-08-13 |
RU2266131C2 (en) | 2005-12-20 |
JP2004511256A (en) | 2004-04-15 |
AU7283101A (en) | 2002-04-29 |
KR100455899B1 (en) | 2004-11-08 |
KR20020031475A (en) | 2002-05-02 |
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