KR101572322B1 - Screening methods for therapeutic agents of obesity or metabolic diseases - Google Patents

Abstract

The present invention relates to a method for screening a therapeutic agent for obesity or metabolic diseases using DNMT1, comprising the steps of: culturing cells; Treating the cell with a candidate substance; Measuring methylation of the adiponectin promoter in the cell; And confirming that the methylation of the adiponectin promoter is reduced as compared with the control. In addition, there is provided a therapeutic agent for obesity or metabolic diseases comprising a DNMT1 activity inhibitor.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention [0001] The present invention relates to screening methods for treating obesity or metabolic diseases,

The present invention relates to a method for screening a therapeutic agent for obesity or metabolic diseases using DNMT1.

Epigenetic regulation, including DNA methylation and histone modification, is an important mechanism in the regulation of gene expression in eukaryotic cells [1, 2]. Accumulated evidence suggests that epigenetic modification is an important part of adapting to environmental changes. For example, nutritional status regulates DNA methylation of genes such as hepatocyte nuclear factor 4α (HNF4α), pancreatic and duodenal homeobox 1 (PDX1), and peroxisome proliferator-activated receptor α (PPARα) [3- 5]. In addition, deficiency of methyl / folate inhibits DNA methylation, whereas replacement of methyl donor increases DNA methylation [6, 7]. In addition, high-fat diets (HFD) alter the expression of various genes associated with insulin sensitivity and inherit metabolic imbalances inherited in the next generation [8, 9].

Adiponectin is a gene specifically expressed in adipocytes and regulates systemic energy homeostasis such as glucose and lipid metabolism [10, 11]. In metabolic tissues, adiponectin improves glucose utilization, fatty acid oxidation and insulin sensitivity [11, 12]. In addition, adiponectin inhibits obesity-induced immune responses and has anti-atherogenic effects [13-15]. Interestingly, adiponectin expression and its serum levels have been reduced in obesity-associated metabolic diseases such as obesity and insulin resistance, type 2 diabetes and cardiovascular disease [16-20]. In contrast, supplementation of adiponectin improved insulin sensitivity and metabolic markers in obese animal models [21]. However, it remains unclear how adiponectin gene expression is reduced in metabolic diseases.

In adipocytes, a variety of factors are involved in the regulation of adiponectin gene expression. For example, peroxisome proliferator-activated receptor gamma (PPARy) ligands such as thiazolidinediones (TZDs) enhance adiponectin gene expression and subsequent enhanced insulin sensitivity [22-24]. However, increased adipocyte size, inflammatory response and oxidative stress act as inhibitors of adiponectin expression [20, 25-28]. Among the various regulatory steps, transcriptional regulation is a major determinant of serum adiponectin levels. Previously known transcriptional regulation of adiponectin is achieved by the binding of transcription factors to cis -regulatory factors in the adiponectin promoter. For example, PPARγ binds to the PPAR response element (PPRE) in the adiponectin promoter and increases adiponectin promoter activity and adiponectin mRNA [22, 29]. In addition, mouse and human adiponectin promoters have two binding sites for sterol regulatory element binding protein 1c (SREBP1c), and SREBP1c is attached to this region to promote the promoter activity of adiponectin gene [29]. Another major adipogenic transcription factor, CCAAT / enhancer binding protein α (C / EBPα), increases adiponectin gene expression by binding to the C / EBPα response element in the adiponectin promoter [30-32]. Thus, previous studies have focused on finding transcription factors that regulate adiponectin expression, and studies have examined how epigenetic changes such as DNA methylation and histone modification affect adiponectin expression in obese conditions I did.

We examined the role of DNA methylation in the expression of adiponectin gene and found that hypermethylation of the adiponectin promoter inhibits adiponectin transcription and leads to chromatin remodeling. Also, DNA methylation in the adiponectin promoter was mainly mediated by DNA methyltransferase 1 (DNMT1), and DNMT1 expression was improved in adipocytes of obese animals. Moreover, the promotion of adiponectin expression by DNMT1 inhibition reduced the inflammatory response and increased insulin sensitivity. The present invention provides a method for screening for a therapeutic agent for obesity or metabolic diseases through the expression of adiponectin gene by a substance that inhibits DNA methylation.

[1] JC Rice and CD Allis, "Histone methylation versus histone acetylation: new insights into epigenetic regulation," Curr Opin Cell Biol , vol. 13, pp. 263-73, Jun 2001.

[2] R. Jaenisch and A. Bird, "Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals," Nat Genet , vol. 33 Suppl, pp. 245-54, Mar. 2003.

[3] I. Sandovici, NH Smith, MD Nitert, M. Ackers-Johnson, S. Uribe-Lewis, Y. Ito , et al . , "Maternal diet and aging alter the epigenetic control of a promoter-enhancer interaction at the Hnf4a gene in rat pancreatic islets," Proc Natl Acad Sci USA, vol. 108, pp. 5449-54, Mar 29 2011.

[4] JH Park, DA Stoffers, RD Nicholls, and RA Simmons, "Development of type 2 diabetes induced intrauterine growth retardation in rats associated with progressive epigenetic silencing of Pdx1," J Clin Invest, vol. 118, pp. 2316-24, Jun 2008.

[5] BR Carone, L. Fauquier, N. Habib, JM Shea, CE Hart, R. Li , et al . , "Paternally induced transgenerational environmental reprogramming of metabolic gene expression in mammals," Cell , vol. 143, pp. 1084-96, Dec 23 2010.

[6] J. Champier, F. Claustrat , N. Nazaret, M. Fevre Montange, and B. Claustrat, "Folate depletion changes gene expression of fatty acid metabolism, DNA synthesis, and circadian cycle in male mice," Nutr Res , vol. 32, pp. 124-32, Feb 2012.

[7] RD Wilson, JA Johnson, P. Wyatt, V. Allen, A. Gagnon, S. Langlois , et al . , "Pre-conceptional vitamin / folic acid supplementation 2007: the use of folic acid in combination with a multivitamin supplement for the prevention of neural tube defects and other congenital anomalies," J Obstet Gynaecol Can, vol. 29, pp. 1003-26, Dec 2007.

[8] SA Langie, S. Achterfeldt, JP Gorniak, KJ Halley-Hogg, D. Oxley, FJ van Schooten , et al . , "Maternal folate depletion and high-fat feeding from weaning affects DNA methylation and DNA repair in brain offspring," FASEB J, Apr 19 2013.

[9] SF Ng, RC Lin, DR Laybutt, R. Barres, JA Owens, and MJ Morris, "Chronic high-fat diet in fathers programs beta-cell dysfunction in female rat offspring," Nature , vol. 467, pp. 963-6, Oct 21 2010.

[10] PE Scherer, S. Williams, M. Fogliano, G. Baldini, and HF Lodish, "A novel serum protein similar to C1q, produced exclusively in adipocytes," J. Biol Chem . , Vol. 270, pp. 26746-9, Nov 10 1995.

[11] T. Yamauchi, J. Kamon, Y. Minokoshi, Y. Ito, H. Waki, S. Uchida , et al . , "Adiponectin stimulates glucose utilization and fatty acid oxidation by activating AMP-activated protein kinase," Nat Med . , Vol. 8, pp. 1288-95, Nov 2002.

[12] T. Yamauchi, Y. Nio, T. Maki, M. Kobayashi, T. Takazawa, M. Iwabu , et al . , "Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions," Nat Med . , Vol. 13, pp. 332-9, Mar 2007.

[13] S. Lim, BK Koo, SW Cho, S. Kihara, T. Funahashi, YM Cho , et al . , "Association of adiponectin and resistin with cardiovascular events in Korean patients with type 2 diabetes: the Korean atherosclerosis study: a 42-month prospective study," Atherosclerosis , vol. 196, pp. 398-404, Jan 2008.

[14] K. Tsubakio-Yamamoto, F. Matsuura, M. Koseki, H. Oku, JC Sandoval, M. Inagaki , et al . , "Adiponectin prevents atherosclerosis by increasing cholesterol efflux from macrophages," Biochem Biophys Res Commun , vol. 375, pp. 390-4, Oct 24 2008.

[15] N. Maeda, I. Shimomura, K. Kishida, H. Nishizawa, M. Matsuda, H. Nagaretani , et al . , "Diet-induced insulin resistance in mice lacking adiponectin / ACRP30," Nat Med . , Vol. 8, pp. 731-7, Jul 2002.

[16] Y. Arita, S. Kihara, N. Ouchi, M. Takahashi, K. Maeda, J. Miyagawa , et al . , "Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity," Biochem Biophys Res Commun , vol. 257, pp. 79-83, Apr 2 1999.

[17] M. Ryo, T. Nakamura, S. Kihara, M. Kumada, S. Shibazaki, M. Takahashi , et al . , "Adiponectin as a biomarker of the metabolic syndrome," Circ J, vol. 68, pp. 975-81, Nov 2004.

[18] Y. Yamamoto, H. Hirose, I. Saito, K. Nishikai, and T. Saruta, "Adiponectin, an adipocyte-derived protein, predicts future insulin resistance: a two- J Clin Endocrinol Metab , vol. 89, pp. 87-90, Jan 2004.

[19] T. Yatagai, S. Nagasaka, A. Taniguchi, M. Fukushima, T. Nakamura, A. Kuroe , et al . , "Hypoadiponectinemia associated with visceral fat accumulation and insulin resistance in Japanese men with type 2 diabetes mellitus," Metabolism , vol. 52, pp. 1274-8, Oct 2003.

[20] M. Kumada, S. Kihara, S. Sumitsuji, T. Kawamoto, S. Matsumoto, N. Ouchi , et. al . , "Association of hypoadiponectinemia with coronary artery disease in men," Arterioscler Thromb Vasc Biol , vol. 23, pp. 85-9, Jan 1 2003.

[21] T. Yamauchi, J. Kamon, H. Waki, Y. Terauchi, N. Kubota, K. Hara , et al . , "The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity," Nat Med . , Vol. 7, pp. 941-6, Aug 2001.

[22] M. Iwaki, M. Matsuda, N. Maeda, T. Funahashi, Y. Matsuzawa, M. Makishima , et al . , "Induction of adiponectin, a fat-derived antidiabetic and antiatherogenic factor, by nuclear receptors," Diabetes , vol. 52, pp. 1655-63, Jul 2003.

[23] JG Yu, S. Javorschi, AL Hevener, YT Kruszynska, RA Norman, M. Sinha , et al . , "The effect of thiazolidinediones on plasma adiponectin levels in normal, obese, and type 2 diabetic subjects," Diabetes , vol. 51, pp. 2968-74, Oct 2002.

[24] Y. Kanatani, I. Usui, K. Ishizuka, A. Bukhari, S. Fujisaka, M. Urakaze , et al . , "Effects of pioglitazone on suppressor of cytokine signaling 3 expression: potential mechanisms for its effects on insulin sensitivity and adiponectin expression," Diabetes , vol. 56, pp. 795-803, Mar 2007.

[25] T. Pischon, CJ Girman, GS Hotamisligil, N. Rifai, FB Hu, and EB Rimm, "Plasma adiponectin levels and risk of myocardial infarction in men," JAMA , vol. 291, pp. 1730-7, Apr 14 2004.

[26] N. Ouchi, M. Ohishi, S. Kihara, T. Funahashi, T. Nakamura, H. Nagaretani , et al . , "Association of hypoadiponectinemia with impaired vasoreactivity," Hypertension , vol. 42, pp. 231-4, Sep 2003.

[27] M. Adamczak, A. Wiecek, T. Funahashi, J. Chudek, F. Kokot, and Y. Matsuzawa, "Decreased plasma adiponectin concentration in patients with essential hypertension," Am J Hypertens , vol. 16, pp. 72-5, Jan 2003.

[28] S. Furukawa, T. Fujita, M. Shimabukuro, M. Iwaki, Y. Yamada, Y. Nakajima , et al . , "Increased oxidative stress in obesity and its impact on metabolic syndrome," J Clin Invest , vol. 114, pp. 1752-61, Dec 2004.

[29] JB Seo, HM Moon, MJ Noh, YS Lee, HW Jeong, EJ Yoo , et al . , "Adipocyte determination- and differentiation-dependent factor 1 / sterol regulatory element-binding protein 1c regulates mouse adiponectin expression," J Biol Chem . , Vol. 279, pp. 22108-17, May 21 2004.

[30] SK Park, SY Oh, MY Lee, S. Yoon, KS Kim, and JW Kim, "CCAAT / enhancer binding protein and nuclear factor-Y regulate adiponectin gene expression in adipose tissue," Diabetes , vol. 53, pp. 2757-66, Nov 2004.

[31] L. Qiao, PS Maclean, J. Schaack, DJ Orlicky, C. Darimont, M. Pagliassotti , et al . , "C / EBPalpha regulates human adiponectin gene transcription through an intronic enhancer," Diabetes , vol. 54, pp. 1744-54, Jun 2005.

U.S. Patent Application No. 11/566441 relates to the inhibition of DNA methyltransferases such as DNMT1 and DNMT3b2. The present invention suggests a compound and a method for inhibiting DNMT1 and DNMT3b2. In U.S. Patent Application No. 11/791834, the present invention provides a method for improving or reducing ischemic injury, reperfusion injury, and myocardial infarction by administering an inhibitor of histone acetylase removal enzyme or an inhibitor of DNA methyltransferase enzyme (DNMT) .

Gerda Egger et al. Suggest that inhibitors of various enzymes, such as specific DNA methyltransferases and histone deacetylases, that regulate posterior genetic manipulation can prevent tumor formation for certain malignant tumors (Nature 429, pp 457 -463, May 27, 2004). Giovanni L. Gravina et al. Have proposed DNA methyltransferase (DNMT) as a progeny genetic modulator. The use of DNMT inhibitors provides an improved approach to cytotoxicity or radiation targeting DNA-protein complexes. Preclinical cancer models have demonstrated that DNMT inhibitors can be effective in the treatment of other types of cancer (Mol Cancer. 2010 Nov 25; 9: 305).

It is an object of the present invention to provide a method for screening a therapeutic agent for obesity or metabolic diseases.

It is still another object of the present invention to provide a therapeutic agent for obesity or metabolic diseases comprising a DNMT1 activity inhibitor.

In order to achieve the above object, a first aspect of the present invention is a method for culturing a cell, comprising: culturing a cell; Treating the cell with a candidate substance; Measuring methylation of the adiponectin promoter in the cell; And confirming that the methylation of the adiponectin promoter is reduced as compared with the control.

The methylation analysis may be performed using any one or more selected from the group consisting of an immunochemical method, a method using a radioisotope material, a method based on a difference in molecular weight due to electrophoresis, a method using a fluorescent dye or a bisulfite sequencing method A method for screening a therapeutic agent for obesity or a metabolic disease.

More specifically, the present invention further provides a method for screening a therapeutic agent for obesity or metabolic diseases, which further comprises confirming that the adiponectin expression level is increased.

More specifically, the present invention further provides a method for screening a therapeutic agent for obesity or metabolic diseases, which further comprises confirming that the expression amount of DNMT1 is decreased.

A second aspect of the present invention provides a therapeutic agent for obesity or metabolic diseases comprising a DNMT1 activity inhibitor. More specifically, the DNMT1 activity inhibitor is a substance that reduces gene expression of DNMT1, decreases protein expression of DNMT1, or shortens the protein lifetime of DNMT1, and provides a therapeutic agent for obesity or metabolic diseases. More specifically, the DNMT1 activity inhibitor comprises RG108 or 5-azacytidine.

The amount of adiponectin that is associated with obesity or metabolic disease can be regulated to be used for the screening of therapeutic agents for obesity or metabolic diseases.

Since DNMT1 is involved in the methylation of the adiponectin promoter, it can be used as a therapeutic agent for obesity or metabolic diseases through the inhibitor of DNMT1 activity.

FIG. 1 shows mRNA levels of adiponectin, PPARγ2, C / EBPα, and TNFα in adipocytes from normal chow diets (NCD) and high fat diets (HFD).
Figure 2 shows the CpG dinucleotide present in the adiponectin promoter.
Figure 3 shows the results of bisulfite sequencing of the adiponectin promoter R2 region in adipocytes of normal or high fat diabetic rats. The black circle represents methylated cytidine (hereinafter, 'C'), and the white circle represents C that is not methylated.
Figure 4 is a quantification of the results of Figure 3.
Figure 5 shows the correlation between the degree of methylation of the R2 region and the amount of adiponectin expression (mRNA level).
Figure 6 shows the results of bisulfite sequencing of the adiponectin promoter R1 region in adipocytes of normal or high fat diabetic rats.
Figure 7 is a quantification of the results of Figure 6.
Fig. 8 shows the correlation between the degree of methylation of the R1 region and the amount of adiponectin expression (mRNA level).
FIG. 9 shows mRNA levels of adiponectin, PPARγ2, TNFα and MCP-1 in adipocytes of wild type (WT) and db / db rats.
Figure 10 shows the results of bisulfite sequencing of the adiponectin promoter R2 region in normal or db / db rat adipocytes.
Fig. 11 shows the results of Fig. 10 quantified.
Figure 12 shows the correlation between the degree of methylation of the R2 region and the amount of adiponectin expression (mRNA level).
Figure 13 shows the results of bisulfite sequencing of the adiponectin promoter R1 region in normal or db / db rat adipocytes.
FIG. 14 shows the result of FIG. 13 quantified.
Fig. 15 shows the correlation between the degree of methylation of the R1 region and the amount of adiponectin expression (mRNA level).
Figure 16 shows the expression levels of DNMT1 and adiponectin in adipocytes (ADs) and SVCs of adipose tissue.
FIG. 17 is a graph showing the amount of mRNA of DNMT1, adiponectin and PPARγ in lipid precursor cells (Pre) and adipocytes (ADs).
18 is a measurement of the amount of protein of DNMT1, adiponectin, PPARγ, and β-actin in adipose precursor cells (Pre) and adipocytes (ADs).
FIG. 19 shows the expression levels of DNMT1 and adiponectin in adipocytes of normal (NCD) and high fat diets (HFD) in adipocytes or adipocytes of normal (WT) and db / db rats.
20 shows the expression pattern of adiponectin after inhibiting DNMT1 expression using siRNA in SVCs.
21 shows the expression pattern of adiponectin after inhibiting DNMT1 expression using siRNA in 3T3-L1 adipose cell line.
Figure 22 shows changes in adiponectin at the protein level. 23 shows the results of observing the methylation change on the adiponectin promoter by bisulfite sequencing when inhibiting the expression of DNMT in the 3T3-L1 adipose cell line.
Fig. 24 is a result of quantifying the results of Fig. 23. Fig.
25 shows the expression pattern of adiponectin after overexpressing DNMT1 in a 3T3-L1 adipocyte cell line.
Figure 26 shows changes in adiponectin at the protein level.
Figure 27 shows methylation changes on the adiponectin promoter observed with bisulfite sequencing when DNT1 overexpression in 3T3-L1 adipose cell line.
Fig. 28 shows the results of Fig. 27 quantified.
FIG. 29 shows changes in expression levels of adiponectin, DNMT1, MCP1, and iNOS after treatment with 10 ng / ml of TNF? For 24 hours in 3T3-L1 cells differentiated into adipocytes.
FIG. 30 shows DNA methylation changes of the adiponectin promoter R2 region by TNF? By bisulfite sequencing.
Fig. 31 shows the results of Fig. 30 quantified.
Figure 32 shows changes in body weight after daily injections of Vehicle (Veh), RG108, 5-aza-CR, and rogiglitazone in abdominal cavity of db / db rats for one month.
FIG. 33 shows the weight gain after daily injections of Vehicle (Veh), RG108, 5-aza-CR, and rogiglitazone into abdominal cavity of db / db rats for one month.
Fig. 34 shows the results of measuring the major organ weights after daily injections of Vehicle (Veh), RG108, 5-aza-CR, and rogiglitazone in abdominal cavity of db / db rats for one month.
FIG. 35 shows fasting blood glucose levels after daily injections of vehicle (Veh), RG108, 5-aza-CR, and rogiglitazone in abdominal cavity of db / db mice for one month.
FIG. 36 shows the results of vehicle (Veh), RG108, 5-aza-CR and rogiglitazone injected daily for one month in abdominal cavity of db / db rats and then measuring fasting insulin levels.
FIG. 37 shows triglyceride (TG) of serum after Vehicle (Veh), RG108, 5-aza-CR, and rogiglitazone were injected into abdominal cavity of db / db rats for one month each month.
FIG. 38 shows serum free fatty acid (FFA) levels measured after daily injections of Vehicle (Veh), RG108, 5-aza-CR, and rogiglitazone in abdominal cavity of db / db rats for one month.
FIG. 39 shows AST of serum (Veh), RG108, 5-aza-CR, and rogiglitazone after intraperitoneal injection of db / db rats for one month.
Figure 40 shows the result of glucose tolerance test.
41 shows the area under the graph of the glucose load test.
Figure 42 shows the amount of serum added to adiponectin.
Figure 43 shows DNA methylation changes of the adiponectin promoter R2 region in adipocytes by bisulfite sequencing.
FIG. 44 shows the result of FIG. 43 quantified.
Figure 45 shows human adiponectin promoter R2 region and CpG dinucleotide position.
Figure 46 shows the results of the bisphosphite sequencing of the adiponectin promoter R2 region in SVCs and adipocytes (ADs) in adipose tissue of humans.
FIG. 47 shows the results of FIG. 45 quantified.
Figure 48 shows the correlation between the degree of methylation of the R2 region and BMI.

Hereinafter, specific embodiments of the present invention will be described. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The scope of rights of the present invention extends to those parts of the technical field of the present invention which are obvious to those skilled in the art to easily substitute or change by using the prior art based on the matters described in the claims.

Example  1. Cell culture.

3T3-L1 cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% bovine calf serum (BCS). 3T3-L1 cells were grown in 100% confluency to induce differentiation into adipocytes. Ten days later, 10% fetal bovine serum (FBS), 0.5 mM 3-isobutyl-1- Methylxanthine (IBMX), 1 [mu] M dexamethasone, and 1 [mu] g / ml insulin for 2 days. The cells were then grown in DMEM medium supplemented with 10% FBS and 1 μg / ml insulin for 2 days, followed by DMEM medium supplemented with 10% FBS every other day. Stromal vascular cells (SVCs) were cultured for two days in DMEM medium supplemented with 10% FBS. To induce differentiation into adipocytes, SVCs were incubated with 10% FBS, 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, 850 nM insulin, 125 nM indomethacin, 1 nM triiodothyronine (T3) lt; RTI ID = 0.0 > uM < / RTI > They were then grown for 2 more days in DMEM medium containing 10% FBS, 850 nM insulin, 1 nM T3, and 1 μM rogglitaxin.

Example  2. Transient transduction ( Transient transfection ).

Each siRNA (20 μM), pcDNA3.1-myc-His, and pcDNA3.1-DNMT1 were transferred to SVCs or 3T3-L1 cells using a microporator.

Example  3. RNA  Separation and quantitative Real time - PCR .

Total RNA was extracted with Iso-RNA lysis reagent and cDNA was synthesized with RevertAid M-MuLV reverse transcriptase. The amount of relative mRNA CFX96 ^TM Real-time system was used and calibrated with the amount of cyclophilin mRNA and calculated.

Example  4. Genomic DNA  Extraction Bisulfite  Sequencing.

DNA was extracted with phenol-chloroform extraction method. Cells are lysed with lysis buffer (50 mM Tris-Cl (pH 8.0), 50 mM EDTA (pH 8.0), 100 mM sodium chloride, 2% SDS, 20 ug / ml RNase) for one hour at 37 ° C. And then reacted with 10 占 퐂 / ml proteinase K at 50 占 폚 for three hours. Then, phenol and chloroform are used to remove other cytoplasmic substances, and ethanol is used to precipitate the DNA. Bisulfite conversion was performed using an EpiTect bisulfite kit. The adiponectin promoter portion was amplified by PCR using the primers designed by Meti Primer Software (www.urogene.org/methprimer/index1.html) with the converted DNA. The PCR conditions were as follows. After a reaction at 95 ° C for 7 minutes, a cycle of 95 ° C for 1 minute, 55 ° C for 30 seconds, and 72 ° C for 1 minute was repeated 35 times, and an additional reaction was added at 72 ° C for 10 minutes. The PCR products were put into bacteria using a TOPO TA cloning kit. The clones were sequenced using the sequencing service.

Example  5. Western Blasting .

Cells were resuspended in TGN buffer (150 mmol / l sodium chloride, 50 mmol / l Tris, pH 7.5, 0.2% Nonidet P40; 1 mmol / l phenylmethylsulfonyl fluoride; 100 mmol / l sodium fluoride; 1 mmol / l Ordovana (Na ₃ VO ₄ ; 10 μg / ml aprotinin; 2 μg / ml pepstatin A; and 10 μg / ml leupeptin). Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (PVDF). Membrane is blocked with 5% nonfat milk and attached with primary antibody. Next, proteins were visualized using a secondary antibody coupled with horseradish peroxidase and chemiluminescence.

Example  6. Animal experiment.

All animal experiments were approved by Seoul National University Animal Experimental Ethics Committee. 6-week-old or 8-week-old C57BLKS / J- Lepr ^db / Lepr ^db (db / db) male, C57BLKS / J-m + male, and 8 weeks old C57BL / 6J male rats were purchased. All animals were raised in a group of 12 light and 12 dark cycles. RG108, 5-aza-CR and rosiglitazone dissolved in DMSO. Each drug was diluted in PBS to 1: 100 and injected into rats. Finally, rats received 1% DMSO (vehicle, control), 5.69 mg / kg / day of RG108, and 0.08 mg / kg / day of 5-aza-CR. 5 mg / kg / day of rosiglitazone was injected. The drug was injected into the abdominal cavity for one month each day. To determine fasting glucose and fasting insulin, the rats were starved for 16 hours and their blood glucose and insulin levels were measured. Glucose tolerance test was performed after starving db / db rats and normal rats (m + / m +) for 16 hours and 6 hours, respectively, after feeding 1 g / kg and 3 g / kg of glucose. Serum triglyceride and free fatty acid were measured to evaluate liver toxicity.

Example  7. Classification of adipose tissue.

The adipose tissue of the testis was removed from the rats and plated in PBS and stained with Krebs-Ringer phosphate buffer (pH 7.4) containing 2% bovine serum albumin (BSA) and type I collagenase (1.75 mg / ml) . The tissue reacted with the enzyme is filtered once with 250 μm nylon mesh and then centrifuged at 3000 rpm for 5 minutes. Wash the upper part of the adipocyte with SVCs submerged in the bottom several times with PBS.

Example  8. Sample of human fat tissue.

Human fat tissue (three obesity or overweight patients, one normal person) was obtained from King's Saud University Obesity Research Center. After adipocyte separation, the degree of methylation of the adiponectin R2 region was analyzed by bisulfite sequencing. All experiments were reviewed by King's Saud University's College of Medicine Ethics Committee.

Example  9. Statistical analysis.

All results were expressed as mean ± standard deviation. Statistical significance was assessed using either Student's t- test or Paired t- test. Differences were considered only when statistical significance was P < 0.05.

Example  10. Fat cells in obese rats Adiponectin  Increased methylation of specific promoter regions.

Consistent with previous reports, adiponectin mRNA expression was reduced in adipocytes from high fat diabetic obese mice (Fig. 1). However, there was no change in the degree of mRNA of PPARγ2 or C / EBPα, a major transcription factor that regulates the expression of adiponectin (FIG. 1). This suggests that the mRNA of adiponectin, which decreases in obesity, can be achieved through other regulatory mechanisms. Epigenetic regulation is one of the important transcriptional control mechanisms, and transcription factors are responsible for accessing the target site. The chromatin structure is varied by epigenetic regulation, such as histone modification and DNA methylation.

To investigate whether postmenopausal changes contribute to decreased adiponectin expression by obesity, we examined the degree of DNA methylation in the adiponectin promoter after extracting adipocytes from normal or high fat diets. There are six CpG dinucleotides in the 1.2 kb upstream of the mouse adeponectin promoter (Figure 2). DNA methylation degree of adiponectin promoter was observed in two regions (Region 1 and Region 2). Region 1 (R1) is relatively close to the transcription start site, and Region 2 (R2) is about 1 kb upstream from the transcription start site (FIG. 2). As shown in FIGS. 3 and 4, it can be seen that adiponectin promoter R2 region is hypermethylated in adipocytes of obese rats fed a high-fat diet as compared to a normal diet. Interestingly, the degree of methylation of the R2 region was negatively correlated with the mRNA expression level of adiponectin (Fig. 5). On the other hand, DNA methylation in the two CpG dinucleotides at the R1 site was not significantly different between the two groups (Figures 6 to 8).

Next, we investigated whether the above phenomenon occurs in obese or obese db / db rats as well as in obese mice. Similar to the results of the high fat diet, adiponectin expression was reduced in db / db mice (Fig. 9) and DNA methylation of the R2 region of the adiponectin promoter was observed to be higher than in WT rats (Figs. 10-12). Whereas DNA methylation of the R1 region was unchanged (Figures 13-15).

These results suggest that DNA methylation of a specific region (R2) on the adiponectin promoter may be a major cause of decreased expression of adiponectin in obese conditions.

Example  11. DNMT1 On by Adiponectin  Regulation of promoter methylation.

DNA methylation is carried out by DNA methyltransferase (hereinafter referred to as 'DNMT') enzyme. There are three DNMTs (DNMT1, DNMT3a, DNMT3b) in mammals. To investigate which DNMT regulates DNA methylation on the adiponectin promoter, we first examined the expression level of DNMT. Among the three DNMTs, the expression of DNMT1 in adipose tissue was decreased in adipocytes compared to SVCs, and the expression was significantly lower in differentiated adipocytes compared to adipocyte precursors (Figs. 16 to 18). In addition, it was observed that the expression level of DNMT1 was increased in adipose cells of db / db rats fed with high fat diet (Fig. 19). The above results suggest that increased DNMT1 in the obese condition can induce DNA methylation of the adiponectin promoter R2 region.

Next, we examined whether DNMT1 can regulate the expression level of adiponectin. When siRNAs were used to inhibit the expression of DNMT1 in SVCs having lipid precursor cells and 3T3-L1 cells as lipid precursor cells, both mRNA and protein of adiponectin were increased (Figs. 20 to 22). In addition, the DNA methylation of the R2 region was significantly reduced (Figures 23 and 24). Conversely, when DNMT1 was overexpressed in 3T3-L1 cells, the expression level of adiponectin was markedly decreased (FIGS. 25 and 26). This reduction in adiponectin expression by DNMTl overexpression was accompanied by an increase in DNA methylation of the adiponectin promoter R2 region (Figures 27 and 28).

These results indicate that DNMT1 is involved in the expression of adiponectin by regulating DNA methylation of the adiponectin R2 region. In addition, DNMT1, which is increased in adipocytes of obese individuals, may be a major cause of decreased adiponectin expression by inducing DNA methylation of specific regions of adiponectin.

Example  12. TNF by Adiponectin  For the promoter DNA  Induction of methylation.

In obesity, adipose tissue is accompanied by a low chronic inflammatory response. This inflammatory response induces an increase in the secretion of inflammatory cytokines in adipose tissue, and TNFα, a typical inflammatory cytokine, is known to decrease adiponectin expression. However, the molecular mechanism for the reduction of TNFα-induced adiponectin expression has not been clearly elucidated. To investigate whether the increase of inflammatory cytokines inhibits the expression of adiponectin through DNA methylation on the adiponectin promoter, 3T3-L1 cells differentiated into adipocytes were treated with TNFα and compared with the level of adiponectin expression and the degree of DNA methylation. As shown in FIGS. 29 to 31, DNMT1 expression was increased when treated with TNFα, and DNA methylation was also increased at the R2 region of the adiponectin promoter. These results suggest that hypermethylation of the R2 region of the adiponectin promoter may be a cause of decreased adiponectin expression by inflammatory cytokines such as TNF [alpha].

Example  13. DNMT  Inhibitor Adiponectin  Improvement of metabolic disease through increased expression.

In order to investigate the effect of inhibition of DNA methylation at the individual level on the recovery of adiponectin expression and the improvement of metabolic diseases, db / db mice were treated with DNMT inhibitor RG108 and methylated non-methylated 5-azacytidine (5-azacytidine, hereinafter referred to as '5-aza-CR'). In the groups of rats injected with RG108 and 5-aza-CR, there was no difference in the body weight change compared to the control group (Figures 32 to 34), but fasting blood glucose and fasting insulin were decreased (Figures 35 and 36), blood triglyceride Fatty acids also decreased (Figs. 37 and 38). In addition, glucose tolerance test showed that glucose intolerance was improved in both groups compared to the control group (FIGS. 40 and 41). While toxicity did not appear (Fig. 39). These results suggest that the inhibition of DNMT activity or the inhibition of DNA methylation may improve metabolic diseases.

Next, we examined whether these improvements were due to decreased DNA methylation on the adiponectin promoter and increased expression of adiponectin. As shown in Figure 42, both RG108 and 5-aza-CR increased the amount of adiponectin in the blood. In addition, adiponectin DNA methylation in adipocytes was also reduced compared to the control (Fig. 43). This suggests that the effect of DNMT inhibitors is due to increased expression of adiponectin by decreasing DNA methylation on the adiponectin promoter.

These results suggest that DNMT inhibitors increase the expression of adiponectin, which is a potential therapeutic agent for obesity and metabolic diseases.

Example  14. People's Adiponectin  Promoter R2  Of fat cells in obese people DNA And methylation .

A human adiponectin promoter has high base sequence homology with the rat adiponectin promoter R2 region. To determine whether these three CpG dinucleotides were negatively correlated with adiponectin production, we also obtained adipose tissue from a different body mass index (BMI), followed by adipocytes and SVCs DNA methylation of the adiponectin promoter R2 region was examined (Fig. 46). As in the case of rats, DNA methylation of the R2 region in adipocytes was found to have a positive correlation with obesity (Figs. 47 and 48). This result suggests that DNA methylation at the R2 region of the human adiponectin promoter can regulate adiponectin expression. Thus, increased DNA methylation in the R2 region can be one of the major factors in the reduction of adiponectin expression in both mice and humans.

Claims (9)

Culturing the cells;
Treating the cell with a candidate substance;
Measuring methylation of the adiponectin promoter in the cell; And
And confirming that the methylation of the adiponectin promoter is reduced as compared to the control. The method according to claim 1, wherein the methylation analysis is performed using an immunochemical method, a method using a radioisotope material, a method based on difference in molecular weight according to electrophoresis, a method using a fluorescent dye or a method for confirming using bisulfite sequencing And a method for screening a therapeutic agent for obesity or metabolic disease. The method for screening a therapeutic agent for obesity or metabolic diseases according to claim 1, wherein the cell is an animal cell. The method for screening a therapeutic agent for obesity or metabolic diseases according to claim 3, wherein the animal cell is an adipocyte. The method for screening a therapeutic agent for obesity or metabolic diseases according to claim 1, further comprising the step of confirming that the amount of adiponectin expression is increased. The method according to claim 1, further comprising the step of confirming that the expression level of DNMT1 is decreased. A therapeutic agent for obesity or metabolic diseases comprising a DNMT1 activity inhibitor. 8. The therapeutic agent for obesity or metabolic diseases according to claim 7, wherein the DNMT1 activity inhibitor is a substance that decreases gene expression of DNMT1, decreases protein expression of DNMT1, or shortens the protein life of DNMT1. 8. The therapeutic agent for obesity or metabolic diseases according to claim 7, wherein the DNMT1 activity inhibitor comprises RG108 or 5-azacytidine.

Images