KR100875493B1 - Composition for inhibiting fat formation, containing clioquinol and its derivatives as active ingredients - Google Patents

Abstract

The present invention relates to a composition for inhibiting fat formation, containing clioquinol and its derivatives as an active ingredient, which is effective in inhibiting fat formation in a concentration dependent and effective manner of clioquinol during adipocyte differentiation, and reducing the expression of fat acid synthase (FAS). It can be usefully used for inhibiting fat formation and for treating and preventing obesity.

Description

Inhibiting composition of lipogenesis containing clioquinol and derivatives etc.

Figure 1 is a picture of fat cells investigated the effect of hypoxia on fat formation.

2 is a graph illustrating the effect of hypoxia on triglyceride and cholesterol formation.

Figure 3a is a result of examining the expression of fatty acid synthase (FAS) in the hypoxic state:

  A: analysis of FAS expression on Hep3B, L6, C2C12 and 3T3-L1 by Northern blot method; And

  B: The degree of FAS expression on Hepa1c1c7 cell line was analyzed by Northern blot method.

FIG. 3B shows the results of changes in hypoxia in wild type Hepa1c1c7 cell line and HIF-1β-deficient Hepa1c1c7 cell line:

  C: graph of the degree of ATP production under normal and hypoxic state on wild-type Hepa1c1c7 cell line;

  D: Gel photograph showing the extent of HIF-1β expression on HIF-1β-deficient Hepa1c1c7 cell line by Western blot; And

  E: Results of investigation of decreased expression of FAS on HIF-1β-deficient Hepa1c1c7 cell line.

4A shows the results of examining the degree of ADD1 / SREBP1c expression under hypoxia:

  A: graph of the expression level of ADD1 / SREBP1c mRNA under normal and hypoxic states by real-time PCR; And

  B: Gel photograph which investigated the expression level of ADD1 / SREBP1c protein under normal oxygen state and hypoxic state.

4b shows the expression level of ADD1 / SREBP1c mRNA in HIF-1β-deficient Hepa1c1c7 cell line:

  C: Northern blot method; And

  D: Western blot method.

5 shows the results of investigating the synthesis of new proteins associated with inhibition of FAS expression under hypoxic conditions:

  A: Gel photograph showing expression of FAS mRNA under normal oxygen and hypoxia after Cycloheximide (CHX) treatment on wild type and HIF-1β-deficient Hepa1c1c7 cell lines;

  B: Gel photograph showing Western blot expression of STRA13 and HIF-1α on 3T3-L1 cell line;

  C: Gel picture examining expression of STRA13 mRNA on 3T3-L1 cell line: and

  D: Gel photograph showing mRNA expression of STRA13 on wild type and HIF-1β-deficient Hepa1c1c7 cell lines.

6A shows the results of investigating the regulation of the FAS promoter under hypoxic conditions:

  A: FAS promoter-luciferase reporter sequence; And

  B: transient transgenic luciferase assay graph under normal and hypoxic conditions.

6B shows the results of the regulation of the ADD1 / SREBP1c promoter under hypoxic conditions:

  C: ADD1 / SREBP1c promoter-luciferase reporter sequence; And

  D: transient transgenic luciferase assay graph under normal and hypoxic states; And

  E: Western blot results under normal and hypoxic conditions.

Figure 7a shows the structure and the amount of IVTT expression of HIF-1α, HIF-1β, STRA13 and ADD1 / SREBP1c.

  A: Diagram showing domain structure of bHLH transcription factor of HIF-1α, HIF-1β, STRA13 and ADD1 / SREBP1c and preparation of IVTT-ADD1, IVTT-HIF-1α, IVTT-HIF-1β and IVTT-ADD1 / SREBP1c Check the gel picture.

7b shows the results of investigating the binding activity of ADD1 / SREBP1c with the FAS promoter in the presence of HIF-1α / β and STRA13:

  B: Gel photograph showing changes in binding activity between GST-ADD1 / SREBP1c and FAS promoter in the presence of STRA13, HIF-1α and HIF-1β;

  C: gel photograph showing the binding activity of IVTT-ADD1 / SREBP1c with FAS promoter in the presence of STRA13, HIF-1α and HIF-1β; And

  D: Gel photograph showing the binding activity between ADD1 / SREBP1c and FAS promoter in the cell nucleus in hypoxic state.

8A is the sequence of the ADD1 / SREBP1c promoter region:

  A: Wild type, SRE variant, E-box variant sequence of the ADD1 / SREBP1c promoter region.

8B shows the results of investigating the binding activity of the ADD1 / SREBP1c and ADD1 / SREBP1c promoters in the presence of HIF-1α / β and STRA13:

  B: Gel photograph showing the binding activity of the ADD1 / SREBP1c and ADD1 / SREBP1c promoters in the presence of ADD1, STRA13, HIF-1α and HIF-1β; And

  C: Gel photograph showing the binding activity of ADD1 / SREBP1c and ADD1 / SREBP1c promoters in the presence of ADD1, STRA13, HIF-1α and HIF-1β, SRE variants and E-box variants.

9A shows the results of examining the binding between bHLH transcriptional activators:

  A: Gel photograph examined by GST pull down system.

9B shows the results of examining the binding between the bHLH transcriptional activators:

  B: Gel photographs examined by immunoprecipitation method (IP) and Western blot in the presence of ADD1, STRA13 and HIF-1α; And

  C: Graph showing the reporter gene expression during ADD1 / SREBP1c, STRA13, HIF-1α transformation.

10 is a cell photograph confirming the effect of inhibiting fat formation by clioquinol (5 μM).

11 is a cell photograph confirming the effect of inhibiting fat formation by clioquinol (1, 2 and 5 μM).

12 is a result confirming the effects of FAS and VEGF expression by clioquinol and its derivatives.

The present invention relates to a composition for inhibiting fat formation containing clioquinol and derivatives thereof as an active ingredient. Specifically, the composition for inhibition of the present invention effectively inhibits fat formation and inhibits the expression of fatty acid synthase (FAS).

Under hypoxic conditions, cells cannot maintain the oxygen respiration required for oxidative phosphorylation by mitochondria, resulting in a decrease in ATP production. Many types of oxygen-tolerant cells have addressed the risk of energy deficiency due to increased anaerobic metabolism as well as reduced demand for energy (Hochachka, PW 1986, Science, 231, 23-241). Many studies show that such adaptation mechamism depends primarily on anaerobic metabolism due to increased activity of glycolytic enzymes. In contrast, there is evidence that reduced demand for ATP is also important for adaptation. Extensive protein synthesis was rapidly inhibited in hepatocytes that were adapted to oxygen deprivation, which reduced the demand for ATP (Hochachka, PW et al ., 1996, Proc. Natl. Acad. Sci. USA 93: 9493-9498 ). These results suggest that hypoxic cells reduce anabolic processes, such as ATP-intensive protein and lipid synthesis, and we believe that this process occurs before the reduction of the actual ATP level and feedforward. It was assumed that regulation took place. It has also been reported that adipocyte differentiation is inhibited in hypoxia (Yun. Z. et al., 2002, Dev Cell. 2: 331-341).

The hypoxic state induces the expression of many genes by increasing the level of hypoxia-inducible factor 1α (hIF-1α), a basic helix-loop-helix (bHLH) -PAS transcription factor. HIF-1α has a functional heterodimer form with HIF-1β (bHLH-PAS protein). HIF-1β was found to be an aryl hydrocarbon receptor nuclear tranlocator (Arnt), which was found to be an aryl hydrocarbon receptor (AHR) pair (Li, H., et. al . , 1996, J. Biol. Chem, 271: 21262-21267; Wang, GL et al ., 1995, J. Biol. Chem. 270: 1230-1237). Highly conserved HIF-1β is stable, while HIF-1α is extremely unstable, which is regulated by oxygen. Translated HIF-1α protein in normal oxygen state is ubiquitinated and rapidly degraded by the ubiquitin proteosome metabolism (Huang, LE, et. al . , 1996, J. Biol. Chem. 271: 32253-32259; Kallio, PJ, et al ., 1999, J., Biol. Chem. 274: 6519-6525). HIF-1α contains an oxygen-dependent degradation domain (Huang, LE, et al ., 1998, Proc. Natl. Acad. Sci. USA 95: 7987-7992), there is a highly conserved region that contains sites that bind to the tumor suppressor pVHL (von Hippel-Lindau protein) (Cockman, ME, et. al ., 2000, J. Biol. Chem. 275: 25733-25741. pVHL constitutes the assembly of a complex that activates the ubiquitin E3 ligase, which ubiquitizes HIF-1α as a target for degradation. Recent findings suggest that pVHL is defective with conserved proline residues on the ODD (oxygen dependent degradation domain) domain. Hydroxide is in the process of molecular oxygen and Fe ^{2 +} required (Ivan, M., et al ., 2001, Science 292: 464-468; Jaakkola, P., et al . , 2001, Science 292: 468-472).

Lipogenesis refers to the process by which glucose is transformed into triglycerides by several enzymes and occurs in the liver and adipose tissue. Glucose is converted to acetyl-CoA by glycolytic enzymes, acetyl-CoA to malonyl-CoA by ACC (acetyl-CoA carboxylase), and malonyl-CoA to FAS ( Fatty acid is converted into fatty acid by fatty acid synthase, fatty acid is converted to fatty acyl-CoA by glycerol-P-transferase, and fatty acyl-CoA is converted to triglyceride by other fat-forming enzymes (Kersten, S. , 2001, EMBO, Rep. 2: 282-286). Fatty acid synthase (FAS), a fat-forming enzyme responsible for the endogenous synthesis of fatty acids, was found to be one of the products regulated by hormone and nutrient promotion at the level of transcription and activity (Bennett, MK, et. al ., 1995, J. Biol. Chem. 270: 25578-25583; Wang, Y., et al . , 2004, J. Nutr. 134: 1032-1038).

Insulin and sterols have long-term effects on FAS gene expression (Assimacopoulos-Jeannet, FS, et al., 1995, Metabolism 44: 228-233), and are usually transcription factors, ADD1 / SREBP1c (adipocyte determination, and differentiation-dependent factor). 1 / sterol regulatory element binding protein 1c) (Horton, JD, 1999, J. Clin . Invest . 103: 1067-1076; Shimomura, I., et al . , 1999, J. Biol . Chem . 274: 30028-30032). ADD1 / SREBP1c belongs to the bHLH leucine zipper family and is synthesized by binding to the endoplasmic reticulum membrane as a 125 kDa precursor protein. ADD1 / SREBP1c is cleaved by sterol-dependent continuous two-step proteolysis and separated from the endoplasmic reticulum membrane, where 68 kDa mature transcription factors, including the N-terminal region of ADD1 / SREBP1c, are transferred into the nucleus (Brown, MS , et al ., 1997, Cell , 89: 331-340). Nuclear migration by protein cleavage is initiated primarily by reduction of sterols (Wang, X., et al ., 1994, Cell 77: 53-62) and polyunsaturated fatty acids (Yahagi, N., et. al . , 1999, J. Biol. Chem . 274: 35840-35844). In addition, the levels of ADD1 / SREBP1c mRNA and protein were expressed in AMP-activated protein kinase (AMPK) and fasting intake (Gosmain, Y., et. al . , 2005, J. Lipid Res . 46: 697-705) and insulin (Chen, G., et al . , 2004, Proc . Natl . Acad . Sci . USA , 101: 11245-11250; Yoshikawa, T., et al ., 2001, Mol . Cell . Biol . 21: 2991-3000). Phosphorylation of AMPK by metformin activates phosphorylation of ACCs to inhibit activity and cause a decrease in ADD1 / SREBP1c expression (Zhou, G., et al ., 2001, J. Clin. Invest. 108: 1167-1174).

Clo quinol (Clioquinol: hereinafter "CQ" hereinafter) is Zn ^{2 +,} Cu ^{2 +,} and use sikimyeo selectively chelated by the same metal ions as Ca ^{2 +,} to adjust the effect of the heavy metal ion in the enzyme activity and protein formation do. PKa value of CQ is Cu ^{2 +,} 15.8; Zn ^{2 +,} 12.5; Ca ^{2 +,} 8.1; A Mg ^{2 +, 8.6 (Agrawal, YK} et al ., J Pharm Sci . 75: 190-192, 1986). CQ is hydrophobic and crosses the blood brain barrier. CQ has recently been reassessed as a prototype metal-protein attenuating compound that reduces metalloprotein precipitation and subsequent oxidative stress in Alzheimer's disease, Parkinson's disease and Huntington's disease. CQ was spotlighted as an antibiotic in the mid-1900s but was recovered in Japan as a cause of myeloid-visual neuropathy (Cherny, RA, Neuron 30: 665-676, 2001; Kaur, D. et. al ., Neuron 37: 899-909, 2003; Nguyen T. et al ., Proc Natl Acad Sci USA 102: 11840-11845, 2005). In a recent study of APP2576 transgenic mice, an animal model of Alzheimer's disease, CQ has been reported to reduce amyloid beta plug and amyloid serum levels without adverse effects (Doraiswamy, PM et. al ., Lancet Neorul 3: 431-434, 2004). In a recent phase 2 clinical trial involving 36 Alzheimer's patients, CQ slowed cognitive decline and significantly reduced amyloid beta levels (Ritchie CW et. al ., Arch Neurol 60: 1685-1691, 2003).

CQ also induces cell death in several human cancer cell lines. Addition of copper or zinc to CQ-treated cancer cell lines did not prevent cell death due to CQ, but rather increased cell death. An increase in zinc level was observed by fluorescent labeling of CQ-treated cells, suggesting that CQ causes cell death by activation of zinc ionophores (Ding, WO, et al ., Cancer Res . 65: 3389-3395, 2005). To date, the relationship between CQ and fat formation is unknown.

The present inventors completed the present invention by confirming that CQ and its derivatives inhibited fat formation while investigating the effect of HIF-1 on fat formation.

An object of the present invention is to provide a composition for inhibiting fat formation and health food for preventing and inhibiting obesity comprising clioquinol or a derivative thereof as an active ingredient.

In order to achieve the above object, the present invention provides a composition for inhibiting lipogenesis (lipogenesis) comprising clioquinol or a derivative thereof containing an active ingredient.

In addition, the present invention provides a health food for the prevention and inhibition of obesity containing an active ingredient clioquinol or a derivative thereof.

In addition, the present invention provides a composition for inhibiting fat acid synthase (FAS) comprising clioquinol or a derivative thereof as an active ingredient.

The present invention also provides a composition for inhibiting fat formation, comprising STRA13 protein as an active ingredient.

In addition, the present invention provides a composition for inhibiting fat formation, comprising HIF-1α protein as an active ingredient.

Hereinafter, the present invention will be described in detail.

The present inventors examined the fat formation of 3T3-L1 cell line, which is a progenitor cell under hypoxic and normal oxygen conditions. As a result, it was confirmed that the fat formation is inhibited when the hypoxic state as known in the prior art (see Fig. 1). In addition, as a result of examining the effects on the formation of triglycerides and cholesterol under the hypoxic state, it was observed that the production of triglycerides and cholesterol is inhibited in the hypoxic state than in the normal oxygen state (see Fig. 2). Then, the expression level of FAS in normal and hypoxic state was examined in various cell lines. As a result, it was observed that mRNA expression of FAS decreases in the hypoxic state (see FIG. 3A). The amount of ATP under hypoxic conditions was also measured. As a result, it was measured that the amount of ATP was reduced under the hypoxic state than the normal oxygen state (see C of FIG. 3B). This suggests that, under hypoxic conditions, anabolic enzymes such as FAS can be reduced to prevent assimilation before ATP is actually reduced. We then prepared a HIF-1β-deficient cell line and compared the mRNA expression levels of FAS on the cell line with wild-type cell lines. As a result, it was confirmed that the expression of FAS mRNA suppressed due to hypoxia on HIF-1β-deficient cell line is lost. This suggests that HIF-1β is an essential molecule for reducing hypoxic expression of FAS.

Lipid biosynthesis gene expression is initiated by potential fat biosynthesis activators such as ADD1 / SREBP1c. ADD1 / SREBP1c belongs to the family helix-loop-leucine zipper (bHLH-LZ). Using real-time PCR, the present inventors confirmed that the mRNA amount of ADD1 / SREBP1c was reduced in the HepG2 cell line, which is human hepatocytes (see A of FIG. 4A), and the ADD1 / SREBP1c protein level was also reduced (FIG. 4B). B). Northern analysis and RT-PCR results show that ADD1 / SREBP1c is reduced under hypoxic conditions in wild-type cells, but not in HIF-1β-deficient Hepalclc7 cells. This suggests that HIF-1β is required for hypoxic reduction of ADD1 / SREBP1c (see C of FIG. 4B). Later, Western blot confirmed that hypoxia failed to decrease the expression of ADD1 / SREBP1c in HIF-1β deficient cells (see FIG. 4B). This suggests that HIF-1β is required for hypoxic reduction of ADD1 / SREBP1c (see C and D in FIG. 4B).

To investigate the association of hypoxic inhibition of FAS with the synthesis of new proteins, we treated cycloheximide on Hepalclc7 cells and HIF-1β-deficient Hepalclc7 cells prior to hypoxic exposure (see FIG. 5). FAS mRNA levels in hypoxic cells were not reduced by cycloheximide treatment in wild-type Hepalclc7 cells. However, in HIF-1β-deficient cell lines, FAS expression did not change in both hypoxic and cycloheximide treatment groups. This suggests that hypoxic inhibition of FAS requires the synthesis of new proteins. The hypoxic state induces the synthesis of many new proteins. In addition, by inhibiting ubiquitination, HIF-1α protein is stabilized 1 hour after hypoxic exposure (Miyazaki, K. et. al . , 2002, J. Biol. Chem . 277: 47014-47021; Sun, H., and R. Taneja, 2000, Proc . Natl . Acad . Sci . USA 97: 4058-4063; Yun, Z., et al , Dev . Cell 2: 332-341). Through this, it can be seen that HIF-1α / β double complexes function as transcriptional activators and increase the expression of their target genes. HIF-1 also activates the expression of specific transcriptional inhibitors. In the hypoxic state, the basic helix-loop-helix protein, STRA13 / DEC1 / SHARP2, is HIF-1 dependent and functions as an inhibitor in the expression of several genes including PPARγ2, c-Myc, and STRA13 (Azmi, S. et al. , et al . , 2003, J. Biol . Chem . 278: 20098-20109; St-Pierre, B., et al ., 2022, J. Biol . Chem . 277: 46544-46555). STRA13 binds to E-Box to form homozygotes and inhibit target genes (St-Pierre, B., et al . , 2022, J. Biol . Chem . 277: 46544-46555). Western blot results showed that hypoxia increased levels of both HIF-1α and STRA13 (see B and C in FIG. 5) and that expression of STRA13 was dependent on HIF-1 (see D in FIG. 5).

Experimental Results As HIF-1 inhibited the activity and amount of ADD1 / SREBP1, we investigated changes in ADD1 / SREBP1c activity on the FAS promoter in the presence of HIF-1α or STRA13. We transiently transformed the plasmid expressing ADD1 / SREBP1c with the FAS promoter reporter plasmid containing the upper region (-220 bp to +25 bp) of the rat FAS gene (A in FIG. 6A), and ADD1 / SREBP1c. Overexpression of was found to increase the expression of the reporter gene (see FIG. 6B). In the presence of overexpressed ADD1 / SREBP1c, the hypoxic state still inhibits FAS expression, meaning that the hypoxic state not only reduces the amount of ADD1 / SREBP1c but also inhibits its activity, thereby inhibiting the expression of FAS. Transforming HIF-1α and STRA13 together with ADD1 / SREBP1c reduces FAS expression in normal cells, indicating that HIF-1α and STRA13 inhibit the activity of ADD1 / SREBP1c on the FAS promoter.

The ADD1 / SREBP1c promoter contains a sterol regulatory element sequence, and ADD1 / SREBP1c binds to its promoter and increases its transcription. We transiently transformed the plasmid encoding ADD1 / SREBP1c with a reporter plasmid comprising the upper region (2.7 kb) of the mouse ADD1 / SREBP1c gene, or transiently transformed with HIF-1α or STRA13, followed by ADD1. The change in / SREBP1c promoter activity was investigated (see C in FIG. 6B). Overexpression of ADD1 / SREBP1c increased the expression of the reporter gene (see D in FIG. 6B). Even with overexpression of ADD1 / SREBP1c, the activity of the ADD1 / SREBP1c promoter was reduced in hypoxia. Transformation of HIF-1α or STRA13 with ADD1 / SREBP1c inhibited the ADD1 / SREBP1c promoter more than in steady state cells, indicating that HIF-1α and STRA13 inhibited the ADD1 / SREBP1c promoter (see D in FIG. 6B). ). We transformed HIF-1α or STRA13 into HepG2 cells to measure ADD1 protein. As shown in E of FIG. 6B, it was confirmed that overexpression of HIF-1α or STRA13 inhibited the amount of ADD1 / SREBP1c than in steady state cells, thereby confirming that HIF-1α and STRA13 are related. Overexpression of HIF-1α in hypoxic cells further inhibits ADD1 / SREBP1c.

The inventors then investigated whether HIF-1α or STRA13 inhibited the binding of ADD1 / SREBP1c to DNA because it has the bHLH domain required to bind to the E-box sequence (-CANNTG-) complex-dependently. (See A of FIG. 7A). ADD1 / SREBP1c is a specific bHLH-leucine zipper protein with two types of DNA sequence specificities. ADD1 / SREBP1c specifically binds not only to the E-box sequence but also to the SRE (sterol regulatory element) sequence. Most SREBPs (sterol regulatory binding proteins) recognize and bind to SRE. In contrast, ADD1 / SREBP1c binds to and activates a promoter comprising a FAS promoter and an E-Box (see A of FIG. 6A and C of FIG. 6B) (Magana, MM, et. al . , 1997, J. Lipid Res . 38: 1630-1638; Magna, MM, and TF Osborne. 1996, J. Biol . Chem . 271: 32689-32694). Two independent and specific SREBP binding sites are on either side of the E-Box sequence on the FAS promoter (Magna, MM, and TF Osborne. 1996, J. Biol. Chem. 271: 32689-32694). As can be seen in the in vitro EMSA results of B of FIG. 7B, the recombinant GST-ADD1 / SREBP1c fusion protein binds to the E-box containing sequence of the FAS promoter (see A of FIG. 7A), but in vitro-translated STRA13 homocomplex Or in vitro-translated HIF-1α / β heterocomplexes did not bind. Instead, their presence prevents ADD1 / SREBP1c from binding to the FAS promoter (see B in FIG. 7B). We used in vitro-translated ADD1 / SREBP1c protein for EMSA. We found that in vitro-transcribed ADD1 / SREBP1c binds to the FAS promoter, but ADD1 / SREBP1c binding is inhibited in the presence of STRA13 and HIF-1α (see C in FIG. 7B). We also used nuclear extracts to investigate the share of the FAS promoter in cells and found that the protein binds to the FAS promoter (see D in FIG. 7B). Addition of the ADD1 / SREBP1c antibody causes the loss of the DNA / protein complex, indicating that ADD1 / SREBP1c is included. Consistent with the results of B and C of FIG. 7B, hypoxic treatment reduces the amount of ADD1 / SREBP1c comprising the DNA / protein complex. These findings indicate that hypoxia-induced HIF-1α / β and STRA13 inhibit ADD1 / SREBP1c binding to the FAS promoter.

The fact that ADD1 / SREBP1c binds to its promoter and activates transcription indicates that the hypoxic state can also inhibit ADD1 / SREBP1c from binding to its promoter, thereby inhibiting the amount of ADD1 / SREBP1c. The ADD1 / SREBP1c promoter contains the SRE complex (-92 to -56 bp of the mouse ADD1 / SREBP1c gene) and it has been found that they are activated by ADD1 / SREBP1c, ie (Amemiya-Kudo, M., et. al ., 2000, J. Biol . Chem . 275: 31078-30185; Kim, JB, et al . , 1995, Mol . Cell . Biol . 15: 2582-2588). The SRE complex also has an E-box, sterol responsive element (SRE), NF-Y and Spl sites (see A in FIG. 8A). EMSA shows that GST-ADD1 / SREBP1c binds to the SRE complex containing oligonucleotides. Interestingly, both the STRA13 homo- and HIF-1α / β heterocomplexes can bind to the SRE complex (down arrow in B of FIG. 8B). Experimental results showed that the binding of the STRA13 homo- or HIF-1α / β heterocomplexes to the ADD1 / SREBP1c promoter was specific for the E-box sequence or the SRE sequence. We added an excess of unlabeled variant oligonucleotides (see A of FIG. 8A). Addition of unlabeled SRE variant oligonucleotides disrupted STRA13 and HIF-1α / β binding, and addition of unlabeled E-box variants failed. These results demonstrate that either the STRA13 homocomplex or HIF-1α / β heterocomplex can bind to the E-box sequence but not to the SRE sequence on the ADD1 / SREBP1c promoter (see C in FIG. 8B). This suggests that STRA13 and HIF-1α / β compete with ADD1 / SREBP1c for binding to E-box in the SRE complex.

In order to investigate whether HIF-1α and STRA13 induced by hypoxic states bind to and inhibit the activity of ADD1 / SREBP1c protein, the inventors of the present invention, GST-ADD1 / SREBP1c fusion protein expressed in bacteria in GST pull down assay and [ ³⁵ P] -labeled HIF-1α, β, STRA13 were used. 9A shows that ADD1 / SREBP1c binds strongly to STRA13, binds to HIF-1α at a weak level, and HIF-1β does not bind to ADD1 / SREBP1c. Co-immunoprecipitation confirmed that STRA13 binds ADD1 / SREBP1c in vivo. In vivo ADD1 / SREBP1c bound very weakly with HIF-1α (see B in FIG. 9B). In order to determine whether ADD1 / SREBP1c and STRA13 or HIF-1α inhibit transcriptional activity by ADD1 / SREBP1c, the present inventors determined that proteins in which ADD1 / SREBP1c and yeast Gal DNA binding domains are fused to HepG2 cells are regulated by Gal4 binding sites. Transformation was performed with the promoter reporter plasmid. ADD1 / SREBP1c binds to the Gal4 binding site to increase the reporter gene. In addition, it was found that expression with STRA13 or HIF-1α inhibits reporter gene transcription. The results show that STRA13 and HIF-1α bind to ADD1 and inhibit the transcriptional activation ability of ADD1 (see C in FIG. 9B). On the one hand HIF-1α induces STRA13, indicating that STRA13 binds to the E-box sequence on the ADD1 / SREBP1c promoter and inhibits the expression of ADD1. In addition, the HIF-1α / β heterocomplex directly binds to the E-box on the ADD1 / SREBP1c primer and binds to the E-box to inhibit ADD1 / SREBP1c.

As described above, the present inventors confirmed that the formation of HIF-1 was related to the inhibition of fat formation in the hypoxic state, and thus the results of the study on the formation of fat using clioquinol and its derivatives to increase the activity of HIF-1α were investigated. Is described below.

Clioquinol (hereinafter referred to as "CQ") or derivatives thereof used in the present invention has a structure as shown in Formula 1 below.

<Formula 1>

R is H or an acetyl group, X ₁ or X ₂ is independently H or a halogen atom.

The halogen atom is preferably F, Cl, Br or I.

Derivatives of CQ (Formula 2) used in the present invention include 5-chloroquinolin-8-yl acetate (Formula 3), 5,7-Dibromo-8-hydroxyquinoline (Formula 4), 8-hydroxyquinoline (Formula 5) and 5 , 7-diiodo-8-hydroxyquinoline (Formula 6) and the like, the chemical formula is as follows, but not limited to all compounds known in the prior art derivatives of CQ can be used in the present invention.

<Formula 2>

<Formula 3>

<Formula 4>

<Formula 5>

<Formula 6>

The present invention provides a composition for inhibiting lipogenesis comprising clioquinol (hereinafter referred to as "CQ") or a derivative thereof including an active ingredient.

The inventors have found that CQ and its derivatives promote the activity of HIF-1α (International Application PCT / KR2007 / 000242). However, the relationship between CQ and its derivatives and fat formation is unknown. It has also been found that adipocyte differentiation is inhibited in hypoxia (Yun. Z. et al., 2002, Dev Cell. 2: 331-341). We investigated the effects of CQ and its derivatives on fat formation. Observation of normal oxygen, hypoxia and fat formation during CQ treatment resulted in significantly lower fat formation than in normal oxygen status, consistent with the previously reported results.Also, hypoxia was inhibited even with CQ (5 μM) treatment. It showed a better anti-lipogenic effect than in the cell line of the state (see Fig. 10). In addition, it was confirmed that fat formation was suppressed in a concentration-dependent manner when the CQ was treated by concentration (see FIG. 11). In addition, since the expression of FAS involved in fat formation is increased while increasing the expression of intracellular VEGF (see FIG. 12), the composition comprising the CQ and derivatives thereof of the present invention as an active ingredient is useful for inhibiting fat formation. Can be.

CQ and derivatives thereof of the present invention can be used as such or in the form of a pharmaceutically acceptable salt. The salt usable in the present invention is not particularly limited as long as it is a pharmaceutically acceptable non-toxic salt, and examples thereof include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid, tartaric acid, lactic acid, citric acid, fumaric acid, Maleic acid, succinic acid, metalsulfonic acid, benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid and the like.

The composition for inhibiting fat formation of the present invention can be used alone or in combination with methods using surgery, hormone therapy, chemotherapy and biological response modifiers for the prevention and treatment of obesity.

The composition for inhibiting fat formation of the present invention may further contain one or more active ingredients exhibiting the same or similar functions to the CQ and derivatives thereof. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary, as antioxidants, buffers And other conventional additives such as bacteriostatic agents can be added. The composition of the present invention may be administered in various oral and parenteral dosage forms in actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc., which are commonly used, may be used. It is prepared by. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The composition of the present invention can be administered orally or parenterally (eg, applied intravenously, subcutaneously, intraperitoneally or topically) according to the desired method, and the dosage is based on the weight, age, sex and health of the patient. The range varies depending on the diet, the time of administration, the method of administration, the rate of excretion and the severity of the disease. The dosage unit may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. The individual dosage preferably contains an amount in which the effective drug is administered once, which usually corresponds to a full, 1/2, 1/3 or 1/4 times the daily dose.

As a result of the toxicity experiment by orally administering the composition for inhibiting fat formation of the present invention, the 50% lethal dose (LD ₅₀ ) by the oral administration toxicity test is determined to be a safe substance of at least 5 g / kg or more. The effective dose is 0.5-6 mg / kg, preferably 3 mg / kg, and can be administered 1-3 times a day. The dose is not necessarily limited to this, and may vary depending on the condition of the patient and the extent of the disease.

The present invention also provides a health food for the prevention and inhibition of obesity containing clioquinol (hereinafter referred to as "CQ") or derivatives thereof as an active ingredient.

When using the CQ of the present invention and its derivatives as food additives, the composition for inhibiting fat formation may be added as it is or used with other foods or food ingredients, and may be suitably used according to conventional methods. Generally, in the preparation of food or beverage, the inhibitory composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less with respect to the raw material. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of food. Examples of foods to which the inhibitory composition of the present invention can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, Teas, drinks, alcoholic beverages and vitamin complexes and the like, and includes all of the health food in the conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the inhibitory composition of the present invention.

In addition to the above, CQ and its derivatives of the present invention are various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the CQ and derivatives thereof of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. Although the ratio of such an additive is not critical, it is generally selected from 0.01 to 0.1 parts by weight per 100 parts by weight of the inhibitory composition of the present invention.

The present invention also provides a composition for inhibiting fat acid synthase (FAS) comprising clioquinol (hereinafter referred to as "CQ") or a derivative thereof as an active ingredient.

The CQ of the present invention or a composition thereof may be usefully used to inhibit the expression of FAS by decreasing the expression of FAS while increasing the expression of VEGF (see FIG. 12).

The present invention also provides a composition for inhibiting fat formation, comprising STRA13 protein as an active ingredient.

The STRA13 protein decreases the activity of ADD1 / SEREBP1c (FIG. 6), thereby reducing the expression of FAS, and thus may be usefully used as a composition for inhibiting fat formation and a composition for inhibiting FAS. The STRA13 protein has the amino acid sequence of SEQ ID NO.

In addition, the present invention provides a composition for inhibiting fat formation, comprising HIF-1α protein as an active ingredient.

The HIF-1α protein decreases the activity of ADD1 / SEREBP1c (FIG. 6), thereby reducing the expression of FAS, and thus may be usefully used as a composition for inhibiting fat formation and a composition for inhibiting FAS. The HIF-1α protein has the amino acid sequence of SEQ ID NO: 18.

Hereinafter, the present invention will be described in detail by examples and formulation examples.

However, the following Examples and Formulation Examples specifically illustrate the present invention, and the contents of the present invention are not limited by Examples and Formulation Examples.

Example 1 Investigation of the Effect of Hypoxia on Lipogenesis

The present inventors investigated the effect of hypoxic state on lipogenesis in 3T3-L1 cell line, which is a preadipocyte. The 3T3-L1 cell line was incubated in Dulbecco's modified Eagle's midium (Dvibegen, USA) containing 10% FBS. DMEM medium containing 10% FBS, 3-isobutyl-1-methylxanthine (500 μM; Sigma, USA), dexamethasone (2 μM; Sigma, USA), and insulin (5 μg / ml; Sigma) in a packed state for differentiation , USA) was treated for 48 hours. After that, the culture was transferred to a steady-state chamber and a low oxygen incubator. As a control group, a cell line cultured in a normal oxygen state (normoxia) was used, and a cell line cultured in a hypoxia state was observed as an experimental group. Cells were incubated at 37 ° C. under 5% CO ₂ , 10% O ₂ , and 85% N ₂ conditions in an anaerobic incubator (0.1% O ₂ ) (Model 1029, Forma Scientific, Inc.) for hypoxic exposure. The culture medium was changed daily and replaced with DMEM medium containing 10% FBS containing 5 μg / ml insulin (Seo, JB et al. , 2004, Mol. Cell. Biol. 24: 3430-3444). Mature adipocytes were washed twice with PBS and fixed with 10% formaldehyde for 1 hour. Oil Red O (0.5% in isopropanol) was diluted with distilled water and left with fixed cells at 37 ° C. for 30 minutes (Seo, JB et al. , 2004, Mol. Cell. Biol. 24: 3430-3444). The intracellular stained lipid droplets were then confirmed under a microscope and photographed.

As a result, it was observed that the 3T3-L1 cell line exposed to the hypoxic state significantly inhibited the formation of adipocytes than the cell line cultured in the normal oxygen state (FIG. 1).

Example 2 Investigation of the Effect on Triglyceride and Cholesterol Formation in the Hypoxic State

3T3-L1 cells were cultured and treated as in Example 1. After culturing for 8 days, the whole cell lysate was prepared, and the levels of triglyceride and cholesterol were measured by a quantitative enzymatic process supplied by Sigma (USA). To detect triglycerides, the cell lysates were reacted with a solution containing lipase (Sigma) (10 minutes to 20 minutes at 37 ° C), and the absorbance of the sample was measured at 540 nm using a spectrophotometer. It was. For cholesterol measurement, the absorbance of the sample was measured at 500 nm by reacting with a solution containing cholesterol esterase and cholesterol oxidase (Sigma) for 10 to 20 minutes at 37 ° C (McGowan). ., MW, et al, 1983 , Clin Chem 29:.... 538-542, Rangaswamy, S., et al, 1997, Circ Res 80:.. 37-44). The amount of triglycerides and cholesterol was averaged to the protein level of cell lysate.

As a result, it was observed that the amount of triglycerides and cholesterol per total protein was inhibited in cell lines exposed to hypoxic states than in normal oxygen states (FIG. 2).

Example 3 Investigation of the Expression Pattern of Fatty Acid Synthase (FAS) in Hypoxic State

In order to investigate the expression pattern of fatty acid synthase (FAS) under hypoxic condition, the inventors of the present invention, Hep3B cell line, human skeletal muscle cell line L6 cell line, mesenchymal cell line C2C12 cell line and mouse Fatty accid synthase (FAS), phospohglycerate kinase-1 (PGK-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18S / 16S rRNA were expressed in adipose precursor cells 3T3-L1. Was investigated.

Hep3B cell lines were cultured using MEM medium containing 10% FBS (Invitrogen, USA), L6 cell lines, C2C12 cell lines and 3T3-L1 cell lines were cultured using DMEM medium containing 10% FBS, cells 100 The cells were cultured to about 70-80% cold in -mm tissue culture plates and treated with each medium containing 0.5% FBS for 16-24 hours prior to treatment. Each cell line was then exposed for 16 hours in hypoxic conditions (0.1% O ₂ ). Then total RNA was extracted. Total RNA was obtained using RNeasy spin column (Qiagen, Inc, USA). Total RNA was transferred to nylon membrane by electrophoresis using a 1% formaldehyde-agarose gel. It was then hybridized with α- [ ³² P] -labeled cDNA, washed, dried and then exposed to Hyperfile MP (Amersham Biosciences, USA) (Hur, E. et al., 2002, Mol. Pharmacol. 62: 975-982). ). PGK-1 (NM_000291) 338-1189 bp, GAPDH (NM_002046) coding region, 18S / 28S was observed stained with Ethidium Bromide. FAS was used as a probe by cutting the DNA sequence of the FAS contained in the pCRII vector used by Seo et al. With the restriction enzyme EcoR1 (Seo JB, et al. , 2004, Mol. Cell. Biol. 24: 3430-3444).

As a result, the hypoxic state inhibited the levels of FAS mRNA on Hep3B cell line, L6 cell line, C2C12 cell line and 3T3-L1 cell line. In contrast, mRNA levels of genes induced expression under hypoxic conditions such as PGK-1 and GAPDH increased (A in FIG. 3A).

Hepa1c1c7 cell line, a wild type mouse liver cancer cell line, was cultured in alpha-MEM medium (Invitrogen, USA). The hypoxic condition was treated under the same conditions as above to investigate mRNA expression of FAS, PGK-1 and 28S / 18S rRNA, and time zones were examined at 0, 1, 2, 4, 8, 16 and 24 hours.

As a result, on the Hepa1c1c7 cell line, FAS decreased the expression of FAS and increased the expression of PGK-1 (FIG. 3A).

<Example 4> ATP measurement investigation in hypoxic state of wild type mouse Hepa1c1c7 cell line

We have been used to detect ATP production levels in normal and hypoxic cells via constant-light signal luciferase assay (ATP Bioluminescence Assay Kit CLS II). Wild-type mouse Hepa1c1c7 cells were cultured in three to 5 × 10 ⁴ in 35 mm tissue culture plates and incubated overnight. After 16 hours, cells were exposed to hypoxia for 6 and 12 hours. Cells were obtained and analyzed according to the methods provided by the manufacturer. Specifically, 25 μl of luciferase solution provided in the CLS II kit was added to the whole diluted cell harvest and measured by 2s-integration on a luminometer (Berthold Lumat LB9501). The molar amount of ATP corresponding to each sample was determined to correspond with the associated luciferase unit based on ATP criteria (10 ⁻⁴ to 10 ⁻¹¹ M ATP). Luciferase assay averaged the concentration of the total protein determined by Bradford analysis based on bovine serum albumin (BSA).

As a result, the inventors confirmed that ATP production levels decreased after 6 hours of exposure to hypoxic state in wild-type Hepa1c1c7 cells (A of FIG. 3B).

Example 5 Investigation of Hypoxic Inhibition in HIF-1β-Deficient Hepa1c1c7 Cell Lines

<5-1> Preparation of HIF-1β-deficient Hepa1c1c7 Cell Line

We prepared a HIF-1β-deficient Hepa1c1c7 cell line in a manner that Whitlock et al. Isolated mutant cell lines that lost dioxin reactivity after treatment with mouse benzo (α) pyrene in mouse hepa1c1c7 cells (Miller, AG). , DIIsrael, and JP Whitlock, Jr. J. Biol . Chem . 258: 3523-3527, 1983). Analysis of the mutant hepa1c1c7, named BpRc1 cells, confirmed the lack of Arnt (same as HIF-1β), a dioxin receptor binding protein (Hyunsung P. Ko, et al, Mol. Cell. Biol . Vol. 10, No. 1, pp 430-436, 1996). We used BpRc1 as hepa1c1c7 cells lacking HIF-1β.

We then examined the expression of HIF-1β on wild-type and HIF-1β-deficient cell lines under defined oxygen and hypoxic conditions using a Western blot method for HIF-1β deficient cell lines. Samples taken from each group were subjected to electrophoresis on 8% SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell Bioscience, USA). In the Western blot, the primary antibody was used as anti-Arnt / HIF-1β antibody (BD Parmingen, USA), and the secondary antibody was mouse Ig conjugated horseradish peroxidase (1: 3,000 dilution) was used. Cell lines were treated for 6 hours in the hypoxic state. As a result, it was confirmed that the expression of HIF-1β-deficient cell line was not observed under normal oxygen or hypoxic state (D in FIG. 3B).

<5-2> HIF -1β-deficient cell line Hypoxia  In state FAS  Expression level investigation

We examined mRNA expression of FAS in HIF-1β-deficient cell lines according to the Northern blot method of Example 3. The cell line was treated for 16 hours in a hypoxic state.

As a result, the expression of FAS could not be observed in the HIF-1β-deficient cell line upon hypoxia treatment (E in FIG. 3B).

<5-3> Hypoxia  Under conditions ADD1 Of SREBP1c Of expression level

The present inventors investigated the expression of the ADD1 / SREBP1c gene under hypoxia in the hep3B cell line, a human liver cancer cell line, by real-time PCR and Western blot.

To treat the cell line in a hypoxic state for 16 hours, to perform real-time RT-PCR 1 μg total RNA to Lee et al. (Lee, YS et al., 2003, Nucleic Acid Res. 31: 7165-7174 Obtained). Real-time RT-PCR was performed using SYBR Green PCR master mix (Applied Biosystems), and the relative amount of each gene was detected by ABI PRISM 7000 (Applied Biosystems). PCR primers used were as follows: forward primer of human ADD1 / SREBP1c mRNA (gcc atg gat tgc act tt: SEQ ID NO: 1) and reverse primer (caa gag agg agc tca atg: SEQ ID NO: 2); Forward primer of 18S rRNA (acc gca gct agg aat aat gga ata: SEQ ID NO: 3) and reverse primer (ctt tcg ctc tgg tcc gtc tt: SEQ ID NO: 4).

After the hypoxic state was treated for 6 hours in the cell line, it was performed by Western blot method as in Example 5-1, and the anti-ADD1 / SREBP1c antibody (BD Pharmingen, USA) was used as the primary antibody. As an antibody, mouse Ig conjugated horseradish peroxidase (diluted 1: 3,000) was used.

As a result, it was confirmed that the expression of ADD1 / SREBP1c mRNA was reduced in the Hep3B cell line during hypoxic treatment (A of FIG. 4A), and at the same time, the expression at the protein level also decreased (B of FIG. 4A).

< Example  6> Hypoxia  Under condition FAS  Investigation of new protein synthesis due to inhibition of expression

<6-1> Hypoxia  Under conditions FAS  Expression inhibition investigation

We treated wild-type and HIF-1β-deficient Hepa1c1c7 cell lines with hypoxia for 12 hours, some of which were treated with 100 μM of CHX (cycloheximide) for 30 minutes before hypoxic treatment. Thereafter, RT-PCR was performed in the same manner as in Example 5-. RT-PCR conditions are as follows. After 5 minutes of initial denaturation at 95 ° C., a denaturation reaction at 95 ° C. for 45 seconds, a primer binding reaction at 56 ° C. for 45 seconds, and a length extension reaction at 72 ° C. for 60 seconds, were repeated 30 times. It was. The primers used were as follows: forward primer of FAS mRNA (tgc tcc cag ctg cag gc: SEQ ID NO: 5) and reverse primer (gcc ccg tag ctc tgg gtg ta: SEQ ID NO: 6), forward primer of 18S rRNA (acc gca) gct agg aat aat gga ata: SEQ ID NO: 3) and reverse primer (ctt tcg ctc tgg tcc gtc tt: SEQ ID NO: 4).

As a result, in hypoxic treatment, wild-type cell lines were stimulated by CHX treatment, but in HIF-1β-deficient cell lines, it was confirmed that the expression of FAS was changed in both hypoxic and CHX treatment groups (FIG. 5A).

<6-2> Hypoxia  Under conditions STRA13 Of expression changes in

The present inventors treated the hypoxic state for 3 hours in the 3T3-L1 cell line and performed the mRNA expression level of STRA13 by Western blot method as in Example 5-1. Anti-STRA13 antibody (Abcam, USA) was used as the primary antibody, and mouse Ig conjugated horseradish peroxidase (1: 3,000 dilution) was used as the secondary antibody.

In addition, the 3T3-L1 cell line was treated for 16 hours in the hypoxic state and mRNA expression level of STRA13 was examined by the Northern blot method as in Example 3.

In addition, after treating wild-type and HIF-1β-deficient Hepa1c1c7 cell lines in hypoxic state for 16 hours, mRNA expression levels of STRA13 were examined by RT-PCR method such as 5-3. The primers used were as follows: forward primer of mouse ADD1 / SREBP1c mRNA (cac ttc atc aag gca gac tc: SEQ ID NO: 7) and reverse primer (cgg tag cgc ttc tca atg gc: SEQ ID NO: 8)

As a result, it can be seen that in the hypoxic state, both the levels of HIF-1α and STRA13 are increased (B and C of FIG. 5), and the expression of STRA13 is dependent on HIF-1 (FIG. 5D).

< Example  7> Hypoxia  Under conditions FAS  Promoter and ADD Of SREBP1c  Regulatory investigation of promoter

<7-1> Temporary Transformation Luciferase  Assay transient transfection  luciferase assay )

We compared the sequences of the FAS promoters of human (H), mouse (M) and rat (R) (A in FIG. 6A). Boxed sequences are sequences selected as regulatory sequences, e-box consensus, hFIRE, hepatic FAS insulin response element, SREBP, ADD1 / SREBP1c binding magnetic flanking E-box (binding) site flanked E-box) and TATA-box. We describe pcDNA3-HIF-1α (human, U22431) (Hunag, LE, et. al ., 1998 , Proc . Natl . Acad . Sci . USA 95: 7987-7992), pcDNA3.1-STRA13 (mouse, AF010305), pcDNA3-ADD1 comprising ADD1 / SREBP1c cDNA (rat, AF286469) labeled myc-his in the bHLH-leucine zipper domain of ADD1 / SREBP1c / SREBP1c (1-403) -myc / hisA (Lee, YS, et al . , 2003, Nucleic Acids Res. 31: 7165-7174) were transformed into 5 × 10 ⁴ 3T3-L1 cell lines at 250 ng each, and at the same time the FAS promoter-driven luciferase reporter plasmid containing the upper regulatory region (-220 bp to +25 bp) of the FAS gene. 200 ng were transformed together. Before obtaining the cells, the transformed cell line was treated for 16 hours in the normal oxygen state or hypoxic state (0.1% O ₂ ). 48 hours after transformation, the cell harvest was prepared and measured by a luminometer (luminometer, Turmer TD-20 / 20, Promega, USA) using a luciferase assay system (Promega, USA). Luciferase activity was averaged using the total protein concentration measured by the Bradford method. Transformation efficiency was confirmed by transforming together pCHO110 (β-glactosidase-encoding plasmid) and measuring β-galactosidase (Hur, E. et. al ., 2002, Mol . Pharmacol . 62: 975-982).

As a result, it was observed that expression of the reporter gene is increased when ADD1 / SERBP1c is overexpressed. In addition, when the abundance of ADD1 / SREBP1c is increased, the hypoxic state appears to inhibit the expression of FAS by not only reducing the amount of ADD1 / SREBP1c but also inhibiting its activity. In addition, when HIF-1α and STRA13 were transformed together with ADD1 / SERBP1c, FAS expression was reduced in normal cells, indicating that HIF-1α and STRA13 inhibited the activity of ADD1 / SREBP1c on the FAS promoter (FIG. B) of 6a.

The ADD1 / SREBP1c promoter contains a sterol regulatory element complex, and to investigate whether it is induced by ADD1 / SREBP1c itself, we have identified a plasmid encoding ADD1 / SREBP1c, pcDNA3-ADD1 / SREBP1c (1 -403) -myc / hisA (Lee, YS, et al . , 2003, Nucleic Acids Res. 31: 7165-7174) 250 ng, pcDNA3-HIF-1α (human, U22431) (Hunag, LE, et al ., 1998 , Proc. Natl . Acad . Sci . USA 95: 7987-7992) 300 ng, pcDNA3.1-STRA13 (mouse, AF010305) were transiently transformed into 5 × 10 ⁴ 3T3-L1 cell lines, respectively, wherein the upstream regulatory region of the mouse ADD1 / SREBP1c gene ( FAS promoter reporter plasmids containing -2.7 kb to + 1 bp) were transformed together to investigate changes in the activity of ADD1 / SREBP1c on the ADD1 / SREBP1c promoter in the presence of HIF-1α or STRA13. Cells were treated for 16 hours in normal or hypoxic state (0.1% O ₂ ) prior to cell harvest. Then, the luciferase assay was performed in the same manner as described above.

As a result, overexpression of ADD1 / SREBP1c increased the expression of the reporter gene. In the hypoxic state, the degree of activation of the ADD1 / SREBP1c promoter by overexpressed ADD1 / SREBP1c was reduced. Transformation of HIF-1α or STRA13 with ADD1 / SREBP1c inhibited promoter activation by ADD1 / SREBP1c than in steady-state cells, suggesting that HIF-1α and STRA13 inhibit promoter activation by ADD1 / SREBP1c. (D in FIG. 6B).

<7-2> Immune Precipitation ( Co - immunoprecipitation ) Research

The present inventors transformed 293 cells with pEBG-ADD1 / SREBP1c along with pCMV-Myc-STRA13 or pCMV-3FLAG-HIF-1α, and whole cell extracts were prepared by Hur et al . (Hur. E., et. al ., 2002, Mol . Pharmacol . 62: 976-982). Immunoprecipitation (IP) was performed to prepare a 300 μg sample in total cell lysate at 1 ° C. for 30 minutes at 4 ° C. and 1 μg of anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and 20 μl of 0.5% ImmunoPure immobilized protein A / G gel (Pierce). , Rockford, IL) and pretreated. The treated extracts were mixed with 2 μg of anti-ADD1 / SREBP1c antibody (Dr. Kim, JB). After adding 15 μl of 0.5% ImmunoPure immobilized protein A / G gel, the mixture was left at 4 ° C. overnight. IP was precipitated and washed four times with PBS and dissolved in SDS sample buffer. Samples were heated for 5 minutes and run on a 7.5% SDS-PAGE gel, and proteins were transferred to nitrocellulose membranes using semi-dry transfer (Trans-Blot SD; Bio-Rad, Hercules, Calif.). The co-immunoprecipitated protein is reacted with an anti-Myc antibody (clone 9E10, Boehringer Mannheim) or an anti-FLAG antibody and enhanced chemiluminescence (Pierce, Rockford, IL) with anti-mouse Ig coupled with horseradish peroxidase (HRP). (Hur, E., et al., 2002, Mol. Pharmacol. 62: 975-982; Yim, S., et al., 2004, Biochem. Biophys. Res. Commun. 322: 9-16) .

As a result, it was confirmed that HIF-1α and STRA13 are related by confirming that the amount of ADD1 / SREBP1c is inhibited in the overexpressed cells of HIF-1α or STRA13 than in the steady state cells. In cells treated in the hypoxic state, overexpression of HIF-1α further inhibited ADD1 / SREBP1c (E in FIG. 6B).

< Example  8> HIF -1α / β and STRA13 In the presence of ADD1 Of SREBP1c of DNA  Binding activity investigation

The present inventors expressed GST (Glutathione S-transferase) and GST-ADD1 / SREBP1c (1-403 amino acid) fusion proteins in E. coli, and purified using glutathione uniflow resin (Amersham, USA) by a method provided by the manufacturer. 1 μg of pcDNA3-ADD1 / SREBP1c (1-403) -myc / hisA, pcDNA3-HA-HIF-1α, pcDNA3-HIF-1β or pcDNA3.1-STRA13 as a template in rabbit reticulocyte lysate (Promega, USA) IVTT ( in in vitro transcription and translation) to express the protein. [ ³⁵ S] -labeled ADD1 / SREBP1c, HIF-1α, HIF-1β or STRA13 protein (15 μL) was added to NETN buffer (20 mM Tris (pH8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P -40 and 1 mM PMSF) were reacted with immobilized GST or GST-ADD1 / SREBP1c for 2 hours at 4 ° C. in 500 μl. The protein was combined with glutathion-uniflow resin, washed three times with 1 ml of NETN buffer at 4 ° C, and eluted by heating in SDS sample buffer. The heated sample was subjected to SDS-PAGE and autoradiography (A in FIG. 7A).

We incubated HepG2 cells 70-80% cold in 100-mm tissue culture plates and incubated for 6 hours at 0.1% O ₂ . Nuclear extracts were performed by the method of Yim et al. (Yim, S., et al ., 2004, Biochem . Biophys . Res . Commun . 322: 9-16). IVTT with 1 μg of pcDNA3-ADD1 / SREBP1c (1-403) -myc / hisA, pcDNA-HA-HIF-1α, pcDNA-HIF-1β or pcDNA3.1-STRA13 as template in rabbit reticulocyte lysate (Promega, USA) (in vitro transcription and translation) was used. GST (glutathione S-transferase) -ADD1 / SREBP1c (1-403 amino acid) fusion proteins were expressed in Escherichia coli (BL21) and purified using the method provided by the manufacturer using glytathione uniflow resin (Amersham, USA). Oligonucleotides for E-box containing sequences of FAS promoters (-76 bp to 51 bp) (Forward: 5'-agt tct gtc agc cca tgt ggc gtg gcc g -3 ': SEQ ID NO: 9, Reverse: 5 '-gta tcg gcc acg cca cat ggg ctg aca g -3': oligonucleotide for SRE complex sequence of SEQ ID NO: 10) (A in FIG. 7A) and ADD1 / SREBP1c promoter wild type sequence (-92 bp to -56 bp) (Forward: 5'-tgc tga ttg gcc atg tgc gct cac ccg agg ggc ggg g-3 ': SEQ ID NO: 11, reverse: 5'-ccc cgc ccc tcg ggt gag cgc aca tgg cca atc agc a-3': sequence Number 12); SRE variant sequence (forward: 5'-tgc tga ttg gcc atg tgc gct aca ccg agg ggc ggg g -3 ': SEQ ID NO: 13, reverse: 5'-ccc cgc ccc tcg gtg tag cgc aca tgg cca atc agc a-3 ': SEQ ID NO: 14); E-box variant sequence (forward: 5'-tgc tga ttg gca aag tgc gct cac ccg agg ggc ggg g-3 ': SEQ ID NO: 15, reverse: 5'-ccc cgc ccc tcg ggt gag cgc act ttg cca atc agc agc a -3 ': SEQ ID NO: 16) (A in Fig. 8a) was synthesized. Oligonucleotides for the FAS promoter and oligonucleotide wild-type promoters for ADD1 / SREBP1c were labeled with α- [ ³² P] -dATP and Clenow enzyme (1.75 pmole). ADD1 / SREBP1c-programmed rabbit reticulocyte lysates, recombinant GST-ADD1 / SREBP1c protein or nuclear extracts were prepared in 20 μL binding reaction buffer (10 mM Tris [pH7.5], 50 mM KCl, containing polydeoxyinosinic-deoxycytidylic acid (1 μg). 2.5 mM EDTA, 0.1% (v / v) Triton X-100, 8.5% (v / v) glycerol, 1 mM DTT, and 0.1% (w / v) non-fat milk) for 30 minutes on ice (ref-NAR). Labeled oligonucleotides (4 × 10 ⁵ cpm, approximately 0.3 pmole) reacted with ADD1 / SREBP1c-programmed rabbit reticulocyte lysate, recombinant GST-ADD1 / SREBP1c protein (5 μg) or nuclear extract (8 μg) for 45 minutes on ice, The reaction mixture was separated on 6% PAGE gel at 4 ° C. In EMSA assays, ADD1 / SREBP1c antibody (1 μg) was added at 4 ° C. and reacted for 2 hours before introducing labeled oligonucleotides (Kim, JB et al . , 1998, J. Clin , Invest. 101: 1-9).

As a result, it was confirmed that the in vitro-translated STRA13 homozygotes or the in vitro-translated HIF-1α / β heterocomplexes did not bind but their presence prevented ADD1 / SREBP1cDNA from binding to DNA (FIG. 7B). B). In addition, it was confirmed that in vitro-translated ADD1 / SREBP1c binds to the FAS promoter, but ADD1 / SREBP1c binding is inhibited in the presence of STRA13 and HIF-1α (C in FIG. 7B). Nuclear extracts were used to investigate the share of the FAS promoter in cells and found that the protein binds to the FAS promoter. The results show that the addition of the ADD1 / SREBP1c antibody causes the loss of the DNA / protein complex, which includes ADD1 / SREBP1c. Consistent with the results of B and C of FIG. 7B, hypoxic treatment reduces the amount of ADD1 / SREBP1c comprising the DNA / protein complex. These findings indicate that hypoxia-induced HIF-1α / β and STRA13 inhibit ADD1 / SREBP1c binding to the FAS promoter.

In addition, EMSA results confirmed that GST-ADD1 / SREBP1c binds to the SRE complex containing oligonucleotides, and that both STRA13 homo- and HIF-1α / β heterocomplexes can bind to the SRE complex (B arrow of FIG. 8B). ). Experimental results showed that the binding between the ADD1 / SREBP1c promoter and the STRA13 homocomplex or HIF-1α / β heterocomplex was specific for the E-box sequence. We added an excess of unlabeled variant oligonucleotides (A in FIG. 8A). Addition of oligonucleotides to unlabeled SRE variants disrupted STRA13 and HIF-1α / β binding, and addition of unlabeled E-box variants failed. These results indicate that the STRA13 homocomplex or HIF-1α / β heterocomplex can bind to the E-box sequence but not to the SRE sequence on the ADD1 / SREBP1c promoter (FIG. 8B C). This suggests that STAR13 and HIF-1α / β compete with ADD1 / SREBP1c for binding to E-box on the SRE complex.

< Example  9> bHLH  Investigation of binding between transcriptional activity

The present inventors expressed GST (Glutathione S-transferase) and GST-ADD1 / SREBP1c (1-403 amino acid) fusion proteins in E. coli, and purified using glutathione uniflow resin (Amersham, USA) by a method provided by the manufacturer. 1 μg of pcDNA3-ADD1 / SREBP1c (1-403) -myc / hisA, pcDNA3-HA-HIF-1α, pcDNA3-HIF-1β or pcDNA3.1-STRA13 as a template in rabbit reticulocyte lysate (Promega, USA) IVTT ( in in vitro transcription and translation) to prepare the protein. [ ³⁵ S] -labeled ADD1 / SREBP1c, HIF-1α, HIF-1β or STRA13 protein (15 μL) was added to NETN buffer (20 mM Tris (pH8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P -40 and 1 mM PMSF) were reacted with GST or GST-ADD1 / SREBP1c bound to resin at 500 μl for 2 hours at 4 ° C. The protein was combined with glutathion-uniflow resin, washed three times with 1 ml of NETN buffer at 4 ° C, and eluted by heating in SDS sample buffer. The heated sample was subjected to SDS-PAGE and autoradiography.

As a result, ADD1 / SREBP1c binds strongly to STRA13, binds to HIF-1α at a weak level, and HIF-1β fails to bind ADD1 / STRBP1c (A in FIG. 9A).

The inventors performed immunoprecipitation in the same manner as in Example 7-2. Specifically, pEBG-ADD1 / SREBP1c was transformed into 293 cell lines with pCMV-Myc-STRA13 or pCMV-3FLAG-HIF-1α. Cells were treated with normal oxygen or hypoxia (0.1% O ₂ ) for 6 hours before cell harvest. IP was then performed using the cell lysate transformed with an anti-ADD1 / SREBP1c antibody (Dr. Kim. JB provided) bound to the resin.

 As a result, it was confirmed that ADD1 / SREBP1c binds relatively strongly with STRA13 and very weakly with HIF-α in vivo (B of FIG. 9B).

Then, the inventors of the present invention, 200 ng of reporter plasmid (gal4-tk-luc) expressed by the Gal4 protein binding base sequence of yeast GAL4-ADD1 / SREBB1c 200 ng, pcDNA3-STRA13 200 ng, pcDNA3-HIF-1α 200 HepG2 cell line was transformed with ng. Then, the expression of the reporter gene luciferase was examined in the same manner as in Example 7-1.

As a result, it was confirmed that Gal4-ADD1 / SREBP1c binds to the Gal4 binding site to increase the reporter gene. In addition, it can be seen that expression with STRA13 or HIF-1α inhibits the transcription of the reporter gene (C of FIG. 9B). This result suggests that HIF-1α induces STRA13, which binds to the E-box sequence of the ADD1 / SREBP1c promoter and the ADD1 / SREBP1c protein to inhibit ADD1 / SREBP1c from activating transcription of its target gene. Able to know. In addition, the HIF-1α / β heterocomplex also directly binds to E-box on the ADD1 / SREBP1c promoter or weakly binds to ADD1 / SREBP1c to inhibit the transcription of the target gene of ADD1 / SREBP1c.

< Example  10> for adipocyte differentiation Clioquinol  Effect investigation

3T3-L1 cell line, which is a preadipocyte, was cultured to fill the plate using DMEM medium (Invitrogen, USA) containing 10% FBS (fetal bovine serum: Invitrogen, USA). Differentiation was then performed while incubating for 8 days in DMEM medium containing 10% FBS, 3-isobutyl-1-nethylxanthine (500 μM), dexamethasone (dexamethasone, 2 μM) and insulin (insulin, 5 μg / ml). The culture medium was replaced at 48 hour intervals with DMEM medium containing 10% FBS and 5 μg / ml insulin. The differentiation process was carried out for 8 days. At this time, the cell line was treated with normal oxygen, hypoxic state and clioquinol (1, 2, 5 μM) to induce differentiation. Hypoxia was incubated at 37 ° C. in an anaerobic incubator (0.1% O _{2 :} Model 1029, Forma Scientific, Inc.) at 5% CO ₂ , 10% H ₂ and 85% N ₂ . In the group treated with clioquinol, the clioquinol was retreated in the replacement medium every time the medium was changed. After 8 days, mature adipocytes were washed twice with PBS and fixed for 1 hour with 10% formaldehyde. Oil red O (0.5% in isopropanol) is diluted with distilled water and used as the final 0.3%. It was added to the fixed cell plate and reacted at 37 ° C for 30 minutes. Intracellular stained lipid droplets were visualized under a light microscope and photographed.

As a result, it was confirmed that the formation of lipid droplets was inhibited when 5 μM of clioquinol was treated in the 3T3-L1 cell line. As a control, it was confirmed that the formation of lipid droplets was suppressed in the 3T3-L1 cell line treated with hypoxia as previously reported (FIG. 10). When the present inventors treated various concentrations of clioquinol (1, 2, 5 μM), as the concentration of clioquinol increased, it was confirmed that the inhibition of fat formation was increased (FIG. 11).

< Example  11> Clioquinol  And genetic change investigation by its derivatives

CQ (50 μM) and its derivatives (5-chloroquinolin-8-yl acetate: 50 μM; 5,7-Dibromo-8-hydroxyquinoline: 5, 10, 50) in the HepG2 cell line, a hepatocyte cell, under normal and hypoxic treatment And 100 μM; and 8-hydroxyquinoline: 5, 10, 50 and 100 μM) were treated for 16 hours or treatment time. The total RNA was then separated by RNeasy spin column (Qiagen, Inc, USA) for reverse transcription, and 1 μg of total RNA was used as a template for RT-PCR. The primers used were forward primers (5′- tgc tcc cag ctg cag gc-3 ′: SEQ ID NO: 5) and reverse primers (5′- gcc ccg tag ctc tgg gtg ta-3) for amplification of FAS (NM_007988). ': SEQ ID NO: 6) was used. Forward primer (5'-cca tga act ttc tgc tgt ctt -3 ': SEQ ID NO: 17) and reverse primer (5'- atc gca tca ggg gca cac ag -3': sequence for amplification of VEGF (AF022375) Number 18) was used. For the amplification of 18S rRNA (X03205) as a control, forward primer (5′- acc gca gct agg aat aat gga ata -3 ′: SEQ ID NO: 3) and reverse primer (5′- ctt tcg ctc tgg tcc gtc) tt-3 ′: SEQ ID NO: 4) was used. RT-PCR conditions are as follows. After 5 minutes of initial denaturation at 95 ° C., a denaturation reaction at 95 ° C. for 45 seconds, a primer binding reaction at 56 ° C. for 45 seconds, and a length extension reaction at 72 ° C. for 60 seconds, were repeated 30 times. It was. The above conditions were repeated 30 times and PCR products were electrophoresed on 1% agarose gel to confirm the results.

As a result, it was confirmed that clioquinol and its derivatives decrease the expression of FAS while increasing the expression of VEGF (FIG. 12).

< Example  12> Clioquinol  Acute Toxicity Test of Oral Administration of Infant or Derivatives thereof

The present inventors performed the following experiment to determine the acute toxicity of clioquinol or its derivatives (5-chloroquinolin-8-yl acetate, 5,7-Dibromo-8-hydroxyquinoline and 8-hydroxyquinoline). Acute toxicity test was performed using 6 weeks-old SPF SD. Clioquinol or its derivatives (5-chloroquinolin-8-yl acetate, 5,7-Dibromo-8-hydroxyquinoline and 8-hydroxyquinoline) were each suspended in 0.5% methylcellulose solution and dosed at a dose of 5 g / kg / 15 ml. Oral administration. Hematology and blood biochemical tests were performed by observing the mortality, clinical symptoms, and weight changes of the animals after the administration of the test substance.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with the test substance, and no toxic changes were observed in weight changes, blood tests, blood biochemistry tests, and autopsy findings.

As a result, the rats showed no toxicity change up to 5 g / kg, and the minimum lethal dose (LD ₅₀ ) for oral administration was found to be a safe substance of 5 g / kg or more.

Examples of preparations for the compositions of the present invention are illustrated below.

< Formulation example  1>: Preparation of pharmaceutical preparation

1. Preparation of powder

2 g of clioquinol

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

2. Preparation of Tablets

Clioquinol 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.

3. Preparation of Capsule

5-chloroquinolin-8-yl acetate 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

4. Manufacture of rings

5,7-Dibromo-8-hydroxyquinoline 1 g

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

5. Preparation of Granules

8-hydroxyquinoline 150 mg

Soy extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.

< Formulation example  2>: Manufacture of food

Foods containing clioquinol of the present invention were prepared as follows.

1. Preparation of Cooking Seasonings

20 to 95 parts by weight of clioquinol of the present invention was prepared for cooking spices for health promotion.

2. Preparation of Tomato Ketchup and Sauce

0.2 ~ 1.0 parts by weight of clioquinol of the present invention was added to tomato ketchup or sauce to prepare tomato ketchup or sauce for health promotion.

3. Manufacturing of Flour Foods

0.5 to 5.0 parts by weight of clioquinol of the present invention was added to wheat flour, and the mixture was used to prepare bread, cake, cookies, crackers and noodles to prepare foods for health promotion.

4. Preparation of soups and gravy

0.1-5.0 parts by weight of clioquinol of the present invention was added to soups and broth to prepare meat products for health promotion, soups and noodles of noodles.

5. Preparation of Ground Beef

10 parts by weight of clioquinol of the present invention was added to the ground beef to prepare a ground beef for health promotion.

6. Manufacture of Dairy Products

5-10 parts by weight of clioquinol of the present invention was added to milk, and various dairy products such as butter and ice cream were prepared using the milk.

7. Manufacture of wire

Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.

Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.

The grains, seeds and clioquinol prepared above were prepared by combining the following ratios.

Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),

Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),

Clioquinol (3 parts by weight),

Ganoderma lucidum (0.5 parts by weight),

Foxglove (0.5 part by weight)

< Formulation example  3>: Manufacture of beverage

1. Manufacture of health drinks

Instant sterilization by homogeneously mixing clioquinol with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Health drinks were prepared by packaging in small packaging containers such as plastic bottles.

3. Preparation of Vegetable Juice

5 g of clioquinol of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

4. Preparation of Fruit Juice

1 g of clioquinol of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.

The composition for inhibiting fat formation of the present invention effectively inhibits fat formation in a concentration-dependent manner of clioquinol during adipocyte differentiation, and is useful for inhibiting fat formation and treating and preventing obesity by reducing the expression of fat acid synthase (FAS). Can be.

<110> UNIVERSITY OF SEOUL FOUNDATION OF INDUSTRY-ACADIMIC COOPERATION <120> Inhibiting composition of lipogenesis containing clioquinol and          derivatives <130> 7p-01-31 <160> 18 <170> KopatentIn 1.71 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> human ADD1 / SREBP1c mRNA forward primer <400> 1 gccatggatt gcacttt 17 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> human ADD1 / SREBP1c mRNA reverse primer <400> 2 caagagagga gctcaatg 18 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 18S rRNA forward primer <400> 3 accgcagcta ggaataatgg aata 24 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18S rRNA reverse primer <400> 4 ctttcgctct ggtccgtctt 20 <210> 5 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> FAS mRNA forward primer <400> 5 tgctcccagc tgcaggc 17 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FAS mRNA reverse primer <400> 6 gccccgtagc tctgggtgta 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse ADD1 / SREBP1c forward primer <400> 7 cacttcatca aggcagactc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> mouse ADD1 / SREBP1c reverse primer <400> 8 cggtagcgct tctcaatggc 20 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> E-box oligonucleotide <400> 9 agttctgtca gcccatgtgg cgtggccg 28 <210> 10 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> E-box loigonucleotide <400> 10 gtatcggcca cgccacatgg gctgacag 28 <210> 11 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> SRE complex oligonycleotide <400> 11 tgctgattgg ccatgtgcgc tcacccgagg ggcgggg 37 <210> 12 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> SRE complex oligonucleotide <400> 12 ccccgcccct cgggtgagcg cacatggcca atcagca 37 <210> 13 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> SRE mutant oligonucleotide <400> 13 tgctgattgg ccatgtgcgc tacaccgagg ggcgggg 37 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> SRE mutant oligonucleotide <400> 14 ccccgcccct cggtgtagcg cacatggcca atcagca 37 <210> 15 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> E-box mutant oligonucleotide <400> 15 tgctgattgg caaagtgcgc tcacccgagg ggcgggg 37 <210> 16 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> E-box mutant oligonucleotide <400> 16 ccccgcccct cgggtgagcg cactttgcca atcagca 37 <210> 17 <211> 411 <212> PRT <213> Artificial Sequence <220> <223> STRA13 <400> 17 Met Glu Arg Ile Pro Ser Ala Gln Pro Pro Pro Thr Cys Leu Pro Lys   1 5 10 15 Ala Pro Gly Leu Glu His Gly Asp Leu Ser Gly Met Asp Phe Ala His              20 25 30 Met Tyr Gln Val Tyr Lys Ser Arg Arg Gly Ile Lys Arg Ser Glu Asp          35 40 45 Ser Lys Glu Thr Tyr Lys Leu Pro His Arg Leu Ile Glu Lys Lys Arg      50 55 60 Arg Asp Arg Ile Asn Glu Cys Ile Ala Gln Leu Lys Asp Leu Leu Pro  65 70 75 80 Glu His Leu Lys Leu Thr Thr Leu Gly His Leu Glu Lys Ala Val Val                  85 90 95 Leu Glu Leu Thr Leu Lys His Val Lys Ala Leu Thr Asn Leu Ile Asp             100 105 110 Gln Gln Gln Gln Lys Ile Ile Ala Leu Gln Ser Gly Leu Gln Ala Gly         115 120 125 Asp Leu Ser Gly Arg Asn Leu Glu Ala Gly Gln Glu Met Phe Cys Ser     130 135 140 Gly Phe Gln Thr Cys Ala Arg Glu Val Leu Gln Tyr Leu Ala Lys His 145 150 155 160 Glu Asn Thr Arg Asp Leu Lys Ser Ser Gln Leu Val Thr His Leu His                 165 170 175 Arg Val Val Ser Glu Leu Leu Gln Gly Gly Ala Ser Arg Lys Pro Leu             180 185 190 Asp Ser Ala Pro Lys Ala Val Asp Leu Lys Glu Lys Pro Ser Phe Leu         195 200 205 Ala Lys Gly Ser Glu Gly Pro Gly Lys Asn Cys Val Pro Val Ile Gln     210 215 220 Arg Thr Phe Ala Pro Ser Gly Gly Glu Gln Ser Gly Ser Asp Thr Asp 225 230 235 240 Thr Asp Ser Gly Tyr Gly Gly Glu Leu Glu Lys Gly Asp Leu Arg Ser                 245 250 255 Glu Gln Pro Tyr Phe Lys Ser Asp His Gly Arg Arg Phe Ala Val Gly             260 265 270 Glu Arg Val Ser Thr Ile Lys Gln Glu Ser Glu Glu Pro Pro Thr Lys         275 280 285 Lys Ser Arg Met Gln Leu Ser Glu Glu Glu Gly His Phe Ala Gly Ser     290 295 300 Asp Leu Met Gly Ser Pro Phe Leu Gly Pro His Pro His Gln Pro Pro 305 310 315 320 Phe Cys Leu Pro Phe Tyr Leu Ile Pro Pro Ser Ala Thr Ala Tyr Leu                 325 330 335 Pro Met Leu Glu Lys Cys Trp Tyr Pro Thr Ser Val Pro Val Leu Tyr             340 345 350 Pro Gly Leu Asn Thr Ser Ala Ala Ala Leu Ser Ser Phe Met Asn Pro         355 360 365 Asp Lys Ile Pro Thr Pro Leu Leu Leu Pro Gln Arg Leu Pro Ser Pro     370 375 380 Leu Ala His Ser Ser Leu Asp Ser Ser Ala Leu Leu Gln Ala Leu Lys 385 390 395 400 Gln Ile Pro Pro Leu Asn Leu Glu Thr Lys Asp                 405 410 <210> 18 <211> 826 <212> PRT <213> Artificial Sequence <220> <223> HIF-1 alpha <400> 18 Met Glu Gly Ala Gly Gly Ala Asn Asp Lys Lys Lys Ile Ser Ser Glu   1 5 10 15 Arg Arg Lys Glu Lys Ser Arg Asp Ala Ala Arg Ser Arg Arg Ser Lys              20 25 30 Glu Ser Glu Val Phe Tyr Glu Leu Ala His Gln Leu Pro Leu Pro His          35 40 45 Asn Val Ser Ser His Leu Asp Lys Ala Ser Val Met Arg Leu Thr Ile      50 55 60 Ser Tyr Leu Arg Val Arg Lys Leu Leu Asp Ala Gly Asp Leu Asp Ile  65 70 75 80 Glu Asp Asp Met Lys Ala Gln Met Asn Cys Phe Tyr Leu Lys Ala Leu                  85 90 95 Asp Gly Phe Val Met Val Leu Thr Asp Asp Gly Asp Met Ile Tyr Ile             100 105 110 Ser Asp Asn Val Asn Lys Tyr Met Gly Leu Thr Gln Phe Glu Leu Thr         115 120 125 Gly His Ser Val Phe Asp Phe Thr His Pro Cys Asp His Glu Glu Met     130 135 140 Arg Glu Met Leu Thr His Arg Asn Gly Leu Val Lys Lys Gly Lys Glu 145 150 155 160 Gln Asn Thr Gln Arg Ser Phe Phe Leu Arg Met Lys Cys Thr Leu Thr                 165 170 175 Ser Arg Gly Arg Thr Met Asn Ile Lys Ser Ala Thr Trp Lys Val Leu             180 185 190 His Cys Thr Gly His Ile His Val Tyr Asp Thr Asn Ser Asn Gln Pro         195 200 205 Gln Cys Gly Tyr Lys Lys Pro Pro Met Thr Cys Leu Val Leu Ile Cys     210 215 220 Glu Pro Ile Pro His Pro Ser Asn Ile Glu Ile Pro Leu Asp Ser Lys 225 230 235 240 Thr Phe Leu Ser Arg His Ser Leu Asp Met Lys Phe Ser Tyr Cys Asp                 245 250 255 Glu Arg Ile Thr Glu Leu Met Gly Tyr Glu Pro Glu Glu Leu Leu Gly             260 265 270 Arg Ser Ile Tyr Glu Tyr Tyr His Ala Leu Asp Ser Asp His Leu Thr         275 280 285 Lys Thr His His Asp Met Phe Thr Lys Gly Gln Val Thr Thr Gly Gln     290 295 300 Tyr Arg Met Leu Ala Lys Arg Gly Gly Tyr Val Trp Val Glu Thr Gln 305 310 315 320 Ala Thr Val Ile Tyr Asn Thr Lys Asn Ser Gln Pro Gln Cys Ile Val                 325 330 335 Cys Val Asn Tyr Val Val Ser Gly Ile Ile Gln His Asp Leu Ile Phe             340 345 350 Ser Leu Gln Gln Thr Glu Cys Val Leu Lys Pro Val Glu Ser Ser Asp         355 360 365 Met Lys Met Thr Gln Leu Phe Thr Lys Val Glu Ser Glu Asp Thr Ser     370 375 380 Ser Leu Phe Asp Lys Leu Lys Lys Glu Pro Asp Ala Leu Thr Leu Leu 385 390 395 400 Ala Pro Ala Ala Gly Asp Thr Ile Ile Ser Leu Asp Phe Gly Ser Asn                 405 410 415 Asp Thr Glu Thr Asp Asp Gln Gln Leu Glu Glu Val Pro Leu Tyr Asn             420 425 430 Asp Val Met Leu Pro Ser Pro Asn Glu Lys Leu Gln Asn Ile Asn Leu         435 440 445 Ala Met Ser Pro Leu Pro Thr Ala Glu Thr Pro Lys Pro Leu Arg Ser     450 455 460 Ser Ala Asp Pro Ala Leu Asn Gln Glu Val Ala Leu Lys Leu Glu Pro 465 470 475 480 Asn Pro Glu Ser Leu Glu Leu Ser Phe Thr Met Pro Gln Ile Gln Asp                 485 490 495 Gln Thr Pro Ser Pro Ser Asp Gly Ser Thr Arg Gln Ser Ser Pro Glu             500 505 510 Pro Asn Ser Pro Ser Glu Tyr Cys Phe Tyr Val Asp Ser Asp Met Val         515 520 525 Asn Glu Phe Lys Leu Glu Leu Val Glu Lys Leu Phe Ala Glu Asp Thr     530 535 540 Glu Ala Lys Asn Pro Phe Ser Thr Gln Asp Thr Asp Leu Asp Leu Glu 545 550 555 560 Met Leu Ala Pro Tyr Ile Pro Met Asp Asp Asp Phe Gln Leu Arg Ser                 565 570 575 Phe Asp Gln Leu Ser Pro Leu Glu Ser Ser Ser Ala Ser Pro Glu Ser             580 585 590 Ala Ser Pro Gln Ser Thr Val Thr Val Phe Gln Gln Thr Gln Ile Gln         595 600 605 Glu Pro Thr Ala Asn Ala Thr Thr Thr Thr Ala Thr Thr Asp Glu Leu     610 615 620 Lys Thr Val Thr Lys Asp Arg Met Glu Asp Ile Lys Ile Leu Ile Ala 625 630 635 640 Ser Pro Ser Pro Thr His Ile His Lys Glu Thr Thr Ser Ala Thr Ser                 645 650 655 Ser Pro Tyr Arg Asp Thr Gln Ser Arg Thr Ala Ser Pro Asn Arg Ala             660 665 670 Gly Lys Gly Val Ile Glu Gln Thr Glu Lys Ser His Pro Arg Ser Pro         675 680 685 Asn Val Leu Ser Val Ala Leu Ser Gln Arg Thr Thr Val Pro Glu Glu     690 695 700 Glu Leu Asn Pro Lys Ile Leu Ala Leu Gln Asn Ala Gln Arg Lys Arg 705 710 715 720 Lys Met Glu His Asp Gly Ser Leu Phe Gln Ala Val Gly Ile Gly Thr                 725 730 735 Leu Leu Gln Gln Pro Asp Asp His Ala Ala Thr Thr Ser Leu Ser Trp             740 745 750 Lys Arg Val Lys Gly Cys Lys Ser Ser Glu Gln Asn Gly Met Glu Gln         755 760 765 Lys Thr Ile Ile Leu Ile Pro Ser Asp Leu Ala Cys Arg Leu Leu Gly     770 775 780 Gln Ser Met Asp Glu Ser Gly Leu Pro Gln Leu Thr Ser Tyr Asp Cys 785 790 795 800 Glu Val Asn Ala Pro Ile Gln Gly Ser Arg Asn Leu Leu Gln Gly Glu                 805 810 815 Glu Leu Leu Arg Ala Leu Asp Gln Val Asn             820 825  

Claims (19)

Clioquinol or a pharmaceutical composition for preventing and inhibiting obesity comprising a derivative thereof having the structure of Formula 1 as an active ingredient: <Formula 1>

R is H or an acetyl group, X ₁ or X ₂ is independently H or a halogen atom. delete The pharmaceutical composition for preventing and inhibiting obesity according to claim 1, wherein the halogen atom is F, Cl, Br or I. The method of claim 1, wherein the derivative of clioquinol is chloroacetoxy quinoline (5-chloroquinolin-8-yl acetate), brooxyquinoline (5,7-diiodo-8-hydroxyquinoline), 5,7- Dibromo-8-hydroxyquinoline (5,7-dibromo-8-hydroxyquinoline) or 8-hydroxyquinoline (8-hydroxyquinoline) characterized in that the pharmaceutical composition for the prevention and inhibition of obesity. The pharmaceutical composition for preventing and inhibiting obesity, according to claim 1, which inhibits ubiquitination or enhances HIF-1α transcriptional activity of clioquinol or its derivative HIF-1α (hypoxia-inducible factor 1 alpha). Health foods for the prevention and inhibition of obesity comprising clioquinol or derivatives thereof having the structure of Formula 1 as an active ingredient: <Formula 1>

R is H or an acetyl group, X ₁ or X ₂ are independently H or halogen source character. delete 7. The health food for preventing and suppressing obesity according to claim 6, wherein the halogen atom is F, Cl, Br or I. The method of claim 6, wherein the derivative of clioquinol is chloroacetoxy quinoline (5-chloroquinolin-8-yl acetate), brooxyquinoline (5,7-diiodo-8-hydroxyquinoline), 5,7- Dibromo-8-hydroxyquinoline (5,7-dibromo-8-hydroxyquinoline) or 8-hydroxyquinoline (8-hydroxyquinoline) characterized in that the health food for the prevention and inhibition of obesity. delete delete delete delete delete delete delete delete delete delete

Images