KR101985577B1 - Use of CRY1 for treatment and screening target of therapeutic agents of diabetes mellitus - Google Patents

Abstract

The present application discloses the use of CRY1 as a therapeutic agent for diabetes. In addition, the present disclosure discloses a method for screening a glycemic control agent or a diabetes therapeutic agent using a novel molecular mechanism based on the SREBP1c-CRY1-FOXO1 glucose biosynthesis regulatory pathway. The therapeutic agent according to the present invention can simultaneously inhibit glucose biosynthesis without increasing fat biosynthesis.

Description

Use of CRY1 for treatment and screening target of therapeutic agents of diabetes mellitus}

The present application is a technique related to diabetes treatment and therapeutic drug development.

Diabetes is a type of metabolic disease such as insufficient insulin secretion or normal function. It is characterized by high blood glucose, which increases the concentration of glucose in the blood, and causes various symptoms and signs and releases glucose from urine. . Diabetes is divided into type 1 and type 2, which is caused by the inability to produce insulin at all. Type 2 diabetes, which is relatively low in insulin, reduces insulin resistance. Lack of insulin function, which prevents cells from burning glucose effectively). Type 2 diabetes seems to be caused by environmental factors such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of diet, but it can also be caused by defects in certain genes, pancreatic surgery, infections, drugs, etc. have.

Insulin is a major anabolic hormone secreted at the time of ingestion, and is responsible for energy metabolism in the body. Insulin inhibits glucose biosynthesis and increases fat biosynthesis in the liver, thereby storing surplus energy. In diabetes mellitus, which is represented by insulin resistance, an interesting phenomenon occurs. The insulin signaling pathway does not function and the inhibition of glucose biosynthesis disappears, and glucose is continuously produced in the liver and released into the blood. This increases the concentration of lipids in the blood and results in selective insulin resistance resulting from fatty liver.

More than 90% of diabetics have type 2 diabetes. In the case of current type 2 diabetes, medications of metformin and thiazolidinedione (TZD) have shown considerable usefulness, but they have not been able to treat the underlying cause of diabetes such as insulin resistance. Several side effects have also been reported, requiring the development of more potent and safer drugs that can fundamentally address insulin resistance.

Cryptochrome Circadian Clock 1 (CRY1) is a blue light-sensitive flavoprotein that is involved in the biological clock rhythm of plants and animals.

Republic of Korea Patent No. 2013-0027059 relates to a urine label for detecting bladder cancer, CRY1 as a bladder cancer marker is disclosed.

Korean Patent Laid-Open No. 2015-0106492 relates to a composition for treating insulin resistance-related diseases comprising a substance that activates FPR2, which maintains phosphorylation of Akt, which is important for insulin signaling, and inhibits phosphorylation of PKC-theta. It is disclosed to improve the resistance.

The present application seeks to provide a therapeutic agent for type 2 diabetes and a therapeutic screening method based on a novel molecular mechanism.

In one aspect, the present application provides a pharmaceutical composition for treating diabetes, including type 1 or 2 diabetes, comprising the CRY1 gene or a protein encoded by the gene.

In one embodiment in particular the diabetes in which the composition according to the invention can be used is type 2 diabetes.

In another embodiment, a type 2 diabetic patient in which the composition according to the present invention can be used is accompanied by selective insulin resistance, in which selective metabolism is still increased, but glucose biosynthesis is not suppressed. However, SREBP1c is increased while CRY1 is due to a decrease.

In another aspect, the present invention also provides a composition for controlling blood glucose, particularly a hypoglycemic agent, in particular a hypoglycemic agent through inhibiting glucose biosynthesis, including the CRY1 gene, or type 1 or 2 diabetes mellitus comprising a protein encoded by the gene. do.

In another aspect, the present disclosure also provides a method for screening glucose biosynthesis inhibitors that targets the SREBP1c-CRY1-FOXO1 signaling pathway.

In one embodiment the method comprises the steps of providing a cell or animal model with reduced or lacking CRY1 expression or activity; A second step of treating said cells with a test substance that is expected to increase the expression or activity of CRY1; A third step of measuring FOXO1 protein concentration in cells treated with the test substance; And; And a fourth step of selecting the FOXO1 protein as a candidate for type 2 diabetes if the amount of the FOXO1 protein is decreased in comparison with the FOXO1 protein in a control group not in contact with the test substance.

Another embodiment includes measuring the expression of PEPCK and / or G6Pase, an enzyme involved in glucose biosynthesis that is promoted by FOXO1 in lieu of or in addition to a third step, in which case the fourth step In the candidate material is a decrease in the expression of the PEPCK or G6Pase in the cells in contact with the test material compared to the control cells not in contact with the test material.

In the method according to the present invention, cells or animals in which the expression of CRY1 is decreased may be increased in the expression of SREBP1c, which is a higher-level protein in addition to the reduction of CRY1.

The compounds selected by the screening method according to the present application can inhibit glucose biosynthesis, and thus can be usefully used for glycemic control, hypoglycemia, and diabetes treatment.

In another aspect, the present application provides a kit for regulating the CRY1 gene, or a protein encoded by the gene, or an SREBP1c-CRY1-FOXO1 signaling pathway in hepatocytes or animals in vitro containing the gene.

Provided are a kit for inhibiting glucose biosynthesis in hepatocytes or animals in a CRY1 gene, a protein encoded by the gene, or in vitro comprising the gene.

In another aspect, the present invention provides a method of regulating the SREBP1c-CRY1-FOXO1 pathway or a method of inhibiting glucose biosynthesis by regulating the pathway, the method comprising treating the CRY1 gene, or a protein encoded by the gene, in hepatocytes or animals in vitro. In the above method, in particular, the hepatocytes and animals have increased SREBP1c expression and reduced expression or activity of CRY1 protein.

It has been found herein that SREBP1c can inhibit glucose biosynthesis via the SREBP1c-CRY1-FOXO1 pathway and also inhibit glucose biosynthesis by overexpression of CRY1 in diabetic animal models. Therefore, CRY1 may be usefully used as a new anti-diabetic agent that can inhibit glucose biosynthesis at the same time without increasing fat biosynthesis and as a target of therapeutic development.

1 shows that CRY1 is stimulated by feeding and exposure to insulin according to one embodiment of the present disclosure. (a, b) C57BL / 6 mice were fasted for 24 hours and then reingested for 12 hours. Both ingested mice and re-ingested mice were ZT 3 sacrificed. CRY1 mRNA (a) and CRY1 protein (b) in the liver were measured using qRT-PCT and Western blot normalized to TBP mRNA levels, respectively. pSREBP1, precursor SREBP1; nSREBP1, nuclear SREBP1. Data are shown as mean ± sd. N = 3. ^* P <0.05, ^** P <0.01 (Student's t-test) for each group. (C) CRY1 gene expression was measured using qRT-PCR after 12 h insulin exposure. TBP mRNA levels were used for qRT-PCR normalization. Data are shown as mean ± sd. N = 3. ^* P <0.05, ^** P <0.001 (Student's t-test) for each group.
2 shows that CRY1 gene expression according to an embodiment of the present application is directly activated by SREBP1c. (a, b) Mouse primary hepatocytes were infected with adenovirus with Ad-MOCK or Ad-SREBP1c. CRY1 mRNA (a) and CRY1 protein (b) levels were measured using qRT-PCT and Western blot normalized to TBP mRNA levels, respectively. N = 3. ^* P <0.05, ^** P <0.01 (Student's t-test) for each group. (C) Luciferase activity of the WT CRY1 promoter and the 3XSRE mutant promoter was measured after co-transfection with an expression plasmid encoding SREBP1c or MOCK in HEK293T cells. Luciferase activity was normalized to β-gal activity. TSS, Transcription Start Site; SRE, Sterol Regulatory Elements. Data are shown as mean ± sd. N = 5. ^** P <0.01 (Student's t-test) for each group. (d) ChIP assay showing CRY1 promoter occupied by SREBP1c in H4IIE cells. (e) Mouse primary hepatocytes were isolated from SREBP1c ^{− / −} and SREBP1c ^{+ / +} mice. SREBP1c and CRY1 protein levels in the presence of insulin (10 nM) were measured by Western blot. (f) SREBP1c ^{− / −} and SREBP1c ^{+ / +} mice were fasted for 24 hours and then reingested for 12 hours. Both ingested mice and re-ingested mice were ZT 3 sacrificed. SREBP1c and CRY1 mRNA levels were measured by qRT-PCR and normalized to TBP mRNA levels. Data are shown as mean ± sd. N = 3-4 for each group. ^* P <0.05, ^** P <0.01 (Student's t-test).
3 shows that the SREBP1c-CRY1 signaling pathway regulates hepatic glucose production according to an embodiment of the present disclosure. (a) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-SREBP1c. Glucose oxidase (GO) was used to measure relative glucose production. Data are shown as mean ± sd. N = 3. ^** P <0.05 (Student's t-test) for each group. (b) C57BL / 6 mice were infected with Ad-G6Pase-luc and either Ad-MOCK or Ad-SREBP1c. G6Pase promoter activity was measured by optical in vivo image and photon density. (c, d) C57BL / 6 mice were infected with Ad-MOCK or Ad-SREBP1c to perform a pyruvate tolerance test (PTT) (c). All mice were subjected to PTT on ZT3 at the expense of ZT3. The results were converted to AUC (d). Data are shown as mean ± sd. N = 5. ^* P <0.05, ^** P <0.01 (Student's t-test) for each group. (e, f) PTT (e) was performed in SREBP1 ^{− / −} and SREBP1c ^{+ / +} mice. All mice were subjected to PTT on ZT3 at the expense of ZT3. The results were converted to AUC (f). Data are shown as mean ± sd. N = 5. ^* P <0.05, ^** P <0.01 (Student's t-test) for each group. (g) H4IIE cells were infected with siCON or siCRY1. Relative mRNA levels were measured by qRT-PCR and normalized to cyclophilin mRNA levels. Data are shown as mean ± sd. N = 3. ^* P <0.05, ^** P <0.001 (Student's t-test) for each group. (h) Mouse early hepatocytes were infected with Ad-MOCK or Ad-CRY1. Relative mRNA levels were measured by qRT-PCR and normalized to TBP mRNA levels. Data are shown as mean ± sd. N = 3. ^* P <0.05 for each group (Student's t-test). (i, j) PTT was performed on CRY1 ^{+ / +} and CRY1 ^{− / −} mice (i). All mice were subjected to PTT on ZT3 at the expense of ZT10. The results were converted to AUC (j). Data are shown as mean ± sd. N = 7. ^* P <0.05, ^** P <0.01 (Student's t-test) for each group. (k) Mouse primary hepatocytes isolated from CRY1 ^{+ / +} and CRY1 ^{− / −} mice were infected with Ad-MOCK or Ad-SREBP1c. Relative glucose production was measured using a GO (glucose oxidase) kit. Data are shown as mean ± sd. N = 8. ^*** P <0.001 versus Ad-MOCK, ^### P <0.001 versus CRY1 ^{+ / +} (Student's t-test) for each group. (l, m) PTT (l) in SREBP1c ^{+ / +} mice injected with Ad-MOCK and SREBP1c ^{− / −} mice injected with Ad-MOCK or Ad-CRY1. Results were converted to AUC (m). All mice were subjected to PTT on ZT3 at the expense of ZT10. The results were converted to AUC (j). Data are shown as mean ± sd. N = 7-10 per group. ^** P <0.01, ^*** P <0.001 versus SREBP1c ^{+ / +} , Ad-MOCK, ^# P <0.05, ^### P <0.001 versus SREBP1c ^-/- , Ad-MOCK (Student's t-test).
4 shows that CRY1 regulates FOXO1 protein levels. (a, b) Mouse primary hepatocytes were infected with adenovirus with Ad-MOCK or Ad-CRY1. The expression profile of FOXO1 was analyzed by protein level (a) using Western blot and mRNA level using qRT-PCR. Data are shown as mean ± sd. N = 4. ^* P <0.05 for each group (Student's t-test). (c, d) H4IIE cells were infected with siCON or siCRY1. Immunocytochemical Analysis of Endogenous FOXO1. DAPI, 4 ', 6-diamidino-2-phenylindole. Scale bar, 10 μm. Endogenous FOXO1 and CRY1 protein levels were analyzed using Western blot (d). (e, f) Expression patterns of FOXO1 protein in livers of CRY1 ^{+ / +} and CRY1 ^{− / −} mice were analyzed by Western blot and qRT-PCT (f). Relative mRNA levels were measured using qRT-PCR to normalize to TBP mRNA levels. Data are shown as mean ± sd. N = 4. ^* P <0.05 for each group (Student's t-test). (g) CRY1 and FOXO1 protein expression in SREBP1c ^{+ / +} and SREBP1c ^{− / −} mouse livers was analyzed by Western blot. (h) H4IIE cells were co-transfected with siCRY1 and / or siFOXO1. The relative mRNA levels were measured using qRT-PCR to normalize to cyclophilin mRNA levels. Data are shown as mean ± sd. N = 3. ^# P <0.05, ^## P <0.01 versus siCRY1, ^* P <0.05, ^** P <0.01, ^*** P <0.001 versus siCON (Student's t-test).
5 shows that long-term insulin treatment stimulates CRY1 expression and inhibits hepatic glucose angiogenesis. (a, b) Mouse primary hepatocytes were treated with 10 nM insulin for different time periods. Protein level (a) was measured by Western blot. mRNA level (b) was analyzed by qRT-PCR. Data are shown as mean ± sd. N = 3. ^* P <0.05 for each group, ^*** P <0.001 versus siCON (Student's t-test). (ce) Mouse primary hepatocytes isolated from CRY1 ^{+ / +} and CRY1 ^{− / −} mice were treated with 10 nm insulin for different periods of time. Protein levels (c) were analyzed by Western blot and relative mRNA levels (d) were measured by qRT-PCR and normalized to TBP mRNA levels. Data are shown as mean ± sd. N = 3. ^* P <0.05, ^** P <0.01, ^*** P <0.001 versus CRY1 ^{+ / +} control (Student's t-test) for each group. Relative glucose production (e) was measured using a GO (glucose oxidase) kit. Data are shown as mean ± sd. N = 5. ^* P <0.05 for each group, ^** P <0.01 versus CRY1 ^{+ / +} control (Student's t-test). (f) Mouse primary hepatocytes were infected with Ad-MOCK and Ad-CRY1 and then treated with insulin (10 nM) or insulin (10 nM) and AKTVIII (5 μm) for 12 hours. Protein levels were measured by Western blot. (gi) C57BL / 6 mice were infected with Ad-MOCK or Ad-CRY1 to perform PTT with or without the AKT inhibitor MK2206. MK2206 (30 mg kg ⁻¹ ) was orally administered for 10 minutes prior to PTT. All mice were subjected to PTT on ZT3 at the expense of ZT10. The results were converted to AUC (h). Data are shown as mean ± sd. N = 5-7 for each group. ^## P <0.01, ^### P <0.001 versus Ad-MOCK + vehicle control, ^* P <0.05, ^** P <0.01, versus Ad-MOCK + MK2206 control (Student's t-test).
FIG. 6 shows that CRY1 promotes ubiquitin-mediated FOXO1 degradation. (A) Mouse primary hepatocytes were infected with adenovirus with Ad-MOCK or Ad-CRY1. The cells were treated with 20 μm MG132 or vehicle for 4 hours. Total cell lysates were analyzed by Western blot using the indicated antibodies. (b) HEK293T cells were transfected with GFP-CRY1 and / or FOXO1-MYC expression vectors. Co-immunoprecipitation and western blot with anti-MYC antibodies were performed in the presence of the indicated antibodies. IP, immunoprecipitation. (c) COS-1 cells were co-transfected with plasmids encoding FOXO1-MYC, GFP-CRY1, and Ubiquitin-HA. After transfection, the cells were treated with MG132 (20 μm) for 6 hours and the cell lysates were immunoprecipitated with anti-MYC antibody and then subjected to western blot in the presence of the indicated antibody. (d) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-CRY1. After infection, the cells were treated with MG132 (20 μm) for 4 hours. Nuclear and cytoplasmic fractions were separated and western blots were performed in the presence of the indicated antibody. (e) COS-1 cells were co-transfected with plasmids encoding nFOXO1-MYC, GFP-CRY1, and Ubiquitin-HA. After transfection, the cells were challenged with MG132 (20 μm) for 6 hours. The cell lysates were immunoprecipitated in the presence of anti-MYC antibody. IP, immunoprecipitation.
Figure 7 shows that CRY1 is involved in MDM2-mediated FOXO1 ubiquitination. (a) Mouse primary hepatocytes were infected with Ad-MOCK or Ad-CRY1 and / or siCON or siMDM2. Total cell lysates were analyzed by Western blot in the presence of the indicated antibody. (b) HEK293T cells were transfected with FLAG-MDM2, nFOXO1-MYC, and GFP-CRY1 expression vectors. Total cell lysates were co-immunoprecipitated in the presence of anti-MYC antibody followed by Western blot in the presence of the indicated antibody. IP, immunoprecipitation. (c) COS-1 cells were co-transfected with nFOXO1-MYC, FLAG-MDM2, FLAG-CRY1, and Ubiquitin-HA encoding plasmids. After transfection, the cells were challenged with MG132 (20 μm) for 6 hours. Cell lysates were immunoprecipitated with anti-MYC antibodies. IP, immunoprecipitation. (d) COS-1 cells were co-transfected with nFOXO1-MYC, FLAG-MDM2, Ubiquitin-HA, and siCRY1 encoding plasmids. The cells were challenged with MG132 (20 μm) for 6 hours. Cell lysates were immunoprecipitated with anti-MYC antibodies. IP, immunoprecipitation.
8 shows that CRY1 alleviates your life cycle in a diabetic mouse model. (ac) to 8-week-old male mice STZ ^- when ZT 3 were sacrificed one week after it was injected (150mg kg ^1). Blood glucose levels (a) were measured at ad libitum in ZT 3. After all mice were sacrificed at ZT 3, liver protein levels (b) were analyzed by Western blot. mRNA levels (c) were measured by qRT-PCR analysis to normalize to TBP mRNA levels. Data are shown as mean ± sd. N = 3. ^* P <0.05, ^** P <0.01, ^*** P <0.001 (Student's t-test) for each group. (df) STZ-injected mice were infected with MOCK or CRY1 encoding adenovirus (adenoviral dose of 2109 viral particles per mouse). Blood glucose levels (d) were randomly measured in ZT3. After all mice were sacrificed in ZT 3, mRNA levels (e) were normalized to TBP mRNA levels as measured by qRT-PCR analysis. Liver protein levels (f) were analyzed by Western blot. Data are shown as mean ± sd. N = 4. ^* P <0.05, ^** P <0.01, (Student's t-test) for each group. (g, h) 10-week old male db / + and db / db mice were sacrificed when optionally ZT3. The relative mRNA levels (g) of various liver genes were measured by qRT-PCR analysis to normalize to TBP mRNA levels. Data are shown as mean ± sd. N = 4. ^* P <0.05 for each group (Student's t-test). Protein level (h) was measured by Western blot. (il) PTT (i) was performed by infecting 10-week-old male db / db mice via the tail vein with MOCK or CRY1 encoding adenovirus (adenoviral dose of 21010 viral particles per mouse). All mice were PTT performed on ZT 3 at the expense of ZT 10. The results were converted to AUC. Blood glucose level (j) was randomly measured in ZT 3. After all mice were sacrificed in ZT 3, liver protein levels (k) were analyzed by Western blot. mRNA levels (l) were normalized to TBP mRNA levels after analysis by qRT-PCR. Data are shown as mean ± sd. N = 5. ^* P <0.05, ^** P <0.01, ^*** P <0.001 (Student's t-test) for each group.
Figure 9 shows that the SREBP1c-CRY1 axis inhibits hepatic glucose angiogenesis by promoting FOXO1 degradation. After prolonged insulin action, the SREBP1c-CRY1 axis inhibits hepatic glucose angiogenesis through nuclear FOXO1 degradation. During the assimilation state, increased CRY1 promotes MDM2-mediated FOXO1 degradation and thereby insulin continuously inhibits hepatic glucose production.

Herein we identified the SREBP1c-CRY1-FOXO1 pathway, in which SREBP1c, a transcription factor activated by insulin and involved in promoting fat biosynthesis and inhibiting glucose biosynthesis, regulates CRY1 gene expression and CRY1 inhibits glucose biosynthesis by regulating FOXO1 protein levels. In addition, since CRY1 overexpressed in the diabetic mouse model alleviates hyperglycemia, the use of CRY1 can simultaneously suppress glucose biosynthesis without increasing fat biosynthesis.

In one embodiment, the present application relates to a composition for treating type 1 or type 2 diabetes comprising a Cry1 (Cryptochrome Circadian Clock 1) gene or a protein or a functional equivalent thereof, or a substance activating its expression.

Cryptochrome Circadian Clock 1 (CRY1) included in the composition according to the present invention is a kind of flavoprotein that is sensitive to blue light in plants, but it is known that its role is not largely known in animals, and is only involved in biological clock rhythm. . The gene or protein sequence of CRY1 included in the composition according to the present invention can be used in various forms and derived so long as the effect according to the present invention is achieved. In one embodiment CRY1 derived from mammals, in particular humans, or a functionally equivalent variant thereof, is used and its gene and protein sequences are known (Gene ID: 12952, NCBI Reference Sequence: NP — 031797.1).

Genes according to the present invention include genomic DNA, cDNA and chemical synthetic DNA. Genomic DNA and cDNA can be prepared according to methods known in the art. Genomic DNA, for example, extracts genomic DNA from cells having the gene of interest and constructs a genomic library (vectors may be used, for example, plasmids, phages, cosmids, BACs, PACs, etc.) and viewed the library. Colony or plaque hybridization is performed using a probe prepared on the basis of the DNA encoding the protein of the present invention, or primers specific for the DNA encoding the protein of the present invention are prepared and PCR (Polymerase Chain Reaction) using the same is performed. It can also manufacture by doing. For example, cDNA synthesizes a cDNA based on mRNA extracted from a cell having a CRY1 gene, inserts the synthesized cDNA into a vector such as λZAP, prepares a cDNA library, and expands the cDNA library. Can be prepared by colony or plaque hybridization or by PCR.

As used herein, the term "functionally equivalent variant" refers to a variant compared to a wild type protein or gene sequence thereof derived from a particular species, but having the efficacy identified herein. Thus, for example, the CRY1 protein of the present invention or a gene encoding the same may be a mutants encoding a protein derived from a human sequence consisting of an amino acid sequence in which one or more amino acids are substituted, deleted, added and / or inserted, Derivatives, alleles, variants and homologues. Furthermore, for example, variations in nucleotide sequences may not be accompanied by variations in amino acids in proteins (degeneracy variants), and such degeneracy mutants are also included in the gene of the present invention.

Genes encoding the CRY1 protein and proteins functionally equivalent thereto are known in the art, for example PCR methods (Saiki et al., Science, 230: 1350-1354, 1985; Saiki et al., Science, 239: 487-491, 1988). For example, those skilled in the art will be able to isolate primers from a variety of mammals having high homology with the CRY1 gene by designing primers capable of specific hybridization thereof from known base sequences of the CRY1 gene.

The gene isolated as described above has high homology with the amino acid sequence of the human-derived CRY1 protein at the amino acid level. High homology refers to the identity of at least 50%, more preferably at least 70%, more preferably at least 90% (eg, at least 95%) sequences throughout an amino acid sequence. The homology of amino acid sequences or nucleotide sequences is based on BLAST (Proc. Natl. Acad. Sci. USA, 90, 5873-5877, 1993), a program called BLASTN or BLASTX (Altschul et al, J. Mol. Biol. , 215, 403-410, 1990) and specific methods are known on the following website (http: //www,ncbi.nlm.nih.gov.).

The CRY1 protein or gene included in the composition according to the present invention includes all of full length or truncated forms. In addition, there may be sequence variations according to a specific individual, region, environment, etc., even if they are derived from the same host, for example, humans, and of course, some sequences have been modified (deleted, substituted, added), but all functionally equivalent variants are present. It can be used in the present invention. In one embodiment it is of human origin and is as previously mentioned.

When the composition according to the present invention comprises the CRY1 gene or a variant thereof, the gene encoding the protein may express the CRY1 gene in target cells, for example, diabetic mast cells derived from mammals, according to known methods such as recombinant DNA technology. As long as it is possible, it may be used, for example, as a linear DNA, a plasmid vector or other DNA delivery vector. Such vectors are preferably non-viral and are not particularly limited as long as they can express the CRY1 gene in eukaryotic cells, and reference may be made to those described in the Examples herein.

In addition, the CRY1 gene of the present invention may be introduced into an expression vector used in a gene therapy system or the like, for example, a viral vector, and then included in a viral particle as a carrier according to a known method. The viral vector is not limited as long as the protein of the present invention can be introduced into a desired cell or tissue, and is preferably an adenovirus vector, and the viral vector is capable of expressing a gene contained therein in a eukaryotic cell. It is not particularly limited.

When the composition according to the present invention comprises a protein or a functionally equivalent variant thereof, for example, the vector may be purified and used after transfection into an appropriate cell in a cell culture system according to a known method. Such proteins of the invention include, for example, purified proteins, water soluble proteins, or those in fusion with protein or amino acid residues in a form bound to a carrier for delivery or administration to a target cell.

Diabetes in which the composition according to the present invention is used is a symptom of high blood glucose concentration, and includes type 1 and type 2 diabetes. Type 1 diabetes is a disease in which pancreatic beta cells that secrete insulin cause insulin secretion in the blood. Type 2 diabetes secretes insulin in pancreatic beta cells, but insulin receptors in liver, adipocytes and muscles. The lower signal causes a problem that the insulin signal is not transmitted even though there is insulin, but in such a situation, the pancreatic beta cells may be overloaded, resulting in a breakdown of the beta cells, thus preventing insulin from being secreted. In other words, type 2 diabetes has some insulin secretory function in the pancreas, but the insulin resistance is increased due to various insulin resistance due to various causes, resulting in hyperglycemia and relative insulin secretion disorder. In the present study, we observed the phenomenon of inhibiting glucose biosynthesis by overexpression of CRY1 in both type 1 and type 2 diabetic mice. The present application can be used as type 1 diabetes as well as type 2 diabetes glycemic regulators, blood sugar lowering agents.

The composition according to the invention can be used especially for type 2 diabetes, which exhibits insulin resistance by inhibiting glucose biosynthesis without increasing fat biosynthesis. Specifically, SREBP1c, whose expression is increased by insulin, regulates CRY1 gene expression, and CRY1 inhibits glucose biosynthesis by regulating FOXO1 protein levels.

In one embodiment, type 2 diabetes in which the composition according to the invention is used is particularly increased in hyperinsulinemia, especially SREBP1c as defined herein, while CRY1 can be used for diabetes due to reduction, a kind of selective insulin resistance.

Selective insulin resistance is a condition in which hyperlipidemia occurs due to excessive insulin, resulting in increased blood lipid levels and fatty liver, but high blood sugar. The reason for this has not been identified previously, and the CRY1 protein or gene according to the present invention may alleviate the hyperglycemia of such selective resistance insulin symptoms.

As used herein, the term "treatment" refers to controlling irregular blood sugar caused by diabetes, in particular to alleviate high blood sugar or to return or maintain blood sugar in the normal range.

In this respect, the present application also relates to a pharmaceutical composition for glycemic control of a diabetic patient comprising the CRY1 gene, or a protein encoded by the gene.

Glucose control refers to lowering or maintaining hyperglycemia in the normal range of blood glucose.

The composition of the present application may further contain at least one active ingredient exhibiting the same or similar function in addition to CRY1, or a compound for maintaining / increasing the solubility and / or absorption of the active ingredient. In addition, the pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, drug treatment and biological response modifiers for the treatment or prevention of kidney cancer.

The composition of the present invention may be prepared by including at least one pharmaceutically acceptable carrier in addition to the above-mentioned active ingredient. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, seeding, and one or more of these components, as necessary. Other conventional additives such as buffers and bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations, pills, capsules, granules, or tablets such as aqueous solutions, suspensions, emulsions, and the like, and may act specifically on target organs. Target organ specific antibodies or other ligands may be used in combination with the carriers so as to be used. Furthermore, it may be suitably formulated according to each disease or component using an appropriate method in the art or using the method disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA). Can be.

The method of administration of the composition of the present application is not particularly limited thereto, and known administration methods may be applied, and parenteral administration (for example, intravenous, subcutaneous, intraperitoneal, or topical) or oral, depending on the desired method. In the case of parenteral administration, it can be administered through a patch-type, nasal / respirator attached to the skin, and administration by intravenous injection is preferable to obtain a rapid therapeutic effect. The dosage may vary widely depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Parenteral administration may be preferred for protein preparations comprising the CRY1 gene or polypeptide, but does not exclude other routes and means. For typical drugs, dosage units include, for example, about 0.01 mg to 100 mg but do not exclude the below and above ranges. The daily dose may be about 1 μg to 10 g, and may be administered once to several times a day.

Herein, glucose production is reduced by SREBP1c overexpression in mouse primary hepatocytes, SREBP1c regulates glucose production through the expression of CRY1, and CRY1 enhances the binding of a protein called MDM2, which can degrade FOXO1 and FOXO1, thereby enhancing FOXO1. The SREBP1c-CRY1-FOXO1 signaling pathway was found to promote the degradation of. FOXO1 inhibits the regulation of the expression of Phosphoenolpyruvate carboxykinase (PEPCK) and G6Pase (Glucose-6-phosphatase), which are major enzymes of glucose biosynthesis. do.

Thus, in another aspect, the present disclosure is directed to a method for screening a prophylactic or therapeutic agent for type 2 diabetes that targets the Sterol Regulatory Element Binding Transcription Factor (SREBP1c) -CRY1-FOXO1 signaling pathway.

In one embodiment the method comprises a first step of providing a liver cell or animal having reduced expression or activity of CRY1; A second step of treating said cells with a test substance that is expected to increase the expression or activity of CRY1; A third step of measuring FOXO1 protein concentration in cells treated with the test substance; And; And a fourth step of selecting a glucose biosynthesis inhibitor or a candidate for treating diabetes when the FOXO1 protein amount is reduced compared to the FOXO1 protein amount in a control group not in contact with the test substance.

The cells or animals to which the method according to the present invention is applied may be used to exhibit the characteristics or symptoms of diabetes, in particular cells and / or animals in which SREBP1c and / or CRY1 is reduced in expression or activity.

SREBP1c and / or CRY1 expression is expression at the gene and protein levels, with reduced expression of cells compared to normal cells, especially normal hepatocytes, which levels are reduced by those skilled in the art based on the disclosure herein and the level of the art. Can be judged. Activity of SREBP1c and / or CRY1 means that the protein is expressed, but due to a mutation or the like, it does not exhibit or decrease the desired activity, and those skilled in the art can determine a reduced level based on the disclosure herein and the level of the art. can do.

The animal model used in the method according to the present invention may be a type 1 or type 2 diabetes model mouse, which is known in the art, for example, streptozotocin-induced type 1 diabetic mice or naturally occurring 2 Db / db mice (C57BLKsJ-db / db mice), a type diabetes model.

Cells used in the method according to the present invention is a cell capable of synthesizing glucose having the above-mentioned conditions, such cells are cells derived from the animal model described above, in particular cells capable of synthesizing glucose, in particular primary hepatocytes, or cell lines Including but not limited to H4IIE, HepG2.

In another embodiment according to the present application, a cell or animal that can be used in the method according to the present application may be an animal or cell in which the expression of SREBP1c, which is an upper level of CRY1, is increased but the expression or activity of CRY1 is decreased.

In the method according to the present invention, the molecular target is CRY1, and a substance which increases its expression or activity may be used as a test substance for diabetic therapeutic agents or glucose biosynthesis inhibition screening. A substance that increases the expression of CRY1 means an increase in gene and / or protein levels.

The test substance used in the method of the present invention is a substance which is expected to modulate the expression and / or activity of the target gene or protein described above. For example, for the purpose of screening a drug, the compound may have a low molecular weight therapeutic effect. have. For example, compounds of about 1000 Da in weight such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may form part of a compound library, and the number of compounds constituting the library may vary from tens to millions. Such compound libraries include peptides, peptoids and other cyclic or linear oligomeric compounds, and small molecule compounds based on templates such as benzodiazepines, hydantoin, biaryls, carbocycles and polycycle compounds (such as naphthalene, phenoty) Azine, acridine, steroids, and the like), carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocycles (such as triazine, indole, thiazolidine, etc.), but these are merely illustrative. It is not limited to this.

The method according to the present application can be measured by the change in the expression level of the FOXO1 protein after the test material treatment to show the desired effect.

Forkhead box protein O1 (FOXO1) is a protein of the SREBP1c-CRY1-FOXO1 pathway identified herein and is a transcription factor related to glycemic control in insulin signaling, and human nucleic acid and protein sequences are known as NM_002015 and NP_002006.2, respectively. have. Here we find that CRY1 promotes the degradation of FOXO1 through MDM2 and eventually inhibits glucose synthesis, resulting in a type 2 diabetes therapeutic based on the SREBP1c-CRY1-FOXO1 mechanism that eventually lowers the protein concentration of FOXO1.

In another embodiment, a test substance can be selected by measuring the expression of PEPCK or G6Pase, an enzyme involved in glucose biosynthesis in which expression is promoted by FOXO1 in addition to the third step or instead of the FOXO1 of the third step. . In this case, the candidate material in the fourth step of the present method is a decrease in the expression of the PEPCK or G6Pase in the cells in contact with the test material compared to the control cells not in contact with the test material.

The nucleic acid and protein sequences of Phosphoenolpyruvate carboxykinase (PEPCK) are known as (Gene ID: 18534, NP_035174.1) and the nucleic acid and protein sequences of G6Pase (Glucose 6-phosphatase), respectively (Gene ID: 14377, NP_032087.2). Is known.

Determination of the amount of FOXO1 protein or expression of PEPCK or G6Pase herein includes quantitative and / or qualitative analysis, including the detection of presence, absence, and detection of expression levels, which methods are known in the art, and Appropriate methods may be selected for the practice herein.

According to an embodiment of the present disclosure, the detection reagent comprises an antibody, and the measurement of the amount of FOXO1 protein or expression of PEPCK or G6Pase herein is performed using a monoclonal antibody that specifically binds thereto.

Antibodies that can be used herein are polyclonal or monoclonal antibodies, preferably monoclonal antibodies. Antibodies may be commonly used in the art, such as fusion methods (Kohler and Milstein, European Journal of Immunology, 6: 511-519 (1976)), recombinant DNA methods (US Pat. No. 4,816,56) Or phage antibody library methods (Clackson et al, Nature, 352: 624-628 (1991) and Marks et al, J. Mol. Biol., 222: 58, 1-597 (1991)). General procedures for antibody preparation are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; And Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991, which are incorporated herein by reference. For example, the preparation of hybridoma cells producing monoclonal antibodies is accomplished by fusing immortal cell lines with antibody-producing lymphocytes, and the techniques required for this process are well known to those skilled in the art and can be readily implemented. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

Such immunoassays can be performed according to various quantitative or qualitative immunoassay protocols developed in the prior art. The immunoassay format may include radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbant assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunity. Including but not limited to fluorescent staining and immunoaffinity purification. The immunoassay or method of immunostaining is described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984, etc., which is incorporated herein by reference. By analyzing the final signal intensity by the above-described immunoassay process, that is, by performing a signal contrast with a normal sample, it is possible to diagnose whether the disease occurs.

Methods for the analysis of the amount and presence of the protein expression patterns are known, for example Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay, radioimmunodiffusion, oukterroni Ouchterlony) immune diffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, and the like. Reagents for detecting protein or nucleic acid levels are known, for example, the former may be an antigen-antibody reaction, a substrate that specifically binds to the marker, a nucleic acid or peptide aptamer, a receptor that specifically interacts with the marker or It can be detected through reaction with a ligand or cofactor, or a mass spectrometer can be used. Reagents or materials that specifically interact with or bind to the markers of the present disclosure may be used with chip or nanoparticles.

According to another embodiment of the present disclosure, expression amount analysis can be performed at the mRNA level if possible. mRNA detection is usually carried out by Northern blot or reverse transcription PCR (polymerase chain reaction). In the latter case, the RNA of a sample is specifically isolated from mRNA, and then cDNA is synthesized therefrom, and then a specific gene or a combination of primers and probes is used to detect a specific gene in the sample. Or it is a method which can determine the expression amount. Such methods are described, for example, in (Han, H .; Bearss, DJ; Browne, LW; Calaluce, R .; Nagle, RB; Von Hoff, DD, Identification of differentially expressed genes in pancreatic cancer cells using cDNA microarray. Cancer Res 2002 , 62, (10), 2890-6).

Candidates compared the amount of FOXO1 protein in the cells in contact with the test material and the control cell not in contact with the test material, the amount of FOXO1 protein in the cell in contact with the test material is the protein of FOXO1 of the control If reduced compared to the amount it can be selected as a candidate for type 2 diabetes treatment.

The type of cell used in the present method and the amount and type of test substance vary depending on the specific test method used and the type of test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, as a candidate, a substance which results in a decrease in the expression or activity of the protein in the presence of the test substance is selected as compared with the control group which is not in contact with the test substance. Compared with the control group, about 99% reduction, about 95% reduction, about 90% reduction, about 85% reduction, about 80% reduction, about 75% reduction, about 70% reduction, about 65% reduction , About 60% reduction, about 55% reduction, about 50% reduction, about 45% reduction, about 40% reduction, about 30% reduction, about 20% reduction, about 10% reduction However, it does not exclude the range beyond this.

In another aspect, the present disclosure relates to a CRY1 gene, or a protein encoded by the gene, or a kit or method for regulating the SREBP1c-CRY1-FOXO1 signaling pathway in an in vitro cell or animal comprising the gene.

In another aspect the invention relates to a kit or method for the control of glucose biosynthesis in a CRY1 gene, or a protein encoded by said gene, or in vitro cells or animals comprising said gene.

For a description of the materials used in the kits and methods and the regulatory mechanism using the same, reference may be made to the foregoing. Methods and kits according to the present application can be usefully used as, but not limited to, blood glucose control agents, hypoglycemic agents, existing drug tests, drug development, research tools of diabetes.

Hereinafter, examples are provided to help understand the present invention. However, the following examples are provided only to more easily understand the present invention, and the present invention is not limited to the following examples.

Example

Animal experiment

C57BL / 6 male mice were purchased from SAMTACO. db / + and db / db mice were purchased from central laboratory animals. SREBP1c deficient mice were received from Dr. J. Horton of the University of texas Southwestern Medical Center. All animals were maintained in a pathogen-free animal room consisting of 12 hours light and 12 hours dark. Tissues of mice were stored at -80 degrees and analyzed. All mouse experiments were performed with permission from SNU IACUC and the Institutional Animal Care and Use Committee at the University of North Carolina.

Reagents and Plasmids

MG132 was purchased from Calbiochem (San Diego, CA, USA). MK2206 was purchased from Selleckchem (S1078). AKTVIII was purchased from Santa Cruz Biotechnology (sc-202048). The following antibodies and where used were purchased: MYC (Cell Signaling, 2276, 1: 1,000 dilution), HA (Cell Signaling, 3724, 1: 1,000 dilution), FOXO1 (Cell Signaling, 2880, 1: 1,000 dilution), phosphor-FOXO1-Ser256 (Cell Signaling, 9461, 1: 1,000 dilution), AKT (Cell Signaling, 9272, 1: 1,000 dilution), and phosphor-AKT-Ser473 (Cell Signaling, 9271, 1: 1,000 dilution), FLAG ( Sigma-Aldrich, F3165, 1: 1,000 dilution), ACTIN (Sigma-Aldrich, A5316, 1: 2,000 dilution), G6Pase (Santa Cruz Biotechnology, sc-33839, 1: 500 dilution), POLII (Santa Cruz Biotechnology, sc- 899, 1: 1,000 dilution), GFP (Santa Cruz Biotechnology, sc-9996, 1: 1,000 dilution), GAPDH (LabFrontier, Co., LF-PA0018, 1: 1,000 dilution), SREBP1 (BD Bioscience, 557036, 1: 1,000 dilution), MDM2 (Abcam, ab16895, 1: 1,000 dilution) and CRY1 (lab made antibody from Dr Aziz Sancar31, 1: 200 dilution). GFP-CRY1 was cloned into pEGFP-N1 vector and FLAG-MDM2 was cloned into pCMV-3 FLAG. Mouse CRY1 promoter was cloned into pGL3-basic vector.

In vivo  imaging system

Ten-week-old C57BL / 6 male mice were infected with adenovirus containing GFP and SREBP1c along with adenovirus containing G6Pase luciferase. After 5 days, 100 mg / kg of firefly D-luciferin was stabbed in the abdominal cavity of the mouse and anesthetized after 5 minutes, and images were obtained using an IVIS100 machine.

Pyruvate  Resistance test

Mice were starved for 16 hours for pyruvate resistance experiments and then injected into the abdominal cavity with pyruvate at a concentration of 2 g / kg. Blood glucose was measured using a free-style blood glucose meter each hour through the blood vessels of the tail.

Cell-based Ubiquitination  Method

FOXO1 WT-MYC, nFOXO1 (ADA) -MYC, GFP-CRY1 (or FLAG-CRY1), FLAG-MDM2, and Ubiquitin-HA were added to COS-1 cells, and MG132 was treated at a concentration of 20 uM for 4 hours. Cells were then crushed using TGN Buffer and then precipitated using myc antibody. After washing the precipitate three times with TGN buffer, the protein was separated using SDS-PAGE. Since the Western blotting was used HA antibody.

ChIP  assay

4% formaldehyde was used to fix chromatin and protein in the nucleus of H4IIE cells. After fixation, cells were digested and DNA was crushed using sonication. Thereafter, it was precipitated for 2 hours using SREBP1c antibody and control IgG antibody. Precipitated DNA fragments were amplified using the PCR method. ChIP assay primer sequences are as follows: sense, 50-GTCCGAGCCAGCGTAGTAAA-30, antisense, 50-GGATAGCGCGGGCTAGAG-30; negative control primer sense, 50-CCAGCCACTTTGCTGAAGTT-30 and antisense, 50-CTAGACAAGGCTGCCCACTC-30.

Adenovirus  Produce

The cDNAs of SREBP1c and CRY1 were cloned into an adTrack-CMV shuttle vector and recombined into an ad-easy adenoviral vector system. The completed vector was expressed in HEK293A cells to condense the adenovirus produced. For in vivo experiments, 5 x 10 ⁹ pfu (plaqueformation unit) Adenovirus was used.

Mouse primary hepatocyte culture

Primary hepatocytes were isolated from 10-week-old C57BL / 6 male mice. After anesthetizing mice, collagenase was injected using blood vessels of the liver. This separated liver tissue into hepatocytes. After 6 hours, the isolated hepatocytes were attached to the dish. After that, the experiment was carried out using adenovirus or siRNA. Adenovirus was treated at a concentration of 10 PFU for 4 hours and then the media was changed.

Cell breakdown and precipitation

Wash cells with cold PBS then TGN Buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Tween-20 and 0.3% NP-40) or SDS lysis buffer (200 mM Tris-HCl, pH 6.8, 10% glycerol and 4% SDS) were digested with buffer containing 0.1% proteases inhibitor. The by-product of lysed cells was 12,000 r.p.m. After centrifugation for 15 minutes at 4 ℃ 1 ~ 1.5mg of protein was used for immunoprecipitation. Immunoprecipitation was incubated at 4 ° C. for 2 hours using primary antibody followed by 50% slurry of protein A sepharose. After washing three times, Western blot was performed using SDS-PAGE.

Transient transduction and luciferase  assay

Various DNAs were inserted into HEK293T cells by transient transfection. The method used at this time is calcium-phosphate method. After incubation for 36 hours, luciferase activity and beta-galatosidase activity were measured after cell breakdown using lysis buffer (25 mM Tris-phosphate pH 7.8, 10% glycerol, 2 mM EDTA, 2 mM DTT and 1% Triton X-100). (Promega, Madison, Wis., USA).

RNA Preparation and Quantitative Real Time PCR

Total RNA was extracted with iso-RNA lysis reagent and cDNA was synthesized with RevertAid M-MuLV reverse transcriptase. Relative mRNA levels were measured using the CFX96TM real time system and calculated after correction with TBP or cyclophilin mRNA amounts.

siRNA  Transient transduction

SiRNA Duplex of CRY1, FOXO1, MDM2 Bioneer, Inc. It was purchased from (Daejeon, South Korea). Primary cells were transiently transduced using Lipofectamine RNAi MAX.

Glucose Biosynthesis ASSAY

Glucose biosynthesis in primary hepatocytes was used with a Glucose oxidase assay kit (Sigma-Aldrich, St Louis, MO, USA). Cells were treated with Krebs-Ringer buffer (115 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl ₂ , 1.2 mM NaH ₂ PO ₄ , 2.5 mM CaCl ₂ , 25 mM NaHCO ₃ , 10 mM lactate and 2 mM pyruvate, pH 7.4) at 37 ° C. and 5%. It was measured after incubating for 6 hours under conditions of CO ₂ .

Statistical analysis

The size of the sample was selected at the level having statistical significance based on the slow experiment. Statistical analysis was performed using Student's t-test and expressed as mean ± s.d. Each experiment was performed three or more times independently. The significance of P <0.05 is statistically significant. Samples with technical failures were excluded from the final analysis. Each experiment was not performed randomly. In addition, the experimenter knows the identity of the test sample and performed it.

Example  1. By ingestion and insulin Of CRY1  Up-regulation

In the liver, the peripheral circadian clock genes are regulated by various nutritional and hormonal changes. If the biological clock gene is closely associated with energy homeostasis, the inventors thought that the biological clock gene could affect uptake-dependent liver lipid and sugar metabolism. Since the expression of most biological clock genes fluctuates in a time-dependent manner, the end point of intake and / or reintake experiments was fixed at ZT3. Upon re-ingestion, the expression of lipogenetic genes, including SREBP1c, FASN and ACC, was upregulated in the liver. Consistent with previous studies, expression of glycogenetic genes such as PEPCK and G6Pase was downregulated in post-meal conditions (FIG. 1 a). Interestingly, the level of hepatic CRY1 mRNA was increased in reingested mice (FIG. 1A and FIG. 1A). In addition, the expression of CRY1 protein was significantly increased in the re-ingested mouse liver at ZT 3 (FIG. 1B). Since CRY1 is one of the biological clock core genes, we examined CRY1 gene expression at ZT 22 and ZT 10, the peak and trough times of CRY1 expression, respectively. Upon reingestion, the expression of many biotime genes, including CRY1, BMAL1, CLOCK, PER1, PER2, and Reverbs, changed significantly when ZT22, but mostly not when ZT10. The basal level of CRY1 expression was regulated differently at ZT 22 and ZT 10, whereas reingestion was increased at ZT 22 compared to when CRY1 mRNA and protein levels were ZT 10. These results indicate that CRY1 expression is regulated by nutritional stimulation at certain ZT time points. Because of this finding, we tested whether insulin can increase liver CRY1 gene expression. In primary hepatocytes, CRY1 mRNA levels were increased by insulin similar to SREBP1c mRNA (FIG. 1 c). Taken together, these data show that liver CRY1 expression is upregulated by uptake and insulin.

It has been reported that CRY1 appears to inhibit the angiogenesis of sugars in the liver by interfering with glycocorticoid receptor signaling and glucagon signaling pathways. CRY1 interacts with the α-subunits of glycocorticoid receptors and glucagon receptors that participate in the regulation of sugar angiogenesis. Nevertheless, the specific role of CRY1 during the nutritional state after exposure to insulin is not clearly explained. To test whether CRY1 can inhibit your life cycle by inhibiting glucagon signaling and / or glycocorticoid receptor-dependent pathways, CRY1-overexpressing primary hepatocytes may be used to simulate the condition for glucagon or glycocorticoid stimulation. forskolin), db-cAMP or dexamethasone. In primary hepatocytes, CRY1 partially inhibits your life cycle gene in the presence of forskolin, db-cAMP or dexamethasone, which in addition to the glucagon and glycocorticoid signaling pathways, inhibits hepatic glucose production. It means that there may be another signaling cascade regulated by CRY1. Thus, CRY1 appears to be involved in a variety of regulatory pathways that regulate hepatic glucose angiogenesis in response to various hormones.

Example  2. On SREBP1c  by CRY1  Gene expression regulation

The CRY1 promoter was then analyzed in various species, including monkeys, cattle, sheep, humans, rats, and mice, to examine proteins that regulate insulin-dependent CRY1 gene expression. In addition to E-BOX (CANNTG) in the proximal CRY1 promoter, there are several sterol regulatory elements (SREs), which are also target motifs for the key biological clock proteins BMAL1 and CLOCK. Both SRE and E-BOX motifs are well known binding targets of SREBP1c with double DNA binding specificity. To determine if SREBP1c can regulate CRY1 gene expression, SREBP1c was overexpressed in mouse primary hepatocytes. As shown in Figures a and b, hepatic CRY1 mRNA and CRY1 protein levels were increased by SREBP1c overexpression (a, b in Figure 2), which is the major factor in which SREBP1c upregulates hepatic CRY1 gene expression in post-meal state. Imply that it can be a transcription factor. Next, the effect of ectopic expression of SREBP1c on CRY1 promoter activity was investigated. Expression of wild-type CRY1 promoter-derived luciferase was compared with expression from a promoter with a mutant SRE motif (3XSRE) in HEK293T cells. It was found herein that promoter activity is substantially lost with 3XSRE sequence loss while the E-BOX motif sequence is not (FIG. 2C). In addition, SREBP1c binding to the CRY1 promoter was confirmed by ChIP analysis (d in Fig. 2). As a result, liver SREBP1c reduced G6Pase and PEPCK expression (FIG. 2A).

To investigate whether SREBP1c promotes CRY1 gene expression in the presence of insulin, primary hepatocytes isolated from SREBP1c ^{+ / +} and SREBP1c ^{− / −} mice were challenged with insulin. CRY1 protein levels in SREBP1c ^{+ / +} primary hepatocytes were markedly elevated by insulin, whereas CRY1 protein levels in SREBP1c ^{− / −} primary hepatocytes were increased to a lower extent by insulin (FIG. 2 e). Also SREBP1c ^{+ / +} mice, as opposed to, SREBP1c ^{- / -} did not increase the cross-CRY1 gene expression, even if the re-uptake in the mouse (f of Fig. 2). These data indicate that SREBP1c is a major factor that upregulates liver CRY1 gene expression in the post-meal state.

Example  3. SREBP1c - CRY1  Inhibition of hepatic glycemic process by pathway

SREBP1c overexpression in mouse primary hepatocytes was shown to reduce glucose production (FIG. 3 a). Also in optical in vivo image analysis, liver SREBP1c overexpression significantly inhibited the promoter activity of the in vivo G6Pase gene (FIG. 3B). Thus, expression of the G6Pase and PEPCK genes was inhibited in the liver of SREBP1c overexpressing mice. This finding led to the study of the effect of SREBP1c on in vivo blood glucose levels. Adenovirus overexpression of SREBP1c after pyruvate injection in the PTT test significantly reduced blood glucose levels (FIG. 3 c, d). In addition, the blood glucose was higher in SREBP1c ^{− / −} mice than in SREBP1c ^{+ / +} mice (FIG. 3e, f). These results indicate that liver SREBP1c can inhibit your life process by strongly regulating your life cycle gene expression.

In order to investigate whether CRY1, a new target gene of SRENBP1c, regulates the expression of hepatic glycemic neoplasms, CRY1 expression was inhibited through siRNA (small interfering RNA) in HCCIIE cells. When CRY1 was down-regulated, the expression of G6Pase and PEPCK genes, which are important genes for liver life, increased (FIG. 3G). In contrast, hepatic CRY1 overexpression reduced G6Pase and PEPCK gene expression in mouse primary hepatocytes (FIG. 3H). To confirm this observation, blood glucose levels of pyruvate-induced blood levels were measured in CRY1 ^{+ / +} and CRY1 ^{− / −} mice. As shown in FIG. 3 i, j, blood glucose levels were higher in CRY1 ^{− / −} mice than in CRY1 ^{+ / +} mice. To determine whether CRY1 is a major mediator of hepatic glucose angiogenesis by SREBP1c, glucose production assays were performed in primary hepatocytes from CRY1 ^{+ / +} and CRY1 ^{− / −} mice. As shown in FIG. 3K, SREBP1c overexpression attenuated glucose production in CRY1 ^{+ / +} primary hepatocytes while SREBP1c did not inhibit glucose production in CRY1 ^{− / −} primary hepatocytes. To determine if the SREBP1c-CRY signaling pathway inhibits the actual in vivo hepatic glucose production process, CRY1 was overexpressed adenovirus in the liver of SREBP1 ^{− / −} mice. In PTT, blood glucose levels were higher in SREBP1c ^{− / −} mice than in SREBP1c ^{+ / +} mice, whereas blood glucose levels were decreased in CRY1 overexpressed SREBP1c ^{− / −} mice (l, m in FIG. 3). These data indicate that the SREBP1c-CRY1 signaling pathway can inhibit hepatic glycemic angiogenesis in vivo.

Example  4. On CRY1  by FOXO1  Protein Level Regulation

In order to elucidate the underlying mechanism by which insulin-induced CRY1 inhibits hepatic life processes, the present inventors focused on FOXO1 because the regulatory effects of FOXO1 on insulin signaling and glucose angiogenesis are well established. The level of FOXO1 protein in mouse primary hepatocytes was reduced by CRY1 overexpression (FIG. 4 a) while FOXO1 mRNA levels were not altered (FIG. 4B). These data mean that CRY1 can regulate the protein level, independent of FOXO1 mRNA. In addition, FOXO1 protein levels were increased in hepatocytes with CRY1 inhibition (FIG. 4 c, d). Consistent with these in vitro data, FOXO1 protein levels were higher in CRY1 ^{− / −} mouse livers than in CRY1 ^{+ / +} mouse livers, while FOXO1 mRNA levels were not different between CRY1 ^{+ / +} and CRY1 ^{− / −} mice. (E, f in Figure 4). Compared to SREBP1c ^{+ / +} mice, the levels of FOXO1 protein and G6Pase and PEPCK mRNAs were increased in the liver of SREBP1c ^{− / −} mice while the levels of CRY1 protein were decreased (FIG. 4 g). In order to demonstrate that CRY1 inhibits hepatic glycemic angiogenesis via FOXOl, the effect of CRYl and / or FOXOl inhibition on glycogenic gene expression was investigated. When the FOXO1 gene is downregulated by siRNA, there is no increase in expression of G6Pase and PEPCK genes by CRY1 inhibition, suggesting that FOXO1 may be a downstream factor of CRY1 that regulates uveogenesis. These in vivo and in vitro data mean that CRY1 can inhibit or mitigate hepatic glucose angiogenesis via FOXO1.

Example  5. Insulin-activated On CRY1  by FOXO1  Confirm protein reduction

Activation of angiogenesis per FOXO1-mediated is inhibited by insulin. AKT is a major downstream molecule of insulin-activated signaling, which then translocations from the nucleus to the cytoplasm through association with 14-3-3 proteins (Zhao, X. et al. Multiple elements regulate nuclear / cytoplasmic shuttling of FOXO1: characterization of phosphorylation- and 14-3-3-dependent and -independent mechanisms.Biochem. J. 378, 839849 (2004)). In primary hepatocytes, FOXO1 phosphorylation is rapidly increased by insulin. However, despite a decrease in FOXO1 phosphorylation at later stages of insulin action, liver life cycle programming is continuously and efficiently inhibited. Interestingly, hepatic CRY1 expression increased during the relative late period of insulin action (FIG. 5A).

The translocation of FOXO1 from the nucleus to the cytoplasm by AKT is a well known mechanism by which insulin acutely inhibits liver glucose production. As insulin upregulates CRY1 and sequentially downregulates FOXO1 protein (FIGS. 1C and 4A), these events were studied in chronological order by examining the expression profiles of FOXO1 and CRY1 proteins in insulin-treated primary hepatocytes. As shown in Figure 5a, phosphorylation of AKT and FOXO1 was certainly induced in cells treated with insulin for a relatively short period of time (0-4 h). Consistent with conventional results, short-term insulin treatment reduced the levels of FOXO1 protein, which was mediated by FOXO1 ubiquitination and degradation in cell substrates. In addition, phosphorylation levels of AKT and FOXO1 gradually and substantially decreased by long term (8-12 h) insulin induction. After short-term (0-4h) exposure to insulin, rather than after long-term insulin treatment (8-12h), FOXO1 migration from the nucleus to the cytoplasm was shown to occur. Interestingly, in hepatocytes treated with insulin for an extended period of time (8-12 h), the level of CRY1 protein increased significantly, while the level of total FOXO1 protein continued to decrease, which was inversely proportional to the total amount of FOXO1 protein. Indicates that it is related. Thus, expression of PEPCK and G6Pase genes was downregulated after prolonged (8-12h) or short (0-4h) insulin treatment (FIG. 5B). These data indicate that the continuous reduction of FOXO1 protein may be involved in the inhibition of hepatic uveogenesis during prolonged insulin action.

Next, it was examined whether CRY1 actually regulates FOXO1 protein in insulin-treated hepatocytes. To address this, CRY1 ^{+ / +} and CRY1 ^{− / −} primary hepatocytes were tested. As shown in FIG. 5C, the level of FOXO1 protein was decreased in insulin-treated CRY1 ^{+ / +} primary hepatocytes, while the level of FOXO1 protein was not continuously inhibited in insulin-treated CRY1 ^{− / −} primary hepatocytes. . Later on insulin action, the level of PEPCK and G6Pase mRNA was slightly but substantially increased in CRY1 ^{− / −} primary hepatocytes (FIG. 5 d). In addition, in long-term (8-12h) insulin-treated CRY1 ^{− / −} primary hepatocytes, insulin did not inhibit glucose production (FIG. 5E). Taken together, these data indicate that during long-term insulin action (8-12 h), the FOXO1 protein is reduced and CRY1 can inhibit the expression of liver GI genes.

To determine if increased CRY1 could inhibit liver life processes even in the absence of short-term insulin action (0-4 h), an AKT inhibitor was tested. In primary hepatocytes, insulin increased phosphorylation levels of both AKT and FOXO1, while co-treatment with AKT inhibitor AKTVIII inhibited phosphorylation as expected (FIG. 5F). However, in CRY1-overexpressed hepatocytes, total FOXO1 levels were reduced in insulin treated cells and / or AKT inhibitor treated cells, which means that CRY1 can downregulate FOXO1 protein independently of FOXO1 phosphorylation (FIG. 5, f). To confirm this finding in vivo, mice were tested with another AKT inhibitor, MK2206. As shown in g, h of FIG. 5, administration of MK2206 significantly increased blood glucose levels for pyruvate challenge. However, even in the presence of MK2206 in mice, blood glucose levels were significantly attenuated by adenovirus CRY1 overexpression. The level of FOXO1 protein was significantly increased by MK2206 while FOXO1 protein expression was inhibited in vivo by CRY1 increase (FIG. 5I). Taken together, these data clearly show that CRY1-1 dependent FOXO1 reduction contributes to the suppression of liver life processes independent of AKT activation.

Taken together, the in vivo and in vivo data herein indicate that CRY1-dependent FOXO1 degradation may be one of the major mechanisms that weaken the liver life cycle during prolonged insulin action. Thus, these findings indicate that in the early insulin response, AKT-mediated FOXO1 phosphorylation resulted in acute glucose synthesis, whereas SREBP1c-mediated CRY1 regulation inhibited the angiogenesis of futile sugars in the liver throughout anabolic activity. Involved in a more sustained response to FIG. 9 (FIG. 9).

Example  6. On CRY1  by Of FOXO1 Proteasome  Decomposition Stimulation

Since CRY1 overexpression reduced FOXO1 protein levels but not FOXO1 mRNA levels, it was investigated whether FOXO1 downregulation could be via proteasome degradation. As shown in FIG. 6A, FOXO1 protein reduction by CRY1 overexpression can be alleviated by MG132, which means that the regulation of FOXO1 protein by CRY1 depends, at least in part, on proteasome degradation. do. Examination of the physical interactions between FOXOa and CRY1 protein revealed that CRY1 can interact with FOXO1 protein in co-immunoprecipitation assays (FIG. 6 b). Then, it was examined whether CRY1 can induce FOXO1 degradation via the ubiquitin-proteasome pathway. As shown in FIG. 6C, CRY1 overexpression dramatically promoted FOXO1 poly-ubiquitination, which means that CRY1 can possibly enhance FOXO1 degradation via protein-protein interactions.

To explore the subcellular location of FOXO1 degradation induced by CRY1, the levels of nuclear and cytoplasmic FOXO1 proteins were examined. As shown in d of FIG. 6, the nuclear fraction of the FOXO1 protein was reduced by CRY1 overexpression, while this reduction was prevented by incubation with MG132. At the same time, the level of cell substrate FOXO1 was not altered by CRY1 overexpression, regardless of the presence of MG132. Consistent with these results, polyubiquitination (nFOXO1-MYC) in the nuclear form of the FOXO1 mutant protein was significantly increased by CRY1 (FIG. 6E). Thus, this indicates that degradation of the FOXO1 protein via poly-ubiquitination is stimulated by CRY1 in the nucleus.

Example  7. Of CRY1 MDM2 -medium FOXO1  Identification of decomposition involvement

Among the various ubiquitin E3 ligase of the FOXO1 protein, MDM2 ubiquitin E3 ligase has been shown to be involved in CRY1-mediated FOXO1 degradation. As shown in a of FIG. 7, the levels of FOXO1 protein were significantly recovered by MDM2 inhibition in CRY1-overexpressing cells, suggesting that MDM2 may participate in CRY1-dependent FOXO1 reduction. To investigate the dynamics of CRY1 in MDM2-mediated FOXO1 degradation, we examined whether CRY1 can regulate the subcellular location of MDM2. Wild type CRY1 and cell substrate CRY1 (DNLS-CRY1) did not alter the subcellular location of nuclear MDM2. Instead, it was found that CRY1 strengthens the association between FOXO1 and MDM2 (FIG. 7B).

In another experiment, it was investigated whether CRY1 could modulate MDM2-mediated FOXO1 degradation. As shown in c, d of FIG. 7, MDM2-mediated poly-ubiquitination of the nuclear form of FOXO1 protein was promoted by CRY1 overexpression (FIG. 7 c), whereas FOXO1 polyubiquitination by MDM2 was attenuated by CRY1 inhibition. (D of FIG. 7). These data mean that CRY1 can inhibit FOXO1-mediated liver glucose production by MDM2-induced FOXO1 degradation.

Example  8. Type 1  And Type 2  In the Diabetic Mouse Model On CRY1  Alleviation of hyperglycemia caused by

STZ mouse is a representative model of type 1 diabetes mellitus, and is a mouse in which insulin is not secreted due to a problem in the beta cells of the pancreas that secrete insulin. db / db mouse is a representative model of type 2 diabetes. Insulin is secreted from the beta cells of the pancreas, but insulin signals are not transmitted in the liver, adipocytes, and muscles. do. In such a situation, the beta cells are overloaded so that the beta cells are broken and insulin is not secreted.

In each mouse experiment, we observed a phenomenon in which glucose biosynthesis could be inhibited by overexpression of CRY1 in both insulin secretion and insulin signaling pathways. Rather, it can be a therapeutic target for type 2 diabetes.

Streptozotocin (STZ) -treated insulin-deficient mice had hyperglycemic symptoms as well as glucose angiogenesis programs involving FOXO1 protein levels, with simultaneous increase in PEPCK and G6Pase gene expression (FIG. 8 a-c). CRY1 was overexpressed in livers of STZ-treated insulin-deficient mice because the SREBP1c-CRY1 axis is upregulated by insulin. As shown in d of FIG. 8, CRY1 overexpression reduced blood glucose levels in STZ-treated mice. In addition, CRY1 reduced FOXO1 protein levels as well as PEPCK and G6Pase gene expression (FIG. 8 e, f). These data indicate that CRY1 is one of the major factors that inhibit liver glucose production during insulin action.

Interestingly, in livers of obese animals, such as db / db and diet-induced obese mice, SREBP1c levels were elevated while hepatic glycemic process was not inhibited. To investigate which processes are deregulated in the regulation of liver life processes, mRNA and protein levels for the SREBP1c-CRY1 axis and your life cycle genes in diabetic animals were examined. Similar to previous studies, mRNA levels of SREBP1c and your life cycle genes were high in db / db mice (FIG. 8 g). However, liver CRY1 protein in db / db mice was substantially reduced and the level of MDM2 protein did not change significantly (FIG. 8 h). Similarly, dietary-induced obese mice showed high expression of SREBP1c and glycoprotein genes while CRY1 was not activated. To test this, de-regulated CRY1 protein in diabetic animals could mediate hyperglycemia with increased FOXO1 protein, CRY1 was overexpressed with adenovirus in the liver of db / db mice. As shown in FIG. 8 i, pyruvate-induced blood glucose levels were reduced in CRY1-overexpressing db / db mice. Feeding blood glucose levels were also reduced by CRY1 overexpression (FIG. 8 j). In addition, ectopic CRY1 expression decreased FOXO1 protein levels as well as your life cycle gene expression in db / db mice (k, l in FIG. 8). These results indicate that CRY1 can alleviate hyperglycemia by inhibiting the level of FOXO1 protein in db / db mice and can be used to treat selective insulin resistance.

As one of the major factors of circadian rhythmic regulation, CRY1 expression is affected by various transcription factors and epigenetic regulation. Although CRY1 expression was promoted by insulin and SREBP1c in the in vivo and in vitro data of the present invention, CRY1 mRNA expression was not elevated by reingestion at ZT 10, the trough point during the CRY1 change cycle. This data means that increased SREBP1c may not be sufficient to activate CRY1 when ZT 10. Conversely, at the peak point of the cycle, ZT 22, reingestion increased the levels of CRY1 mRNA and protein strongly, indicating that SREBP1c increases CRY1 expression, perhaps along with other biocycle regulatory factors. Nevertheless, it should be explained whether CRY1 as well as other biological clock genes contribute to regulating your life cycle at ZT22.

On the other hand, we examined whether SREBP1c deficiency could alter liver bioclock gene cycle. In the SREBP1c ^{+ / +} and SREBP1c ^{− / −} mice, the liver bioclock gene cycle was not significantly different. Of ^{course, CRY1 - / - CRY2 - /} - double-mutant mice compared with CRY1 ^{- / -} mice because it indicates less change in the biological clock oscillation, SREBP1c ^{- / -} for the biological clock in mouse liver vibrations induced SREBP1c- The possibility that CRY1 could play a small role could not be ruled out. Furthermore, the SREBP1a and / or SREBP2 activity remaining in SREBP1c ^{− / −} mice is also likely to maintain intact biological clock gene oscillation and further study of this homeostasis is needed in the future. Nevertheless, liver CRY1 gene expression is clearly upregulated by uptake / insulin-mediated SREBP1c.

Consistent with the previous paper, hepatic CRY1 increased at night time (ZT 12-24), when uptake behavior predominantly occurs in nocturnal animals. Although the bioclock cycle did not appear much different in SREBP1c ^{− / −} mice, SREBP1c expression was also increased at night time (ZT 12-24). Unlike the expression profiles of SREBP1c and CRY1, the levels of FOXO1 protein have been shown to decrease at night, suggesting that the bioclock cycle of SREBP1c and CRY1 may contribute to the suppression of traveling glucose production by the regulation of FOXO1. will be.

Since SREBP1c can regulate both glycemic and lipid production processes simultaneously, hepatic SREBP1c can efficiently regulate anabolic pathways by up-regulating fatty acid metabolism and down-regulating sugar metabolism for insulin signaling in the presence of different target genes. It is possible to guess that. The present invention first revealed the role of SREBP1c in CRY1 activation, which is essential for the regulation of hepatic glucose metabolism in the assimilation state. Based on the bioclock vibrational gene expression profile in SREBP1c ^{− / −} mice, SREBP1c does not appear to actively dominate the liver bioclock. Rather, increased expression of hepatic CRY1 during postprandial conditions is preferentially regulated by insulin-activated SREBP1c in a clock-gated manner and ultimately leads to inhibition of glucose production through FOXO1 degradation. (FIG. 9). The present invention provides an important clue to understanding the molecular mechanisms that link liver SREBP1c and glucose homeostasis in physiological pathological conditions.

Although the exemplary embodiments of the present application have been described in detail above, the scope of the present application is not limited thereto, and various modifications and improvements of those skilled in the art using the basic concepts of the present invention defined in the following claims are also provided. It belongs to.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

Claims (9)

A method for screening a diabetes therapeutic or glucose biosynthesis inhibitor that targets the SREBP1c-CRY1-FOXO1 signaling pathway,
A first step of providing a cell or a non-human animal with reduced expression or activity of CRY1;
A second step of treating said cells with a test substance that is expected to increase the expression or activity of CRY1;
A third step of measuring FOXO1 protein concentration in cells treated with the test substance; And;
And a fourth step of screening the FOXO1 protein as a diabetic therapeutic agent or a glucose biosynthesis inhibitor candidate if the amount of the FOXO1 protein is decreased in comparison with the FOXO1 protein in a control group not in contact with the test substance. Way.
The method of claim 1,
The cell or animal in which the expression or activity of CRY1 in the first step is decreased is that the expression of SREBP1c is increased.
The method according to claim 1 or 2,
The animal of step 1 is a type 1 or type 2 diabetes model mouse, and the cell of step 1 is a primary hepatocyte derived from the type 1 or type 2 diabetic mouse, or a glucose comprising an H4IIE or HepG2 cell line. The method is a cell having biosynthesis.
The method of claim 1,
Measuring the expression of PEPCK and / or G6Pase, an enzyme involved in glucose biosynthesis that is promoted by FOXO1 in lieu of or in addition to the third step, and
In this case, in the fourth step, the candidate is a decrease in the expression of the PEPCK or G6Pase in the cells in contact with the test material compared to the control cells not in contact with the test material.


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