KR101910770B1 - A method for screening a therapeutic agent for type 2 diabetes using DSCR1-4, and a composition for diagnosing type 2 diabetes or predicting prognosis - Google Patents

Abstract

The present invention relates to a method for screening a therapeutic agent for type 2 diabetes reducing the binding degree between the down syndrome critical region 1-4 (DSCR1-4) gene and Forkhead box protein O1 (FoxO1), a composition for diagnosis and prognosis of type 2 diabetes, an information providing method for diagnosis and prognosis of type 2 diabetes, and a pharmaceutical composition for prevention or treatment of type 2 diabetes. An increase in the expression level of mRNA and DSCR1-4 genes in the liver cells and liver tissues of mice with induced obesity and diabetes is confirmed. The involvement of the DSCR1-4 genes in regulation of glycogenesis in the liver cells is confirmed in experiments. Accordingly, DSCR1-4 genes are confirmed to be a critical factor with respect to treatment targets of the type 2 diabetes and be useful as a gene marker for treatment of the type 2 diabetes. The DSCR1-4 genes can be variously utilized for pharmaceutical development and screening of the therapeutic agent for type 2 diabetes suppressing expression and activities of genes.

Description

Technical Field [0001] The present invention relates to a method for screening a type 2 diabetes therapeutic agent using DSCR1-4 and a composition for diagnosing type 2 diabetes or predicting the prognosis of a type 2 diabetes using a DSCR1-4, and a composition for diagnosing type 2 diabetes or predicting prognosis}

The present invention relates to a method for screening a therapeutic agent for type 2 diabetes and a composition for diagnosing type 2 diabetes or for predicting the prognosis. More specifically, the present invention relates to a method for screening for a Down syndrome critical region 1-4 (DSCR1-4) gene and FoxO1 protein O1), and a composition for predicting the type 2 diabetes diagnosis or prognosis.

Diabetes mellitus is a disease in which glucose present in the blood is excreted through the urine and is one of the chronic degenerative diseases that are not healed. Due to changes in dietary life and lack of exercise, the metabolism process of the human body has changed dramatically. As a result, chronic degenerative diseases such as diabetes have been increasing. , Complications such as macrovascular complications, microvascular complications, diabetic neuropathy and kidney disease are caused.

In Korea, the prevalence of diabetes is known to be 5-10%, and it is continuously increasing. In the United States, over the last 40 years, diabetes has increased sixfold, and by such an increase, the number of patients is expected to reach 26 million by 2050.

Type 1 diabetes mellitus is caused by insulin secretion deficiency, which is the glucose-regulating hormone in the blood, and is mainly caused by a young age group of about 10 to 20 years. Therefore, diabetes mellitus (juvenile also called diabetes. Type 2 diabetes mellitus occurs mainly after the age of 40 and accounts for most of the diabetic patients in Korea. Type 1 diabetes, unlike type 1, is called adult type diabetes. The cause of the disease is not yet clear, but genetic factors and environmental factors are known to occur together. As a cause of type 2 diabetes, both insulin secretory disorder and insulin action defect (insulin resistance) in target cells are observed in pancreatic beta cells. Insulin secretory disorder refers to a situation in which the insulin secretion is insufficient in the pancreatic beta cells according to blood glucose concentration, which includes both quantitative reduction of insulin-secreting beta cells and functional secretory disorders. Insulin resistance is a condition in which secreted insulin reaches the target organs in the bloodstream, but the insulin action and sensitivity of the target cells are lowered. It is generally considered to be a signal transduction disorder after cell membrane receptor binding, , Obesity, decreased physical activity, and hyperglycemia or dyslipidemia. Insulin resistance requires that a greater amount of insulin be secreted to overcome resistance, and conversely, insulin secretion is not sufficient to cause insulin resistance to be exacerbated by hyperglycemia itself.

As a treatment for diabetes, drug therapy, diet, and exercise therapy are generally used. The goal of treatment is to keep blood sugar close to the normal level to prevent and delay diabetic complications. Currently, oral glucosidase agents, biguanide agents, α-glucosidase inhibitors, and thiazolidinedione drugs are used as oral glucose regulators for the treatment of type 2 diabetes. However, sulfonylureas have been reported to cause side effects such as decreased pancreatic beta cells, gastrointestinal disorders, hypoglycemia, weight gain, and the side effects of the biguanide family Metformin, such as bloating, bloating, diarrhea, anorexia and lactic acidosis In the case of the thiazolidinedione series Rosiglitazone and Pioglitazone, side effects such as hepatotoxicity, cardiac arrest, and fat accumulation are known to cause side effects. Therefore, it is urgent to develop a diabetic therapeutic agent having a new action mechanism without side effects.

In addition, type 2 diabetes is caused by various problems occurring in carbohydrate, lipid metabolism, and insulin signal transduction system. Therefore, in order to develop a new therapeutic agent, a new target protein that regulates such signal transduction and metabolism is desperately needed (Korean Patent No. 10-1625333).

On the other hand, DSCR1 (Down syndrome critical region 1) physically interacts with calcineurin and inhibits the phosphatase activity of calcineurin and is overexpressed in Down syndrome. The DSCR1 gene is located in an important region of Down syndrome in chromosome 21, first identified as calcineurin binding protein and calcineurin inhibitor. The DSCR1 gene can be alternately conjugated to produce three isoforms in humans, such as 1-1L, 1-1S, and 1-4, and all DSCR1 isoforms have seven exons with a calcineurin binding motif. The DSCR1 gene is expressed in several tissues such as brain, spinal cord, kidney, liver, mammary gland, placenta and skeletal muscle. DSCR11 is highly expressed in the heart, brain, muscle and pancreas, and DSCR1-4 is up-expressed in the heart, liver, muscle, pancreas and kidney. According to current research, obesity is common in children and adults with Down syndrome. In addition, DSCR1 overexpressing mice exhibit hyperglycemia and glucose tolerance. However, overexpressed mice have lacked studies to define the physiological role of DSCR1. In addition, the method by which DSCR1 regulates glucose homeostasis remains unclear, and the method by which DSCR1-4 regulates nutritional metabolism is lacking of theoretical evidence in the liver mainly contributing to glucose homeostasis.

In addition, the gluconeogenesis of the liver is regulated by forkhead O box 1 (FoxO1), which directly binds to the G6Pase (6 phosphatase) and PEPCK (phosphoenolpyruvate carboxykinase) promoters to induce transcription. The FoxO1 protein belongs to the forkhead family, which functions as a transcription factor and is regulated by a variety of post-translational modifications including phosphorylation, acetylation, ubiquitination and methylation. However, it is not entirely clear how glucagon stimulates the transfer of transcription factors to the nucleus, such as cAMP response element-binding protein (CREB), FoxO1, and CREB-regulated transcription coactivator2 (CRTC2). Glucagon has been reported to induce intracellular calcium excretion and to increase glucose production. In addition, glucagon stimulates glycosylation by increasing intracellular calcium. Calcium calmodulin-dependent kinase II (CaMKII) not only promotes nuclear FoxO1 promoting glucose production in the liver, but also promotes protein kinase RNA-like endoplasmic reticulum kinase, PERK) - activating transcription factor 4 (ATF4) signaling. Thus, through these studies, it was found that intracellular calcium signaling in the liver is important in maintaining glucose homeostasis. In addition, glucagon promotes cytoplasmic calcium leading to CRTC2 dephosphorylation through calcineurin activation, indicating that DSCR1 can inhibit glycogen synthesis through inhibition of calcineurin.

However, despite the studies demonstrating the role of calcineurin signaling in maintaining glucose homeostasis, there has been a lack of research on the mechanism by which DSCR1-4, an endogenous inhibitor of calcineurin, directly participates in glucose metabolism.

The inventors of the present invention have conducted intensive studies for the treatment of type 2 diabetes and found that the level of DSCR1-4 (Down syndrome critical region 1-4) protein and mRNA expression is increased in hepatocytes and liver tissues of obese and diabetes-induced mice The present inventors have confirmed that DSCR1-4 gene is an important factor for the treatment of type 2 diabetes by experimentally confirming that DSCR1-4 gene is involved in regulation of glycogenesis in hepatocytes. Based on this, the present invention has been completed.

Accordingly, it is an object of the present invention to provide a screening method of a therapeutic agent for type 2 diabetes which reduces the degree of binding of Down syndrome critical region 1-4 (DSCR1-4) gene and FoxO1 (Forkhead box protein O1).

It is another object of the present invention to provide a composition for diagnosing type 2 diabetes or for predicting the prognosis, which comprises an agent for measuring mRNA or protein level of DSCR1-4 gene.

The present invention also provides a method for providing information for diagnosis or prognosis prediction of type 2 diabetes comprising the step of measuring the expression level of the mRNA or protein of the DSCR1-4 gene in a biological sample derived from a subject. The purpose.

It is still another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of type 2 diabetes comprising an expression or activity inhibitor of DSCR1-4 as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention as described above, the present invention provides a screening method for a therapeutic agent for type 2 diabetes mellitus comprising the following steps.

(a) treating the candidate material in cells in vitro;

(b) measuring the degree of binding of the down-syndrome critical region 1-4 (DSCR 1-4) gene to the FoxO1 (Forkhead box protein O1) gene; And

(c) selecting a substance that reduces the degree of binding of the DSCR1-4 gene to FoxO1 as compared to the candidate substance-untreated group as a treatment for type 2 diabetes mellitus.

In one embodiment of the present invention, the cells may be hepatocytes.

In another embodiment of the present invention, the candidate substance may be selected from the group consisting of a compound, a microbial culture or extract, a natural product extract, a nucleic acid, and a peptide.

In another embodiment of the present invention, the nucleic acid is selected from the group consisting of microRNA, siRNA, shRNA, antisense RNA, aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino Lt; / RTI >

In yet another embodiment of the present invention, step (b) may be performed by immunoprecipitation, immunohistochemistry, microarray, northern blotting, western blotting, , Enzyme immunoassay (ELISA), polymerase chain reaction (PCR), and immunofluorescence (immunofluorescence).

In another embodiment of the present invention, in the step (b), when the degree of binding between the DSCR1-4 gene and FoxO1 is decreased, it may be possible to inhibit glucose synthesis by the DSCR1-4 gene to reduce blood glucose.

The present invention also provides a composition for diagnosis or prognosis prediction of type 2 diabetes, comprising a mRNA of a Down syndrome critical region 1-4 (DSCR 1-4) gene or an agent for measuring the level of a protein encoded by the gene.

In one embodiment of the present invention, the mRNA of the DSCR1-4 gene may be composed of the nucleotide sequence of SEQ ID NO: 1.

In another embodiment of the present invention, the protein encoded by the DSCR1-4 gene may comprise the amino acid sequence of SEQ ID NO: 2.

In another embodiment of the present invention, the agent for measuring the mRNA level may be a sense and antisense primer that binds complementarily to the mRNA of the gene, or a probe.

In another embodiment of the present invention, the agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by the gene.

The present invention also relates to a method for diagnosing type 2 diabetes mellitus comprising the step of measuring the expression level of mRNA of DSCR1-4 (Down syndrome critical region 1-4) gene or a protein encoded by the gene in a biological sample derived from a subject, Or prediction of prognosis.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of type 2 diabetes comprising an expression or activity inhibitor of Down syndrome critical region 1-4 (DSCR1-4) as an active ingredient.

The present inventors have confirmed that the inhibition of glycogen synthesis and inhibition of cell signal transduction are inhibited in the hepatocyte when the expression of the DSCR1-4 gene is inhibited and the DSCR1-4 gene is useful as a genetic marker for the treatment of type 2 diabetes And it is expected that it can be used variously for the screening of drugs for the treatment of type 2 diabetes, which inhibits the expression or activity of the genes, and for the development of pharmaceuticals.

1A shows immune blots with increased expression of DSCR1-4 as in Example 1-7 in the liver of wild type (WT) mice after re-ingestion of food compared to fasting mouse liver . At this time, the liver extract was extracted from mice fasted for 24 hours and re-fed for 1 hour.
FIG. 1B is a graph showing that quantitative real-time PCR was performed as in Example 1-8 for liver extracts isolated from mice that were taken by a normal diet or fasted for 24 hours.
FIG. 1C is a graph showing the results of immunoblotting analysis of liver extracts of mice taken in a normal or high-fat diet as in Examples 1-7 using anti-DSCR1 antibodies.
FIG. 1D shows the result of quantitative real-time PCR of the liver extract isolated from mice taken in a normal or high-fat diet, as in Example 1-8.
FIG. 1 (e) is a graph showing the results of quantitative real-time PCR of the liver extract isolated from a mouse or a fasted mouse for 24 hours as in Example 1-8.
1F shows immunoblotting as shown in Example 1-7 showing DSCR1-4 expression in a liver extract of mice expressing obesity as a WT or an appetite suppressant gene, lapatin deficiency (ob / ob).
FIG. 1G shows the result of quantitative real-time PCR performed on the liver extract isolated from WT or ob / ob mice as in Example 1-8.
FIG. 1h shows immunoblotting confirmation as in Example 1-7 showing DSCR1 expression in liver extracts of WT or DSCR1 - / - mice.
FIG. 1I is a graph showing the results of glucose tolerance test (GTT) performed in WT or DSCR1 - / - mice after fasting for 16 hours.
FIG. 1J shows the result of insulin tolerance test (ITT) in WT or DSCR1-4 - / - mice after fasting for 4 hours.
FIG. 1k shows the results of monitoring blood glucose levels in WT or DSCR1 - / - mice re-fed with food for one hour after fasting for 24 hours or fasting for 24 hours.
FIG. 11 is a graph showing the results of monitoring blood glucose levels of WT or DSCR1-4 - / - mice fasted for 14 hours.
FIG. 1m shows the results of pyruvate tolerance test (PTT) in WT or DSCR1-4 - / - mice fasted for 16 hours.
FIG. 1n shows the result of quantitative real-time PCR performed in the liver of WT or DSCR1-4 - / - mouse as in Example 1-8.
FIG. 10 shows the results of comparing mRNA expression levels of lipogenic genes in WT or DSCR1-4 - / - mice.
FIG. 2A shows the results of monitoring body weight in WT or DSCR1-4 - / - mice while consuming the high fat diet.
FIG. 2B is a graph showing the results of glucose tolerance test (GTT) performed in WT or DSCR1-4 - / - mice that fasted for 16 hours and then fed a high fat diet.
FIG. 2C shows the result of insulin resistance test (ITT) performed in WT or DSCR1-4 - / - mice that fasted for 4 hours and then fed the high fat diet.
FIG. 2D shows the result of performing pyruvic acid tolerance test (PTT) in WT or DSCR1-4 - / - mice that fasted for 16 hours and then consumed high fat diet.
FIG. 2 (e) is a graph showing the results of monitoring blood glucose that has been re-fed for one hour after fasting for 24 hours or fasting for 24 hours.
FIG. 2f is a graph showing the results of monitoring blood glucose during fasting for 24 hours in WT or DSCR1-4 - / - mice ingesting high fat diet.
FIG. 2g shows the result of quantitative real-time PCR performed in the liver of WT or DSCR1-4 - / - mice ingesting the high fat diet as in Example 1-8.
FIG. 2h is a graph showing the results of comparing levels of lipogenesis gene expression in WT or DSCR1-4 - / - mice ingesting high fat diet.
FIG. 3A shows the result of reverse transcriptional PCR performed on the extract of hepatocytes treated with 100 nM of glucagon for 4 hours as in Example 1-8. FIG.
FIG. 3B is a graph showing the result of performing glucose assay (Glucose output assay) as in Example 1-4 in hepatocytes administered with vehicle, forskolin (10 μM) or insulin (100 nM) to be.
FIG. 3c is a graph showing the results of immunoblotting of extracts from hepatocytes treated with excipients or glucagon (100 nM) for 2 hours as in Example 1-7.
FIG. 3D shows the results of immunoblot analysis of hepatocytes of WT or DSCR1-4 - / - mice as shown in Example 1-9 using cytoplasmic and nuclear fractions and using the labeled antibody as in Example 1-7 .
Figure 3e shows the activity of hepatic G6Pase-luciferase (Ad-WT G6Pase [-231 / + 57] -Luc) activity in liver of WT or DSCR1-4 - / - mice as in Example 1-6 The live video was confirmed.
FIG. 4A shows the results of insulin resistance test (ITT) in WT or DSCR1 trisomy (DSCR1tri) mice after fasting for 4 hours.
FIG. 4B shows the result of performing pyruvic acid tolerance test (PTT) in WT or DSCR1tri mice after fasting for 16 hours.
Figure 4c shows the results of monitoring blood glucose levels in WT or DSCR1-4tri mice when fasting for 24 hours.
FIG. 4d shows the results of quantitative real-time PCR of liver extracts of WT or DSCR1-4tri mice as in Example 1-8. FIG.
FIG. 4E is a graph showing the results of performing reverse transcription PCR as in Example 1-8 in extracts from WT or DSCR1-4tri hepatocytes treated with 100 nM of glucagon for 4 hours.
FIG. 4f is a graph showing the results of glucose output assay as in Example 1-4 in WT or DSCR1-4tri hepatocytes treated with an excipient, phoscholine (10 μM) or insulin (100 nM) .
Figure 4g shows a live image of liver G6Pase-luciferase (Ad-WT G6Pase [-231 / + 57] -Luc) activity as in Example 1-6 in the liver of WT or DSCR1-4tri mice after fasting for 6 hours It is also confirmed.
Figure 4h shows the results of immunoblotting of extracts from WT or DSCR1-4tri hepatocytes treated with excipients or glucagon (100 nM) for 2 hours as in Example 1-7.
Fig. 4i shows the results of comparing lipogenesis gene expression in WT or DSCR1-4tri mice.
5A is a graph showing the results of immunoblotting of hepatocyte extract treated with excipient or cyclosporin A (Cyclosporin A) at the indicated concentrations for 24 hours as in Example 1-7.
FIG. 5B is a graph showing the results of reverse transcription PCR performed on hepatocyte extract treated with cyclosporin A at the indicated concentration for 24 hours as in Example 1-8. FIG.
FIG. 5c is a graph showing the results of glucose production assay as in Example 1-4 in extracts from hepatocytes treated with excipient, phoscholine or cyclosporin A. FIG.
FIG. 5d shows the result of performing reverse transcription PCR as in Example 1-8 when 100 nM of glucagon is absent or is present in the extract of WT or DSCR1-4 - / - hepatocyte treated with the excipient or cyclosporin A. FIG.
FIG. 5E is a graph showing the results of immunoblotting as in Example 1-7 when 100 nM glucagon is absent or present in the extract from WT or DSCR1-4 - / - hepatocyte treated with the excipient or cyclosporin A. FIG.
Figure 5f shows that glucagon from extracts from WT and DSCR1-4 - / - hepatocytes transfected as in Example 1-5 with non-silencing or si-calcineurin And when reverse transcription PCR is carried out as in Example 1-8, it is not present or exists.
FIG. 5g shows the results of immunoassay as in Examples 1-7 when glucagon is absent or present in the extracts from WT and DSCR1-4 - / - hepatocytes transduced as in Example 1-5 with non-silent or si- Fig. 6 is a graph showing the result of performing blotting.
6A is a graph showing the results of treatment of hepatocytes of WT or DSCR1-4- / - mice with excipients or 100 nM glucagon, followed by fractionation into cytoplasm and nucleus as in Examples 1-9, Fig. 5 shows the result of analysis by immunoblotting.
FIG. 6B shows the results of transfection of 293T cells with plasmids expressing myc-DSCR1-4 as in Example 1-5, and cell lysates were immunoprecipitated and immunoprecipitated as in Examples 1-7 using anti-myc antibodies Showing the result of evaluation by blotting.
FIG. 6C shows the results of treatment of WT hepatocytes with an excipient or 100 nM of glucagon, followed by fractionation into cytoplasm and nuclei as in Examples 1-9, and cell lysates were treated with anti-FOXO1 antibody in Example 1- 7 shows the result of immunoprecipitation and evaluation by immunoblotting.
FIG. 6 (d) is a graph showing the results of immunoprecipitation and immunoblotting of liver extracts of mice fed a normal or high-fat diet, as in Example 1-7, using an anti-FoxO1 antibody.
Figure 6e shows quantitative real-time, as in Examples 1-8, when 100 nM of glucagon is absent or present in the extracts from transplanted WT and DSCR1-4tri hepatocytes as in Example 1-5 with either non-silent or si-FoxO1, Fig. 6 shows the result of performing PCR.
FIG. 6f shows the results of performing Chromatin immune-precipitation (ChIP) on the extracts from WT and DSCR1-4 - / - hepatocytes treated with excipients or glucagon as in Example 1-10, 8 shows the result of quantitative real-time PCR evaluation.
FIG. 6g shows the results of transfection of 293T cells with plasmids expressing myc-DSCR1-4 as in Example 1-5, and extracts from HEK293T cells treated with vehicle or 100 nM glucagon, (ChIP), followed by quantitative real-time PCR as in Example 1-8.
Figure 6h shows a proposed model for a method of modulating glycogen synthesis when DSCR1-4 is fasted or re-ingested.

BRIEF DESCRIPTION OF THE DRAWINGS The invention is capable of various modifications and various embodiments, and particular embodiments are illustrated in the drawings and will be described in detail in the detailed description. It is to be understood, however, that the invention is not to be limited to the specific embodiments, but includes all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

The present invention relates to a method for screening a therapeutic agent for type 2 diabetes by measuring the degree of binding of Foxror1 (Forkhead box protein O1) to DSCR1-4 (Down syndrome critical region 1-4) gene in hepatocytes.

Accordingly, the present invention provides a method for treating a cell, Measuring the degree of binding of the Downskill critical region 1-4 (DSCR 1-4) gene to FoxO1 (Forkhead box protein O1); And selecting a substance that decreases the degree of binding of the DSCR1-4 gene and FoxO1 to the candidate substance-untreated group as a type 2 diabetes therapeutic agent. The present invention also provides a method for screening a therapeutic agent for type 2 diabetes mellitus.

The cell of the present invention may be a hepatocyte. The hepatocyte is a major constituent cell of the liver that constitutes the hepatic lobule. It constitutes about 80% of the whole liver. It contains amino acid, glucose (glucose), fatty acid, triacylglycerol, cholesterol Refers to cells that maintain metabolism, metabolism, hormones, drugs, degradation and detoxification of harmful substances, and maintain homeostasis of the body. The type of the cell is not limited as long as it is a cell originating from a hepatocyte, for example, a mouse hepatocyte can be used, and a person skilled in the art can appropriately select and use it.

The candidate substance of the present invention refers to an unknown substance used in screening to measure the degree of binding of the DSCR1-4 gene to FoxO1, and is preferably a compound, a microorganism culture solution or extract, a natural product extract, a nucleic acid, and a peptide Wherein the nucleic acid is selected from the group consisting of microRNA, siRNA, shRNA, antisense RNA, aptamer, LNA (locked nucleic acid), PNA (peptide nucleic acid), and morpholino But are not limited thereto.

As used herein, the term " microRNA " means a short non-coding RNA consisting of about 22 nucleotide sequences. It is known to function as a post-transcriptional regulator in the process of gene expression. Complementarily binds to a target mRNA having a complementary base sequence to inhibit translation or translation of target mRNAs.

As used herein, the term " siRNA (small interfering RNA) " refers to a short double-stranded RNA capable of inducing RNA interference (RNAi) through cleavage of a specific mRNA. A sense RNA strand having a sequence homologous to the mRNA of the target gene and an antisense RNA strand having a sequence complementary thereto. siRNA is provided as an efficient gene knock-down method or as a gene therapy method because it can inhibit the expression of a target gene.

In the present invention, the term " shRNA (small hairpin RNA) " means a nucleotide consisting of 50-60 single strands and has a stem-loop structure in vivo. In other words, shRNA is an RNA sequence that creates a tight hairpin structure to inhibit gene expression through RNA interference. Long RNAs of 15-30 nucleotides complementary to both loop regions of 5-10 nucleotides form base pairs to form double stranded stems. The shRNA is transfected into a cell via a vector containing the U6 promoter so as to be always expressed, as in Example 1-5, and is usually transferred to a daughter cell so that gene expression inhibition is inherited. The shRNA hairpin structure is cleaved by an intracellular mechanism to become siRNA and bind to RNA-induced silencing complex (RISC). These RISC bind to mRNA and cleave it. The shRNA is transcribed by RNA polymerase.

The step of measuring the degree of binding of the DSCR1-4 gene to the FoxO1 of the present invention may be performed using various methods known in the art for measuring the amount of RNA or protein produced by the binding of the DSCR1-4 gene to FoxO1 Method. ≪ / RTI > Preferably, the expression level of RNA can be measured by PCR, microarray, northern blotting, etc., and the expression amount of the protein can be measured by Western blotting, Can be measured through immunoassay (ELISA), immunoprecipitation, immunohistochemistry, or immunofluorescence. It can be carried out by a person skilled in the art very simply and easily, But is not limited to.

In another aspect of the present invention, there is provided a composition for diagnosing type 2 diabetes mellitus or for predicting prognosis, which comprises an agent for measuring mRNA of DSCR 1-4 (Down syndrome critical region 1-4) gene or a protein level encoded by the gene do.

The DSCR1-4 gene of the present invention may comprise the nucleotide sequence of SEQ ID NO: 1, and the protein encoded by the DSCR1-4 gene may comprise the amino acid sequence of SEQ ID NO: 2.

"Diabetes", a disease to which the present invention is directed, is a type of metabolic disorder such as insufficient secretion of insulin or a failure to function normally. It is characterized by hyperglycemia in which blood glucose concentration is elevated. Causing symptoms and discharging glucose from the urine. Diabetes is divided into type 1 and type 2, type 1 diabetes was formerly called 'child diabetes' and is caused by the inability to produce insulin at all. Type 2 diabetes, which is relatively insufficiently insulin-dependent, is characterized by insulin resistance (insulin resistance that lowers blood sugar and cells do not burn glucose effectively). Type 2 diabetes may be due to environmental factors such as high calorie, high fat, high protein diet, lack of exercise, and stress due to westernization of dietary habits. However, diabetes mellitus may also be caused by defects of specific genes, Infection, or drug.

The term " diagnosis " as used in the present invention means, in a broad sense, judging all aspects of the actual condition of a patient. The contents of the judgment are the pathology, etiology, pathology, severity, detailed aspects of the disease, and the presence or absence of complications. In the present invention, the diagnosis is to judge whether or not the type 2 diabetes develops and the level of progression.

As used herein, the term " prognosis " refers to a prediction of the progression or recovery of a disease, and refers to an outlook or a preliminary evaluation. Prognosis in the present invention refers to, but is not limited to, recurrence, overall survival, or disease-free survival of type 2 diabetes.

The agent for measuring the mRNA level of the DSCR1-4 gene may be, but is not limited to, a sense and antisense primer that binds complementarily to mRNA, or a probe.

As used herein, the term " primer " means a short gene sequence which is a starting point of DNA synthesis, and means an oligonucleotide synthesized for diagnosis, DNA sequencing and the like. The primers may be synthesized to have a length of 15 to 30 base pairs. The primers may be used depending on the purpose of use, and can be modified by methylation, capping or the like by a known method.

As used herein, the term " probe " refers to a nucleic acid capable of specifically binding to mRNA of a length of several hundreds to several hundreds of nucleotides prepared through enzymatic chemical separation purification or synthesis. Radioactive isotopes or enzymes can be labeled to confirm the presence or absence of mRNA and can be designed and modified by known methods.

The agent for measuring the protein level may be an antibody that specifically binds to a protein encoded by the gene, but is not limited thereto.

As used herein, the term " antibody " includes immunoglobulin molecules immunologically reactive with specific antigens and includes both monoclonal and polyclonal antibodies. The antibody also includes forms produced by genetic engineering such as chimeric antibodies (e. G., Humanized murine antibodies) and heterologous binding antibodies (e. G., Bispecific antibodies).

In another aspect of the present invention, there is provided a method for diagnosing type 2 diabetes or for predicting prognosis, comprising the step of measuring an expression level of mRNA of the DSCR1-4 gene or a protein encoded by the gene in a biological sample derived from a subject. Provide information providing method.

The biological sample derived from the subject may include, but is not limited to, tissues, cells, whole blood, blood, saliva, sputum, cerebrospinal fluid and urine.

The expression level of the mRNA may be determined by PCR, reverse transcription polymerase chain reaction (RT-PCR), real-time PCR (Real-time PCR), RNase protection assay (RNase but are not limited to, one or more methods selected from the group consisting of immunoassay, protection assay (RPA), microarray, or northern blotting.

The protein expression level may be measured by Western blotting, radioimmunoassay (RIA), radioimmunodiffusion, enzyme immunoassay (ELISA), immunoprecipitation, or the like using conventional methods known in the art. One or more methods selected from the group consisting of flow cytometry, immunofluorescence, ouchterlony, complement fixation assay, or protein chip, , But is not limited thereto.

As used herein, the term "information providing method for diagnosis or prognosis prediction of type 2 diabetes" is a preliminary step for diagnosis or prediction of prognosis, and provides objective basic information for diagnosis or prediction of type 2 diabetes And the physician's clinical judgment or observations are excluded.

In another aspect of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of type 2 diabetes comprising an expression or activity inhibitor of DSCR1-4 (Down syndrome critical region 1-4) as an active ingredient.

As used herein, the term " prophylactic " means any action that inhibits or delays the onset of type 2 diabetes by administration of a pharmaceutical composition according to the present invention.

As used herein, the term " treatment " means any action that improves or alters the symptoms of type 2 diabetes by the administration of the pharmaceutical composition according to the present invention.

The expression or activity inhibitor may be selected from the group consisting of small interfering RNA (siRNA), short hairpin RNA, miRNA, ribozyme, DNAzyme, peptide nucleic acids, And may preferably be siRNA which inhibits the expression of the DSCR1-4 gene, but is not limited thereto.

The pharmaceutical composition according to the present invention contains as an active ingredient an expression or activity inhibitor of DSCR1-4, and may further comprise a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of application and include, but are not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, And may further contain other conventional additives such as antioxidants and buffers as needed. It may also be formulated into injectable formulations, pills, capsules, granules or tablets, such as aqueous solutions, suspensions, emulsions and the like, with the addition of diluents, dispersants, surfactants, binders and lubricants. Suitable pharmaceutically acceptable carriers and formulations can be suitably formulated according to the respective ingredients using the methods disclosed in Remington's reference. The pharmaceutical composition of the present invention is not particularly limited to a formulation, but may be formulated into injections, inhalants, external skin preparations, and the like.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically) according to the intended method, but preferably can be administered orally, Depends on the condition and the weight of the patient, the degree of the disease, the type of the drug, the administration route and time, but can be appropriately selected by those skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, the term "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit / risk ratio applicable to medical treatment or diagnosis, and the effective dose level will depend on the type of disease, severity, The activity of the compound, the sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or concurrently with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the age, sex, condition, body weight, absorbency, inactivation rate and excretion rate of the active ingredient in the body, type of disease, However, the dosage may be varied depending on the route of administration, the severity of obesity, sex, weight, age, etc. Therefore, the dosage is not limited to the scope of the present invention by any means.

In one embodiment of the present invention, the mouse liver tissue that is obesity-deficient (ob / ob), which is an obesity-induced mouse, and an obesity-inhibiting gene, -4 protein and mRNA expression levels were increased and protein and mRNA expression was increased. In addition, DSCR1-4 gene deficient mice or DSCR1-4 deficient mice were fed a high fat diet to induce obesity and diabetes mellitus Observation of glucose tolerance and insulin resistance in mice showed that glucose tolerance and insulin resistance were improved. Fasting blood glucose was observed as a result of a decrease in fasting blood glucose. In addition, glycogen synthesis was reduced and glycoprotein G6Pase 6 phosphatase) and PEPCK (phosphoenolpyruvate carboxykinase)] was decreased (see Example 2).

In another embodiment of the present invention, when obese or diabetic mice are induced by high-fat diet intake in a mouse deficient in the DSCR1-4 gene, the body weight gain of the mouse is decreased and the sugar tolerance, insulin resistance and sugar production are improved (G6Pase, PEPCK) gene expression was decreased in the fasting state (see Example 3).

In another embodiment of the present invention, when the DSCR1-4 gene is deficient in the liver cells of the mouse, an increase in the expression of glucagon-related glycosyltransferase (G6Pase, PEPCK) is not induced and a decrease in glucose output In the absence of DSCR1-4 gene, phosphorylation of cAMP response element-binding protein (CREB), which is a factor of glycosyltransferase, is decreased. In vivo G6Pase luciferase assay in mice lacking DSCR1-4 gene, G6Pase gene Expression was decreased (see Example 4).

In another embodiment of the present invention, DSCR1 trisomy (DSCR1tri) mice with 3x copies of the DSCR1 transgene gene show insulin resistance, increased hepatic glucose production, increased fasting blood glucose, and increased expression of DSCR1-4 transgenic mice Expression of glycosyltransferase gene was increased in hepatocyte, and expression of gluconeogenous gene was increased in hepatocytes obtained from DSCR1tri mouse. Gene synthesis in DSCR1tri mouse hepatocyte was increased. In vivo G6Pase luminescence analysis The expression of G6Pase was increased in DSCR1tri mice, and it was confirmed that the phosphorylation of CREB, which is important for glycogen synthesis in DSCR1tri mice, is increased (see Example 5).

In another embodiment of the present invention, DSCR1-4 is known to be an endogenous inhibitor of calcineurin. In order to examine the role of calcineurin in the change of hepatic glucose metabolism by DSCR1-4, Treatment with cyclosporin A, an inhibitor of calcineurin A, resulted in an increase in the expression of glycosylated genes and an increase in glucose output, but the increase in calcineurin in liver cells of mice deficient in the DSCR1-4 gene And that the decrease in the expression of the syngeneic gene by the DSCR1-4 gene deficiency is not affected, and it is confirmed that DSCR1-4 acts independently of calcineurin to regulate the syngeneic synthesis by DSCR1-4 (See Example 6).

In another embodiment of the present invention, the glucocorticoid pathway shifts the DSCR1-4 gene into the nucleus and binds to FoxO1, a regulated transcription factor, which is increased in liver tissue of obese-induced mice due to high-fat diet intake . It was confirmed that FoxO1 mediated regulation of DSCR1-4 by DSCR1-4 was confirmed by confirming that the expression of an increased glycosylation gene in DSCR1 trisomy mouse was reduced upon deficiency of FoxO1, (SEQ ID NO: 1), thereby regulating glycosyltransferase synthesis by controlling the expression of the syngeneic gene (see Example 7).

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Example 1. Experimental preparation and method

1-1. Mouse preparation

DSCR1 - / - mice and DSCR1tri (DSCR1 trisomy) mice were provided by Dr. Kwan Hyuk Baek (Sungkyunkwan University School of Medicine). All mice returned to the C57BL / 6J background every 10 generations and maintained a 12/12 hour tone / darkness.

All animal tests are accredited by the International Association of Laboratory Animal Care Assessment and Accreditation of Animal Care International (AAALAC International) and are compliant with the guidelines of the Institute of Laboratory Animal Resources (ILAR). (IACUC) at the Sungkyunkwan University School of Medicine in Seoul, Korea.

1-2. Animal experiment

Eight to 10-week-old male mice were fed the normal diet freely. Blood glucose measurements were taken from tail blood using a glucometer (Roche).

Glucose Tolerance Test (GTT) or Pyruvate Tolerance Test (PTT) were performed by intraperitoneal injection of glucose or sodium pyruvic acid (Sigma) at 2 g / kg body weight after 16 hours of fasting.

The insulin tolerance test (ITT) was performed by intraperitoneal injection of 0.75 Ug / kg body weight of insulin (GIBCO) after 4 hours of fasting.

1-3. Primary hepatocyte isolation and cell culture (primary hepatocyte isolation and cell culture)

Primary hepatocytes were isolated as previously described in 8-10 week mice by the collagenase method.

Primary hepatocytes were cultured in Medium 199 (M199, Sigma) supplemented with 10% fetal bovine serum (FBS, GIBCO), 100 U / ml penicillin, 100 ug / ml streptomycin, 23 mM HEPES and 10 nM dexamethasone (Sigma).

293T cells were obtained from the American Type Culture Collection (ATCC) and cultured in Dulbecco's modified Eagles's medium (DMEM, Welgene) containing 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin at 37 ° C temperature in a humidified atmosphere containing 5% CO2.

1-4. Hepatocyte glucose output assay

Glucose production assays were performed after 8 hours of incubation in Krebs-Ringer buffer containing 20 mM sodium lactate, 2 mM sodium pyruvate and 100 nM dexamethasone (Sigma) . Glucose levels in the Krebs-ringer buffer were measured using a QuantiChromTM Glucose Assay Kit (Bioassay Systems) and normalized to protein concentration.

1-5. Small interference RNA (RNA), plasmids and transfection,

(Target specific si-RNA) (DSCR1-4 (SEQ ID NO: 2)) for small interfering (si) RNA-mediated knockdown of DSCR1-4, calcineurin B and FoxO1. : CTC AGA CTT TAC ACA TAG GAA, calcineurin B: GTG GAC TTC AAA GAA TTC ATT, FoxO1: GCA CCG ACT T TAGA GCA ACC) and negative control siRNA were obtained from Genotech (Korea) Respectively.

For overexpression of DSCR1-4, the pLUX vector and pLUX / DSCR1-4 were obtained from Dr. Hwanhee Baik (Sungkyunkwan University School of Medicine).

Primary hepatocyte 293T cells or HepG2 cells were transfected with si-RNA or pLUX / DSCR1-4, respectively, expressed using Lipofectamine 2000 (Invitrogen) or TransIT-LT1 (MIRUS).

       1-6. In vivo imaging < RTI ID = 0.0 >

Adenovirus expressing the G6Pase promoter driving luciferase (Ad-G6Pase-Luc) was provided by Dr. SH. Ad-G6Pase-Luc was injected into WT, DSCR1-4 - / -, or DSCR1 trisomy mice by tail vein. In vivo luciferase activity was analyzed using an IVIS lumina imaging system after luciferin injection (Califor LifeSciences).

1-7. Immunoblotting and immunoprecipitation (IP)

Cells or liver tissues were lysed on ice for 10 minutes with 150 mM NaCl, 50 mM Tris-Cl, pH 7.4, 1 mM ethylenediaminetetraacetic acid (EDTA), 1% Nonidet P-40 (NP- Deoxycholate, and protease inhibitors (Sigma), and the whole cell lysate was obtained by subsequent centrifugation.

Cell lysates were subjected to SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA) by semi-dry transfer cells (Bio-rad).

After that, the membrane was incubated with anti-DSCR1 (Sigma), anti-phospho-AKT (S473), anti-AKT (Santa Cruz), anti-TORC2 (Calbiochem) , anti-CREB (Santa Cruz), anti-HDAC1 (Santa Cruz), anti-Calcineurin B (UPSTATE), anti-FoxO1 (Santa Cruz and cell signaling) ), anti-FASN (BD Bioscience), anti-phospho-AMPK (Cell signaling), anti-AMPK (Santa Cruz), anti-SREBP1c (Santa Cruz), anti- Santa Cruz) or anti-α-tubulin (Santa Cruz) supplemented with 2% non-fat milk powder. The band was detected using an enhanced chemiluminescence system (Pierce).

For immunoprecipitation, the cell extract was mixed with the antibody immobilized on protein A or G-Sepharose (Amersham) and incubated at 4 ° C for 2 hours.

After washing with lysis buffer, the immunoprecipitates were collected by centrifugation and the protein was boiled in 5X sample buffer. After centrifugation, the supernatant was applied to an SDS-PAGE gel and transferred to a PVDF membrane.

1-8. RNA isolation, reverse transcriptase PCR, and quantitative real-time PCR,

All RNAs in liver tissue or primary hepatocytes were performed using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. RNA was converted to single strand cDNA by revertaid reverse transcriptase (Thermo scientific) with random hexamer primers. 0.2 [mu] g of cDNA was PCR amplified using gene-specific primers. For quantitative real-time PCR, a reaction mixture was prepared using a 2X Power SYBR green PCR master mixture (Applied Biosystems) and 0.5 μM of each primer. The primer set is shown in [Table 1].

[Table 1] Primer used in PCR

1-9. Nucleus cytosol fractionation

To fractionate the cytoplasm and nucleus, hepatocytes were incubated with buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1 mM dithiothreitol, DTT, 0.5% NP-40 and protease inhibitors) And incubated on ice for 15 minutes. And centrifuged at 13,000 rpm for 2 minutes at 4 DEG C to obtain a cytoplasmic lysate. The nuclei were washed with Buffer A and dissolved in Buffer B (20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM DTT and protease inhibitors). After incubation on ice for 10 minutes, the nuclear lysate was obtained by centrifugation at 13,000 rpm for 10 minutes at 4 ° C.

1-10. Chromatin immune-precipitation (ChIP) analysis

Primary hepatocytes and HepG2 cells were cultured 1 day before treatment. Cells were treated with glucagon for 2 hours. The cells were crosslinked by adding 1% formaldehyde to the cells for 10 minutes. Glycine was added to a final concentration of 0.125 M to terminate the cross-linking reaction. The cells were washed with PBS, suspended in SDS lysis buffer, sonicated for 30 seconds at 20% power on a Fisher Scientific Sonic Dismembrator 500, and the DNA was sheared into 200-300 base pairs fragments. DNA-protein complexes were immunoprecipitated for 16 h at 4 ° C using 2 μg anti-FoxO1 or anti-Myc. Protein and / or DNA complexes were captured and washed extensively at 60 ° C for 2 hours using 60 μl of 50% (vol./vol.) Protein A or G agarose (Amersham) / salmon sperm DNA slurry Respectively. Tris-HCl pH 6.5, 5 M NaCl and 0.5 M EDTA for 4 hours at 65 ° C to reverse cross-linking. The DNA was extracted with phenol / CHCl3 and precipitated with ethanol. The precipitated DNA was quantified by real-time PCR as in Examples 1-8. A set of primers such as PEPCK promoter (-484 to -12 base pairs), G6Pase promoter (-231 to +57 base pairs) was used to amplify the PEPCK or G6Pase promoter region containing the IRE.

1-11. Statistical analysis

Data are expressed as mean SEM. The main and interaction effects were analyzed through analysis of variance. Differences between individual group means were analyzed by post-hoc Bonferroni test or unpaired, two tailed t test. A P value of less than 0.05 was considered significant.

Example 2. Function and glucose production of DSCR1-4 in obese or DSCR1-4 gene deficient conditions

2-1. Overexpression of DSCR1-4 gene by obesity and nutrient intake

Glucose homeostasis is largely regulated by the corresponding action and glycosylation, which are generally regulated by nutritional status. To study the metabolic function of DSCR1-4, we examined whether the expression level of DSCR1-4 was altered by nutritional level.

Upon re-ingestion of the mice for 1 hour after fasting overnight, the protein and mRNA levels of DSCR1-4 increased in the liver of mice (Fig. 1a, b).

To determine whether DSCR1-4 is affected by obesity and type 2 diabetes, we used the DSCR1-4 level in the liver of two commonly used obese mouse models, such as diet-induced obesity and genetically induced obesity Respectively. The protein and mRNA levels of DSCR1-4 were about three times higher than those of mice that fed a normal diet (NCD) in the liver of mice receiving high-fat diet (HFD) (Fig. 1c, d). Consistent with this, the liver mRNA levels of DSCR1-4 were significantly increased upon re-ingestion of food compared to fasting mice in high fat diet (HFD) (Fig. 1e). In addition, DSCR1-4 protein and mRNA levels were dramatically increased in the liver of mice expressing obesity with obesity (ob / ob) compared to wild type (WT) (Fig. 1f, 1g). These results indicate that the expression level of DSCR1-4 is related to nutrient mediated metabolism in the liver.

2-2. Reduction of Glucose Production in the Liver by Deficiency of DSCR1-4

If calcineurin regulates intracellular glucose homeostasis by dephosphorylating CREB-regulated transcription coactivator2 (CRTC2) at the fasting and calceinurin is regulated by DSCR1-4, an endogenous inhibitor of calcineurin, DSCR1-4 may affect glucose metabolism. We evaluated glucose tolerance test (GTT) in DSCR1-4 gene deficient (DSCR1-4 - / -) and WT mice to investigate the effect of DSCR1-4 on in vivo glucose metabolism. DSCR1-4 - / - mice were significantly improved in glucose tolerance compared to WT mice (Fig. 1i). In addition, the ability of insulin to reduce blood glucose was slightly improved in DSCR1-4 - / - mice as shown in the insulin resistance test (ITT) (Fig. 1j). These data indicate that the deficiency of DSCR1-4 improves glucose homeostasis.

Next, to determine whether DSCR1-4 affects glucose homeostasis under different nutritional conditions, the blood glucose level of the mice was monitored when fasting or feeding again. DSCR1-4 - / - mice had significantly lower blood glucose levels at 24 hours fasting and 1 hour re-feeding (Fig. 1k, l). We performed pyruvic acid tolerance test (PTT) in view of the fact that pyruvate was converted to glucose by intrahepatic gluconeogenesis and maintained normal blood glucose levels in fasting state. After administration of pyruvic acid, DSCR1-4 - / - mice showed a significant decrease in blood glucose levels compared to WT mice (FIG. 1m). These results indicate that DSCR1-4 deficiency inhibits hepatic glucose production.

To investigate whether DSCR1-4 affects glycosylation, we examined the expression of glycosylation genes in the liver of WT or DSCR1-4 - / - mice. Expression levels of PEPCK (phosphoenolpyruvate carboxykinase) and G6Pase (6 phosphatase) in the fasting state were significantly reduced in the liver of DSCR1-4 - / - mice as compared to WT (Fig. 1n) And the glucose metabolism is improved.

During feeding, circulating insulin inhibits intra-hepatic glucose production through the Irs-Akt-FoxO pathway or the PKA-InsP3R-calcineurin pathway, while circulating insulin inhibits Akt-mediated sterol regulatory factor binding protein 1 (sterol regulatory element-binding protein 1, Srebp1) stimulates lipogenesis, and that over-production of glucose and triglycerides in type 2 diabetes affects hyperglycemia and hypertriglyceridemia, Expression levels of the genes were evaluated. Compared with WT mice, DSCR1-4 - / - mice showed mRNA expression levels of lipogenic genes such as acetyl-CoA carboxylase 1 (ACC1), stearoyl-CoA desaturase-1 (SCD1), fatty acid synthesis (FASN) and SREBP1 , And there was no difference (Fig. Thus, these results demonstrate that decreased glucose production in mice lacking DSCR1-4 is associated with the inhibition of glycosylation gene expression.

Example 3: Inhibition of glucose production by DSCR1-4 deficiency and prevention of obesity-induced obesity

High-fat dietary intake (HFD) leads to insulin resistance, a common feature of type 2 diabetes. To study the role of DSCR1-4 under insulin resistance conditions, we tested whether DSCR1-4 deficiency affected obesity development due to high fat diet (HFD) intake. DSCR1-4 - / - mice showed significantly lower body weight compared to WT mice when high fat diet (HFD) ingestion (Fig. 2a). In addition, DSCR1-4 - / - mice significantly improved glucose tolerance and increased insulin sensitivity at the time of high fat diet (Fig. 2b, 2c). This data indicates that the skin shape of DSCR1-4 - / - mice is related to improved glucose homeostasis.

After pyruvate administration, DSCR1-4 - / - mice ingesting high fat diet (HFD) showed a significant decrease in blood glucose levels compared to WT mice ingesting high fat diet (HFD) (Figure 2d ).

Fasting blood glucose levels were significantly reduced in DSCR1-4 - / - mice fed high fat diet (HFD) compared to WT mice fed high fat diet (HFD) (Fig. 2e, 2f) Is protected from obesity induced by high fat diet (HFD) intake.

We examined whether DSCR1-4 regulates glycogen synthesis and lipogenic gene expression in obese mice. Syngeneic gene expression levels in the liver were significantly reduced in DSCR1-4 - / - mice compared to WT mice fed high fat diet (HFD) (Fig. 2g). However, levels of lipogenic gene expression in the liver between WT and DSCR1-4 - / - mice were similar during HFD delivery (FIG. 2h). As a result, it was found that the deficiency of DSCR1-4 protects against obesity induced by high-fat diet (HFD) intake by inhibiting hepatocyte neogenesis.

Example 4. Reduction of Syngeneic Synthesis by DSCR1-4 Gene Deficiency

Taking into consideration that the deficiency of DSCR1-4 reduces glucose production in vivo, we examined the role of DSCR1-4 in glycogen synthesis. Expression levels of glycosylation genes such as G6Pase (6 phosphatase) and PEPCK (phosphoenolpyruvate carboxykinase) were significantly reduced in DSCR1-4 - / - hepatocytes compared with WT in the treatment of glucagon, Suggesting that it suppresses the expression of myogenous synthetic genes (Fig. 3A). Next, we performed the glucose output assay as in Example 1-4 to evaluate whether DSCR1-4 affects hepatic glucose production.

In the absence of DSCR1-4 deficiency, glucose production was significantly reduced in the treatment of Forskolin or Insulin (Fig. 3b), suggesting that DSCR1-4 deficiency inhibits glucose synthesis by inhibiting the expression of glycans . Considering that CREB-regulated transcription coactivator2 (CRTC2) binds to the cAMP response element-binding protein (CREB) in the fasted state and increases the transcriptional activity of the syngeneic gene, a master activator We further investigated whether modulation of CREB or CRT2 would affect DSCR1-4 synthesis in syngeneic origin. The deficiency of DSCR1-4 did not change the phosphorylation level of CRTC2 in the treatment of glucagon (Fig. 3c). However, the phosphorylation level of CREB was not elevated in the treatment of glucagon in DSCR1-4 - / - hepatocytes compared with that in WT (Fig. 3c). DSCR1-4 deficiency inhibits CREB, but not CRTC2, .

Considering that CREB activated during fasting binds to the glycosylation promoter and induces glycogen synthesis, we investigated whether DSCR1-4 affects nuclei-rich CREB. The deficiency of DSCR1-4 resulted in a decrease in nuclear rich CREB (Fig. 3d), suggesting that deficiency of DSCR1-4 inhibits glycogen synthesis through the regulation of CREB.

To a lack of DSCR1-4 determining an influence on transcription activity of G6pase gene in vivo, and the exemplary WT DSCR1 as in Example 1-6-G6Pase promoter in mice - / indicator luciferase reporter (G6Pase the promoter- directed luciferase reporter (Ad-G6Pase-Luc).

DSCR1-4 - / - mice infected with Ad-G6Pase-Luc significantly reduced the transcriptional activity of the G6pase gene in response to the fasting (Fig. 3e). These observations suggest that the deficiency of DSCR1-4 inhibits glycogen synthesis by reducing the phosphorylation level of CREB but not CREB-regulated transcription coactivator2 (CRTC2).

Example 5. Confirmation of Increase of Glucose Production in Liver by Trisomy of DSCR1-4

Given that the deficiency of DSCR1-4 reduces glycosylation, we have determined whether overexpression of DSCR1-4 enhances glycosylation. ITT and PTT were performed using a DSCR1-4 trisomy (DSCR1-4tri) mouse with a triple copy of the DSCR1-4 transgene gene to assess the effect of DSCR1-4 overexpression on the in vivo hepatic glycogen synthesis. The ITT results indicated that the ability of insulin to reduce blood glucose levels in DSCR1-4tri mice was slightly impaired (FIG. 4A). After administration of pyruvic acid, the blood glucose level of DSCR1-4tri mice was significantly increased as compared to WT mice (Fig. 4B). In addition, DSCR 1-4 rats showed significantly higher blood glucose levels than WT mice after 24 h fasting (Fig. 4c). Similarly, overexpressing mice of DSCR1 showed hyperglycemia. These data and previous studies indicate that DSCR1 can promote hyperglycemia by promoting glucose production.

To determine whether DSCR1-4 regulates glycosylation to increase glucose production, we evaluated the expression of glycosylation genes in DSCR1-4tri liver tissue. The mRNA level of PEPCK was slightly up-regulated in DSCR1-4tri liver tissue compared to that in WT. However, the mRNA levels of G6Pase (6 phosphatase) were not different between DSCR1-4tri and WT liver tissues (Fig. 4d). We evaluated the expression of glycated genes in primary hepatocytes of DSCR1-4tri mice.

Upon glucagon treatment, the level of the syngeneic gene was dramatically increased in DSCR1-4tri hepatocytes compared to WT (Fig. 4e), suggesting that DSCR1-4 mediated and expression increased glycosylation by increased syngeneic synthesis It suggests. In addition, overexpression of DSCR1-4 increased intra-hepatic glucose production during the treatment of Forskolin or Insulin (Fig. 4f). In addition, DSCR1-4tri mice exhibited increased G6Pase transcription activity compared to WT mice at fasting conditions (Fig. 4g). These data indicate that mice with overexpression of DSCR1-4 exhibited hyperglycemia due to increased glucose production.

Next, we examined the phosphorylation levels of cAMP response element-binding protein (CREB) and CREB-regulated transcription coactivator2 (CRTC2) on stimulation of glucagon. Overexpression of DSCR1-4 increased phosphorylation of CREB during glucagon treatment in hepatocytes (Fig. 4h). However, overexpression of DSCR1-4 did not affect phosphorylation of CRTC2 (FIG. 4h). These data suggest that neogenesis per DSCR1-4 mediated enhancement enhances phosphorylation of CREB but not CRTC2. In addition, there was no difference in the expression of lipogenesis gene in the liver of WT mouse and DSCR1-4tri mouse (Fig. 4I). These data indicate that impaired glucose homeostasis in DSCR1-4tri mice is associated with enhancing glycosylation.

Example 6. Confirmation of Synthesized Synthesis of DSCR1-4 (Method Independent of Calcineurin)

Recent studies have shown that DSCR1 activates the CREB pathway by inhibiting the phosphatase activity of calcineurin to prevent dephosphorylation of the cAMP response element-binding protein (CREB). Also, at fasting, calcineurin activates gluconeogenesis through dephosphorylation of CREB-regulated transcription coactivator2 (CRTC2). To determine whether calcineurin is required for DSCR1-4-mediated increased glycosylation, we treated the well-known calcineurin inhibitor, Cyclosporin A, in hepatocytes. Cyclosporin A treatment increased phosphorylation of CREB dose-dependently in WT hepatocytes (Figure 5a). In addition, the level of mRNA of the syngeneic enzyme was increased upon treatment with cyclosporin A in WT hepatocytes, suggesting that cyclosporin A treatment promotes glucose neogenesis (Fig. 5B).

Next, glucose production was measured in the liver in WT primary hepatocytes as in Example 1-4. Forskolin treatment implies that cyclosporin A increases glucose production (Fig. 5c), suggesting that cyclosporin A positively regulates glycosylation through increased phosphorylation of CREB.

DSCR1-4 To evaluate the role of calcineurin in mediating endogenous syngeneic regulation, we examined the expression of glycosylation genes in DSCR1-4 deficient hepatocytes following treatment with cyclosporin A or knockdown of calcineurin. The reduction in intracellular glycosylation due to the deficiency of DSCR1-4 was not affected by cyclosporin A treatment or calcineurin depletion (Fig. 5d-5g). These results indicate that DSCR1-4 mediated neogenesis independently regulates calcineurin.

Example 7. Identification of Physical Interaction Process of DSCR1-4 with FoxO1

We have continued to study mechanisms of how DSCR1-4 deficiency affects hepatic gluconeogenesis. During fasting, glucagon signaling induces glycogen synthesis through the activation of transcription factors such as CREB-regulated transcription coactivator2 (CRTC2) or FoxO1. Regulation of transcription by transcription factors occurs in the nucleus.

DSCR1-4 was examined whether it was transferred to the nucleus upon glucagon treatment. As a result, it was confirmed that DSCR1-4 migrates to the nucleus upon treatment of glucagon (Fig. 6A), and DSCR1-4 can act as a co-transcription factor.

We also investigated whether DSCR1-4 interacts with FoxO1, one of the autosomal transcription factors, to increase the expression of glycated genes in the nucleus. As a result, DSCR1-4 interacted with FoxO1 in DSCR1-4 over-expressing HEK293T cells upon treatment with glucagon (Fig. 6B). Consistently, the treatment of glucagon increased the interactions between endogenous DSCR1-4 (endogenous DSCR1-4) and FoxO1 in hepatocytes (Figure 6c), suggesting that DSCR1-4 interacts with FoxO1 to regulate glycogen synthesis it means.

Given the increased expression of DSCR1-4 in obesity (Figure 1), we examined whether the interaction between DSCR1-4 and FoxO1 is affected by obesity. The interaction of DSCR1-4-FoxO1 was increased in mice fed high-fat diet (HFD) compared to NCD-treated mice (Fig. 6d).

DSCR1-4 To determine the role of FoxO1 in mediating endogenous synaptogenesis, we examined the expression of glycated genes in DSCR1-4tri hepatocytes after knock-down of FoxO1. Upon treatment of glucagon, the DSCR1-4 mediated increase in glycated gene expression was abolished by the deficiency of FoxO1 (Fig. 6E). These results indicate that DSCR1-4 improves synaptic synthesis through FoxO1.

FoxO1 binds to the syngeneic gene promoter to activate the syngeneic synthesis. To determine whether FoxO1 transcriptional activity is mediated by DSCR1-4, we evaluated the occupancy of FoxO1 in the PEPCK and G6Pase (6 phosphatase) promoters in DSCR1-4 - / - hepatocytes. Occurrence of FoxO1 in PEPCK and G6Pase promoters was reduced in DSCR1 - / - hepatocytes compared to WT in glucagon treatment (Fig. 6f).

Next, we examined whether DSCR1-4 binds to the promoter of the syngeneic gene in the FoxO1 binding element. The binding of DSCR1-4 to the PEPCK and G6Pase promoters in HEK293T cells overexpressing DSCR1-4 was increased upon treatment with glucagon (Fig. 6g).

Taken together, it is shown that glucagon induces the induction of migration of DSCR1-4 into the nucleus, thereby increasing the interaction with FoxO1, thereby enhancing the transcription of glycosylation genes such as G6Pase and PEPCK by binding with the glycosyltransferase gene promoter 6h).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Research & Business Foundation SUNGKYUNKWAN UNIVERSITY <120> A method for screening a therapeutic agent for type 2 diabetes          using DSCR1-4, and a composition for diagnosing type 2 diabetes          or predicting prognosis <130> MP16-412 <160> 2 <170> KoPatentin 3.0 <210> 1 <211> 597 <212> RNA <213> DSCR1-4 <400> 1 atggatttta gggactttag ctacaatttt agctccctga ttgcttgtgt ggcaaacgat 60 gatgtcttca gcgaaagtga gaccagggcc aaatttgaat ccctcttcag aacatatgac 120 aaggacacca ccttccagta ttttaagagc ttcaaacgtg tccggataaa cttcagcaac 180 cccttatctg cagccgatgc caggctgcgg ctgcacaaga ccgagttcct ggggaaggaa 240 atgaagttgt attttgctca gactttacac ataggaagtt cacacctggc tccgcccaat 300 cccgacaaac agttcctcat ctcccctccg gcctctcctc ccgttggctg gaaacaagta 360 gaagatgcca cccccgtcat aaattacgat cttttatatg ccatctccaa gctggggcca 420 ggagagaagt atgaactgca tgcagcgaca gaccccactc ccagtgtggt ggtccacgtg 480 tgtgagagtg accaagagaa tgaggaggaa gaggaagaga tggagagaat gaagagaccc 540 aagcccaaaa tcatccagac acggagaccg gagtacacac cgatccacct tagctga 597 <210> 2 <211> 198 <212> PRT <213> DSCR1-4 <400> 2 Met Asp Phe Arg Asp Phe Ser Tyr Asn Phe Ser Ser Leu Ile Ala Cys   1 5 10 15 Val Ala Asn Asp Val Phe Ser Glu Ser Glu Thr Arg Ala Lys Phe              20 25 30 Glu Ser Leu Phe Arg Thr Tyr Asp Lys Asp Thr Thr Phe Gln Tyr Phe          35 40 45 Lys Ser Phe Lys Arg Val Arg Ile Asn Phe Ser Asn Pro Leu Ser Ala      50 55 60 Ala Asp Ala Arg Leu Arg Leu His Lys Thr Glu Phe Leu Gly Lys Glu  65 70 75 80 Met Lys Leu Tyr Phe Ala Gln Thr Leu His Ile Gly Ser Ser His Leu                  85 90 95 Ala Pro Pro Asn Pro Asp Lys Gln Phe Leu Ile Ser Pro Pro Ala Ser             100 105 110 Pro Pro Val Gly Trp Lys Gln Val Glu Asp Ala Thr Pro Val Ile Asn         115 120 125 Tyr Asp Leu Leu Tyr Ala Ile Ser Lys Leu Gly Pro Gly Glu Lys Tyr     130 135 140 Glu Leu His Ala Ala Thr Asp Pro Thr Pro Ser Val Val Val His Val 145 150 155 160 Cys Glu Ser Asp Glu Glu Asn Glu Glu Glu Glu Glu Glu Met Glu Arg                 165 170 175 Met Lys Arg Pro Lys Pro Lys Ile Ile Gln Thr Arg Arg Pro Glu Tyr             180 185 190 Thr Pro Ile His Leu Ser         195

Claims (13)

A screening method for treating type 2 diabetes mellitus comprising the steps of:
(a) treating the candidate material in cells in vitro;
(b) measuring the degree of binding of the down-syndrome critical region 1-4 (DSCR 1-4) gene to the FoxO1 (Forkhead box protein O1) gene; And
(c) selecting a substance that reduces the degree of binding of the DSCR1-4 gene to FoxO1 as compared to the candidate substance-untreated group as a treatment for type 2 diabetes mellitus. The method according to claim 1,
Wherein said cells are hepatocytes. The method according to claim 1,
Wherein the candidate substance is selected from the group consisting of a compound, a microorganism culture solution or extract, a natural product extract, a nucleic acid, and a peptide. The method of claim 3,
Wherein the nucleic acid is selected from the group consisting of microRNA, siRNA, shRNA, antisense RNA, aptamer, locked nucleic acid (LNA), peptide nucleic acid (PNA), and morpholino. Screening method. The method according to claim 1,
The step (b) may be carried out by immunoprecipitation, immunohistochemistry, microarray, northern blotting, western blotting, ELISA, Wherein the measurement is carried out using at least one method selected from the group consisting of enzymatic chain reaction (PCR) and immunofluorescence. The method according to claim 1,
Wherein the decrease in the degree of binding between the DSCR1-4 gene and FoxO1 in the step (b) reduces the glucose uptake by inhibiting glycation by the DSCR1-4 gene,
delete delete delete delete delete delete delete

Images