KR101885946B1 - Biomarker for diagnosing type II diabetes and it use - Google Patents

Abstract

This disclosure discloses pelino-1 biomarkers that are differentially expressed in type 2 diabetes and their uses. The pelino-1 identified herein can be usefully used as a potential diagnostic or diagnostic kit for second diabetes, as a diagnostic kit, and also as a target for screening for the discovery of therapeutic agents.

Description

[0001] Description [0002] BIOMARKER FOR DIAGNOSIS OF DIABETES [0002]

The present invention relates to a biomarker technique capable of diagnosing type 2 diabetes and a type 2 diabetes therapeutic drug screening technique for developing a therapeutic drug using the same.

Type 2 diabetes (non-insulin dependent diabetes mellitus) is a diabetes induced by insulin resistance and is known to be caused by various factors such as obesity, insulin receptor reduction, insulin secretion decline, and genetic factors. Initially, increased insulin secretion in the pancreas by insulin resistance does not reveal much of the symptoms, but if the insulin secretory capacity of the pancreas does not sustain enough insulin resistance, then the symptoms will only appear. Type 2 diabetes accounts for more than 90% of total diabetes. Recently, the activity of modern people is rapidly increasing as lifestyle and obesity increase. Especially, obesity has a positive correlation with type 2 diabetes Diabetes can be increased 90-fold.

Methods for diagnosing diabetes include urine glucose measurement, fasting blood glucose measurement, glucose testing by oral or intravenous injection method, and blood tests such as the measurement of glycated hemoglobin. There are diabetes mellitus method, benedict method, test tape method, etc. However, when diabetes mellitus is present only in postprandial state, there is diabetes mellitus in which diabetes mellitus appears, The diagnosis of diabetes can not be confirmed by proof alone. Fasting blood glucose measurements are diagnosed as diabetes mellitus if the blood glucose level of the fasting capillary before breakfast is 140 mg / dl, but mild cases may be within normal range.

Korean Patent Laid-Open Publication No. 2015-000426 relates to GDF-16 as a diagnostic marker of fasting glucose deficiency disorder and a diagnostic kit for fasting glucose deficiency disorder comprising the same, and proposes a protein marker for diagnosing the pre-dyslipidemia state.

Korean Patent Publication No. 2015-0022878 relates to a diabetic diagnostic marker, which suggests a marker for insulin reduction, particularly a type 1 diabetic diagnostic marker.

However, the existing type 2 diabetes diagnosis method is easy, but it is impossible to accurately and repeatedly measure the blood glucose in the same condition. When the error of the measurement method is considered, the accuracy becomes poor. Therefore, in need. Therefore, there is a need to develop a new technology that can diagnose type 2 diabetes more accurately.

The present invention provides a diagnostic biomarker for type 2 diabetes and its use as a target for the discovery of a therapeutic agent for diabetes.

In one embodiment, the present invention provides a composition for the diagnosis of Type 2 diabetes comprising a reagent for detecting a pelino-1 marker.

The composition according to the present invention can specifically detect type 2 diabetes and can be usefully used for diagnosis of diseases.

In one embodiment, a composition according to the present invention can be used to detect a Pelino-1 marker therefrom, using at least one of adipose tissue, whole blood, serum, plasma, or blood cells obtained from whole blood, Can be used.

The pelino-1 marker according to the present invention can be detected qualitatively or quantitatively at the protein or nucleic acid level, for example at the mRNA level. In one embodiment, the detectable reagent at the protein level comprises a monoclonal antibody, a polyclonal antibody, a substrate, an aptamer, a receptor, a ligand or cofactor, or a reagent for detecting a mass spectrometer. In other embodiments, reagents detectable at the nucleic acid level include reverse transcriptase chain reaction, real-time RT-PCR, RNase protection assay, reagents used in DNA chips or Northern blots.

In another embodiment, the invention also provides a method of detecting pelino-1, comprising: detecting the presence or amount of a pelino-1 marker from a sample; And a step of correlating the presence or amount of the detected marker with the diagnosis or prognosis of the type 2 diabetes of the subject to be examined. In order to provide information necessary for the diagnosis or prognosis of type 2 diabetes, And detecting the marker.

The presence or amount of the marker in the method according to the present invention can be determined at the protein or nucleic acid level.

In the method according to the present invention, the associating step may further use non-marker clinical information including blood glucose, insulin concentration, or ratio of whole macrophages to M1 macrophages in adipose tissue of the subject of type 2 diabetes.

In the method according to the present invention, the associating step is a step of diagnosing the second diabetes when the amount or the presence of the determined marker is reduced by comparing the detection result for each marker determined in the normal control group.

In another aspect, the invention also provides a method of producing a pelino-1 protein or gene, comprising: providing a pelino-1 protein or gene; Contacting the protein with a test substance; And comparing the expression of pelino-1 in a control group not in contact with the protein contacted with the test substance to select a candidate substance as a candidate substance for decreasing the expression, screening for a substance for treating type 2 diabetes mellitus ≪ / RTI >

In the screening method according to the present invention, the Pelino-1 protein or gene may be provided in the form of a cell or an animal model expressing it.

In the screening method according to the present invention, the candidate substance can inhibit the differentiation of M1 macrophages.

The pelino-1 identified herein is differentially expressed in type 2 diabetes, and can be used as a biomarker or a diagnostic kit for diagnosing the possibility or diagnosis of the second diabetes, and is also useful as a target for screening for therapeutic drug discovery Lt; / RTI >

Figure 1 shows the decrease in obesity by high fat diet in pelino-1 deficient mice.
Figure 2 shows a decrease in insulin resistance due to high fat diet in pelino-1 deficient mice.
Figure 3 shows the correlation between the expression of pelino-1 and the diabetes-related markers [blood glucose (left), serum insulin (middle) and M1 macrophages (right)].
Figure 4 shows the expression pattern of the pelino-1 protein in the M1 condition.
FIG. 5 shows that pelino-1 is essential for the differentiation of M1 macrophages to confirm the function in macrophages with or without pelino-1.
Figure 6 shows the expression patterns of other genes involved in the differentiation of M1 macrophages, namely cox-2, CD86 and IL 12a, with or without pelino-1.
Fig. 7 shows the expression patterns of other genes involved in the differentiation of M2 macrophages, namely Fizz 1, Mrc 1 and Ym 1, with or without pelino-1.
Fig. 8 shows the gene expression of M1 macrophages after differentiation of M1-like macrophages using GM-CSF in order to demonstrate that pelino-1 is essential for the differentiation of M1 macrophages.
Fig. 9 shows the M2 gene expression of M2-like macrophages differentiated with M-CSF alone in order to demonstrate that pelino-1 is essential for the differentiation of M1 macrophages.
FIG. 10 is a graph showing the degree of diabetes mellitus according to the expression of pelino-1 gene in obesity (left) and diabetic (right) models. The glucose tolerance test (left) and insulin resistance test Resistance (right).
Fig. 11 shows the correlation between the expression of pelino-1 and obesity.
FIG. 12 shows results obtained by euthanasizing a mouse in which a normal or high-fat diet was consumed for 8 weeks and then obtaining epididymal adipose tissue and confirming the ratio of macrophages and M1 or M2 macrophages in immune cells in adipose tissue by flow cytometry.
Fig. 13 shows the result of RNA expression in epididymal adipose tissue obtained by comforting mice fed with a high-fat diet for 8 weeks and confirming expression of each gene.

We have investigated the relationship between the expression pattern of pelino-1 protein expressed in adipose tissue of type 2 diabetes and its type 2 diabetes using an obesity-induced diabetic model. As a result, pelino- Type diabetes mellitus in patients with type 2 diabetes mellitus.

Accordingly, in one aspect, the present invention relates to a composition for the diagnosis of type 2 diabetes comprising a marker for the diagnosis of type 2 diabetes of pelino-1 or a reagent for detecting pelino-1.

Pelino-1 is a protein that plays a role of ubiquitin ligase (ubiquitin ligase), which plays a role in attaching ubiquitin to a target protein in a cell. It functions in the innate immunity of macrophages, (Moynach PN, The roles of Pellino E3 ubiquitin ligases in immunity, Nat Rev Immunol. 14, 122-31, 2014; Pellino-1 cDNA sequence: NM_020651.3; Protein sequence: NP_065702)

Detection herein includes quantitative and / or qualitative analysis, including detection of presence and absence and expression level detection, and such methods are well known in the art, and those skilled in the art will appreciate that methods suitable for the practice of the present application You can choose from or refer to the following.

The detection of the pelino-1 marker of the present invention includes both quantitative detection and detection at the level of presence, which can be an indicator of the onset and progression of the disease, Diagnosis or prognosis.

The term "sample" or "biological sample" is used herein to refer to any solid or liquid sample obtained from a human or mammal such as a tissue derived from a particular organ, a biological material comprising fat, urine, saliva, But are not limited to, samples. According to one embodiment of the present disclosure, the marker protein of the present disclosure is particularly included in adipose tissue or blood samples.

As used herein, the term " metabolic disease " is a disease caused by lifestyle such as obesity, lack of exercise, hypernutrition, etc., but is not limited to, for example, type 2 diabetes (non-insulin dependent diabetes), hypertension, Or heart disease. According to one embodiment herein, the disease is type 2 diabetes.

The term " diagnosis " in the context of the present invention encompasses determining an object for a second diabetes, i.e., the susceptibility of the subject, determining whether an object currently has a second diabetes, Determining the prognosis of an object that is caught in the subject, or therametrics (e.g., monitoring the status of the object to provide information about the treatment efficacy).

The term "diagnostic marker ", " biomarker ", " marker ", or " diagnostic marker" is used herein to distinguish a diseased specimen from a normal specimen. Or a decrease in the amount of a protein or a biological molecule such as a nucleic acid, a lipid, a glycolipid, a glycoprotein, or the like. The second diabetic diagnostic marker herein is a pelino-1 protein or gene whose expression is increased in diabetic specimens.

As used herein, the terms " detection reagent ", " detecting reagent " are materials capable of detecting the markers herein for quantitative or qualitative determination, for example, at the protein or nucleic acid mRNA level.

Methods for assaying the amount, presence or expression pattern thereof at the protein level are well known and include, for example, Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay, radioimmunodiffusion, Immunoassay, Immunoprecipitation Assay, ComplementFixation Assay, Mass Spectrometry, FACS, Protein Chip, and the like can be performed by using an immunoassay method such as Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, . Reagents that can be detected at the protein level are well known and include, for example, monoclonal antibodies, polyclonal antibodies, substrates that specifically bind to the markers, nucleic acid or peptide aptamers, and specifically interact with the markers Lt; / RTI > receptor or ligand or cofactor or the like. These reagents can be incorporated into nanoparticles or chips as needed.

Methods for the analysis of the amount, presence or expression pattern thereof at the nucleic acid level are well known and include, for example, detection at the RNA level, reverse transcription polymerase chain reaction (RT-PCR) / polymerase Methods using a chain reaction, competitive RT-PCR, real-time RT-PCR RNase protection assay, chip or Northern blot can be used, and these assays are well known and can also be performed using commercially available kits, Appropriate choices may be made for the practice of the present application.

In one embodiment, the detection reagent is an antibody that specifically binds to a protein and can be used for Western blot, tissue immunostaining, ELISA, or flow cytometry. Such methods of immunoassay or immunostaining are 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. By analyzing the intensity of the final signal by the above-described immunoassay process, that is, performing signal contrast with a normal sample, diagnosis of disease occurrence can be made.

In another embodiment, the detection reagent is a reagent for RNA analysis, wherein the marker detection is carried out at the mRNA level. Detection of mRNA is usually performed through Northern blot or reverse transcription PCR (polymerase chain reaction). In the latter case, it is possible to detect a specific gene in a specimen by isolating RNA of the specimen, specifically mRNA, synthesizing cDNA therefrom, and then using a specific primer or a combination of a primer and a probe to detect the presence / Or the amount of expression can be determined. Such a method is described in, for example, 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).

In another embodiment, the detection reagent in the method for measuring the presence and amount or pattern of the marker mRNA according to the present invention by RT-PCR includes, for example, a primer specific to the mRNA of the marker of the present invention. A primer refers to a nucleic acid sequence having a free 3 'hydroxyl group capable of complementarily binding with a template and allowing the reverse transcriptase or DNA polymerase to initiate replication of the template.

The characteristics of metabolic immunological diabetes seen from the viewpoint of immunology from diabetes and other metabolic diseases are as follows: Tumor necrosis factor-α (TNF-α), IL-6 (Interleukin-6 ), And the number and proportion of B cells producing macrophages, CD8 + T cells, and antibodies in adipose tissue are markedly increased, with the highest proportion of macrophages. The macrophages that function as homeostasis are M2 macrophages, the macrophages that induce the inflammatory response are classified as M1 macrophages, the obesity tissues of diabetic patients have increased inflammatory response, and the M1 macrophages Macrophage-mediated inflammation in metabolic disease has been increasing (Sica A, Mantovani A. Macrophage plasticity and polarization in vivo veritas, J Clin Invest, 122, 787-95, 2012; Chawla A, Nguyen KD, Goh YP. , Nat Rev Immunol., 11, 738-49, 2011)

Here we examined the expression of pelino-1 and the markers associated with diabetes through an obesity-induced diabetic model. As a result, the expression of pelino-1 was associated with obesity-induced insulin resistance, and it was found that when the expression of pelino-1 was increased, the blood glucose concentration, insulin concentration and M1 macrophage ratio were increased . In particular, the role of pelino-1 in obesity and diabetes mellitus by high-fat diets has been shown to play a role in the differentiation of M1 macrophages rather than M2 macrophages. The pelino-1 gene has been shown to increase glucose sensitivity and insulin resistance And that the inhibition of the expression of pelino - 1 is essential for the differentiation of M1 macrophages and thus inhibits the differentiation of M1 macrophages in obesity induced by high fat diets.

Accordingly, when pelino-1 is inhibited, it may be useful for the treatment of metabolic diseases, particularly type 2 diabetes, in which obesity due to high-fat diets is a problem.

In another embodiment, the present invention provides a method of detecting a Pelino-1 marker, comprising: detecting the presence or amount of a pelino-1 marker from a sample; And diagnosing and associating the presence or the amount of the detected marker with the type 2 diabetes of the subject to be examined, from the sample of the subject to be examined to provide information necessary for diagnosis or prognosis of type 2 diabetes. The method comprising the steps of:

In the method according to the present invention, the presence or amount of the marker is determined at the protein or mRNA expression level, and the above-mentioned one can be referred to.

The method includes the step of associating a detection result of a marker with a diagnosis or prognosis of type 2 diabetes, wherein the step of associating comprises, depending on the embodiment, determining the amount or presence of the protein of each determined marker Comparing the detection result of the marker, for example, the increase / decrease of the marker, and then diagnosing it based on the comparison. For example, when the value of pelino-1 is significantly increased, for example, in comparison with the value of the control group, information can be provided to diagnose that the disease has occurred in the subject. According to an embodiment of the present invention, the associating step may include comparing a sample of a normal control group with a sample, setting a threshold value for diagnosing the onset of the marker, and comparing the detection result of the target body with the threshold value Can be compared. The threshold setting can be done, for example, by referring to the method described in this embodiment.

In an embodiment of the method according to the present invention, the associating step further comprises non-marker clinical information including blood glucose, insulin concentration, or ratio of total macrophages to M1 macrophages in adipose tissue of the subject to be tested for type 2 diabetes, ≪ / RTI >

Here we examined the expression of pelino-1 and the markers associated with diabetes through an obesity-induced diabetic model. As a result, the expression of pelino-1 was associated with obesity-induced insulin resistance, and it was found that when the expression of pelino-1 was increased, the blood glucose concentration, insulin concentration and M1 macrophage ratio were increased . In particular, in the obese and diabetic models according to the high fat diet, it was confirmed that pelino-1 is essential for the differentiation of M1 macrophages and that the inhibition of pelino-1 expression inhibits the differentiation of M1 macrophages in obesity according to high fat diets.

Accordingly, in another aspect, the present invention provides a method of producing a pelino-1 protein or gene, Contacting the protein with a test substance; And comparing the activity or expression of pelino-1 in a control not contacted with the protein contacted with the test substance to select a candidate substance as a candidate substance for reducing the expression. To a screening method.

The test substance used in the method of the present invention is a substance expected to regulate the expression and / or activity of the pelino-1 protein or gene. For example, for the purpose of screening a drug, a compound has a low molecular weight therapeutic effect . For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such a library of compounds can be prepared by reacting peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles, and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto.

The pelino-1 that can be used in the screening method of the present invention is as mentioned above. Depending on the specific method for screening the pelino-1 protein, various host-derived ones can be used in the present method. . In addition, even the same host, for example, a human, may have sequence variation depending on a specific individual, region, environment, and the like. Of course, this sequence may be modified (deletion, substitution, addition) Can be used in the present invention. In one embodiment, it is human-derived, and such is the same as mentioned above.

Cells expressing the pelino-1 protein may be used in the methods herein, or an obesity-induced diabetic model, such as those used in the examples herein, including such cells, may be used.

Cells and animal models expressing the pelino-1 protein can be prepared using methods known in the art. In one embodiment, the DNA or RNA sequence encoding the protein used in the screening is expressed in an appropriate host cell by using recombinant DNA technology. For example, a plasmid containing the corresponding gene encoding the protein is used in a stable or transient manner after delivery to eukaryotic cells such as insect cells and mammalian cells. The cells are contacted with the test substance under conditions enabling the growth of the cells to be maintained, and then the expression of pelino-1 is examined at the RNA and protein level. Expression of pelino-1 The method of detection at the RNA and protein level is as described above.

In one embodiment according to the present application, the cell expressing the pelino-1 protein can be obtained, for example, by culturing mammalian cells expressing all or a portion of the protein (transient or Stable transfer or endogenous expression) , A test substance was added thereto, and after a certain period of time, western blotting was performed by extracting total proteins from the cells. Compared with the control group (when the test substance was not treated), the expression- It can be selected as a candidate substance.

The amount of the protein used, the type of the cell, and the amount and type of the test substance used in the present method will depend on the specific experimental method used and the kind of the test substance, and those skilled in the art will be able to select an appropriate amount. As a result of the experiment, a substance which causes a decrease in protein expression or activity in the presence of the test substance as compared with the control group not in contact with the test substance is selected as a substance inhibiting the interaction. Less than about 99%, less than about 95%, about 90%, about 85%, about 80%, about 75%, about 70% less, less than about 65% , About 55% reduction, about 50% reduction, about 45% reduction, about 40% reduction, about 30% reduction, about 20% reduction or less.

In another aspect, the present disclosure also relates to a kit for the diagnosis of type 2 diabetes comprising a detection reagent for a pelino-1 protein, or a nucleic acid encoding the same, respectively. Reagents capable of detecting such markers of the present invention may be individually dispensed into a compartmented container and may include instructions for use.

Hereinafter, the present invention will be described more specifically with reference to the following examples, but the scope of the present invention is not limited thereto.

Example

Materials and Methods

Production of mouse obesity-induced diabetic model Pellino1 Hetero or Pellino1 Knockout male mice on C57BL / 6 mice were sacrificed at 8 weeks of age (D12492, Research Diets). The mice were weighed at intervals of one week. After 8 weeks of high-fat diet, the fasting blood glucose level was defined as Fasting Glucose after 4 hours of fasting. In addition, the blood was stored separately to confirm the insulin concentration. After the experiment, mice were euthanized and the proportion of M1 macrophages in adipose tissue was determined.

Flow cytometry To remove the cells from the adipose tissue, the adipose tissue was washed with 1xPBS buffer, and the adipose tissue was cut to a size of about 0.5 mm in length. Thereafter, finely cut fat tissues were added to 100 mM HEPES buffer containing 0.1% collagenase type I (Worthington, USA), and the cells were treated with shaking at 200 rpm at 37 ° C for 20 minutes. After that, the undissolved mackerel was filtered using a filter, and centrifuged at 2000 rpm for 5 minutes to obtain cells. After obtaining the cells, the red blood cells were dissolved in the storage buffer, and the antibodies required for flow cytometry were mixed. The antibodies required for flow cytometry are as follows; Anti-CD11b antibody, anti-F4 / 80 antibody, anti-CD11c antibody, anti-CD206 antibody (all from ebioscience). Each antibody was added to PBS buffer, mixed and reacted at 4 ° C for 30 minutes. After washing with PBS buffer, the remaining cells were analyzed by flow cytometry using LSRII (BD Bioscience) machine according to manufacturer's instructions.

Real-time RT-PCR RNA was isolated from the cells using the SV total RNA isolation kit (Promega, USA) according to the manufacturer's instructions, and DNA complementary to mRNA was prepared using M-MLV RT (Promega) enzyme. Real-time PCR was performed using pelino-1 (Mm00481051_m1) -specific primer (Applied Biosystems, USA) and GAPDH (Mm99999915_g1) was used as an internal control to correct the result. PCR was performed using a 7500 real-time PCR system (Applied Biosystem).

Western blot cells were plated in RIPA buffer supplemented with protease inhibitor and the cells were lysed at 4 ° C for 30 minutes. Centrifugation was then carried out at a rate of 13,000 rpm at 4 DEG C to remove undissolved cellular constituents. Only the supernatant was separated, transferred to a new tube, quantified by Bradford assay, and Western blot was performed. Antibodies used in western blots were as follows; Anti-pelino-1 antibody (Santa Cruz), anti-beta-actin antibody (Cell Signaling). The antibody was reacted for 16 hours while shaking at 4 ° C, and reacted for 1 hour at room temperature for the second antibody.

Culturing and Differentiation of Macrophages The femur and tibia of mice 8-12 weeks old were harvested and bone marrow was obtained. The resulting bone marrow cells were dissolved in a stock solution, and the cells were then dissolved in a culture solution containing 10% FBS and 1% penicillin / streptomycin based on RPMI 1640, SPL) was added with M-CSF (Peprotech) at a concentration of 20 ng / ml and then cultured. After 3 days, the culture solution and M-CSF were added at the same concentration. After the solution was changed, an additional 3 days were further cultured. After that, the cells were washed and adhered to each other, and then the macrophages were differentiated into the culture solution under the following conditions. Negative control: The same amount of PBS was added, M1 condition; LPS (Sigma): 100 ng / ml, IFN-y (Peprotech): 20 ng / ml, M2 condition; IL-4 (Peprotech): 20 ng / ml, IL-13 (Peprotech): 20 ng / ml

Glucose Tolerance Experiments Eighteen weeks of fasting diets were initiated at 8 weeks of age and fasted for 16 hours before the experiment. Thereafter, the body weight was measured, and an aqueous solution of glucose (Sigma) was injected by intraperitoneal injection at a dose of 2 g / kg (glucose mass / mouse body weight). Blood samples were taken at the 0, 15, 30, 60, 90, and 120 minutes after injection of glucose from the tail of the mice.

Insulin tolerance test The mice that had been treated with high fat diet for 8 weeks starting from 8 weeks of age were fasted for 4 hours before the experiment. Thereafter, the body weight was measured, and insulin (Sigma) was injected intraperitoneally under the condition of 0.75 U / kg (insulin unit / mouse body weight). Blood samples were taken at the 0, 15, 30, 60, 90, and 120 minutes after injection of glucose from the tail of the mice.

Statistical analysis All statistical analyzes were performed using the Prism5 (Graphpad) program. The significance is expressed as: *: p <0.05, **: p <0.01, ***: p <0.001.

Example 1. Analysis of pelino-1 expression pattern in an obesity-induced diabetic mouse model

Example 1-1. Reduced obesity by high fat diet in pelino-1 deficient mice

In order to confirm the change in the degree of obesity according to the expression of pelino-1, pelino-1 +/- expressing mouse (Peli1 +/-) and pelino-1 expressing no pelino- High fat diet (HFD) or normal chow diet (NCD) were fed to each mouse (Peli1 - / -) mice and the obesity degree was measured in terms of weight change of the mice.

As shown in FIG. 1, when the high-fat diets were consumed in the normal diet, the weight of the mice that were expressed compared to the mice not expressing pelino-1 was found to be higher in most of the time periods. This is a result indicating that the expression of pelino-1 and the degree of obesity are positively correlated.

Examples 1-2. Reduction of Insulin Resistance by High Fat Diet in Pelino-1 Deleted Mice

It is well known that obesity is closely related to diabetes, which is represented by insulin resistance. When insulin resistance is increased, insulin does not function properly in the body, resulting in an increase in blood glucose concentration. In addition, the increased glucose concentration returns to increase the action to lower the blood glucose concentration, resulting in an increase in the blood insulin concentration. In the results of Example 1-1, the expression of pelino-1 was found to be positively correlated with the obesity degree, and it was confirmed in this experiment how this phenomenon is related to insulin resistance.

As shown in FIG. 2, the blood glucose level (left) and insulin concentration (right) were decreased in the mice lacking pelino-1 as compared with those of the non-pelino-1 mice Respectively. This indicates that obesity-induced insulin resistance, that is, diabetes, is closely related to the expression of pelino-1.

Examples 1-3. Analysis of Correlation between Pelino-1 Expression and Markers Related to Diabetes

It has already been known about diabetes, such as the current blood glucose concentration as a diagnostic marker of diabetes, the insulin concentration correlated to it, and the ratio of the M1 macrophages in adipose tissue, The correlation between the presence of a diagnostic marker and pelino-1 was observed at the protein level by Western blotting and the intensity of the protein band was quantitatively quantified and normalized by dividing the value of beta-actin.

As shown in FIG. 3, the correlation between blood glucose concentration (left), insulin concentration (middle) and M1 macrophage ratio (right) was positively correlated with the expression of pelino-1 When the accuracy was measured by the R-squared value, it was confirmed that the accuracy was high in order of M1 macrophage ratio, insulin concentration, and glucose concentration.

Since these three scales are all closely related to diabetes, the expression of pelino-1 is useful as a biomarker for the diagnosis of diabetes.

Example 2. Characterization of pelino-1 protein in differentiation of macrophages

Example 2-1. Identification of increased expression of pelino-1 protein under M1 conditions

In order to identify the expression pattern of pelino-1 protein in mouse-derived macrophages and differentiation conditions, M1 (classical activation) and M2 (alternative activation) (BMDM) Macrophage activation mechanism, Martinez et al. F1000 Prime Rep. 2014; 6: 13), and cells were obtained and Western blotting was performed on pelino-1.

As shown in FIG. 4, pelino-1 in macrophages derived from the bone marrow of pelino-1 + / + (WT, wild type), +/- (Hetero) Expression was confirmed at the protein level (left). In addition, macrophages derived from the bone marrow of pelino-1 + / + mice were further differentiated under M1 or M2 conditions for 24 hours, and the expression of pelino-1 protein was confirmed by differentiation patterns (right). That is, the expression of pelino-1 protein was increased in the M1 macrophage activation state, considering that M1 macrophages were formed / activated in diabetic patients and animal models (Sica A, Mantovani A. J Clin Invest, 122, 787- And Chawla A, Nguyen KD, Goh YP., Nat Rev Immunol. 11, 738-49, 2011) indicate that pelino-1 can be used as a marker of diabetes.

The protein expression of pelino-1 was confirmed to be influenced by the genotype of mouse used in this experiment. That is, no pelino-1 was detected at the protein level in the KO sample in which pelino-1 was deleted. In addition, when the macrophage was differentiated, it was confirmed that a high amount of pelino-1 protein was expressed when differentiated into M1 under the condition of comparison with the control group. The expression of pelino-1 was not significantly different or slightly decreased when differentiated into M2, compared with the control. This indicates that pelino-1 plays a role of pelino-1 in M1 macrophages more than other states because of its high expression level at the time of differentiation of M1 macrophages, and pelino-1 can be usefully used as a marker of diabetes .

Example 2-2. Pelino-1 is essential for the differentiation of M1 macrophages

In order to confirm the function of macrophages according to presence or absence of pelino-1, macrophages were differentiated into M1 or M2 conditions. After 16 hours, cells were extracted from the cells and gene expression level was confirmed by qRT-PCR Respectively.

As shown in Fig. 5, expression of inos, a typical gene marker of M1 macrophage cells, was confirmed (left). At this time, the average value of the pelino-1 + / + sample of the control group was set to 1, and the values were normalized accordingly. In addition, the expression of arg1 (arginase 1), a typical gene marker of M2 macrophages, was confirmed (right). At this time, the average value of the Pelino-1 + / + samples of the M2 differentiation condition was set to 1 and the values were standardized accordingly.

When M1 and M2 differentiation were induced in macrophages, the expression level of inos in M1, which is a representative gene of M1 differentiation, was greatly increased under the M1 condition, and the expression level of inos was decreased as the expression of pelino- Respectively. Thus, the absence of pelino-1 in the differentiation into M1 macrophages means that the differentiation of M1 macrophages does not occur well, which means that pelino-1 is essential for the differentiation of M1 macrophages.

On the other hand, the expression of arginase1 was increased in the M2 differentiation condition when the differentiation into M2 macrophages was induced, but the expression of arginase1 was not significantly different regardless of the presence or absence of pelino-1. Therefore, it was confirmed that pelino-1 does not play a large role in M2 differentiation.

Since M1 and M2 differentiation of macrophages can not be explained by only one gene, expression patterns of other genes involved in M1 and M2 differentiation were also confirmed to reinforce the above experiment. As shown in FIG. 6, the expression patterns of cox2, cd86, il12a and the like of the M1 genes were different depending on the presence or absence of pelino-1, and the tendency of the inos gene The expression pattern of each gene was found to be significantly reduced when differentiating macrophages with pelino - 1 were matched with M1 expression conditions in accordance with the expression pattern.

Similarly, as shown in FIG. 7, when the expression patterns of other M2 genes such as fizz1, mrc1, ym1 and the like were examined, it was confirmed that the expression of each gene was constant regardless of the expression of pelino-1.

These results indicate that pelino-1 plays a role in the differentiation of M1 macrophages rather than the role in the differentiation of M2 macrophages, and that macrophage-differentiated macrophages lacking M1 macrophage differentiation The phenomenon was confirmed through the expression of various M1 macrophage genes.

Examples 2-3. Further validation of what is essential for the differentiation of M1 macrophages in pelino-1

In order to verify the role of pelino-1 in the differentiation of M1 macrophages, the following experiment was conducted. There are two methods for carrying out experiments on the differentiation of macrophages. This method is a method of differentiating macrophages into M-CSF for 6 days and then further differentiating into M1 and M2, , It is known that macrophages similar to the M1 macrophages are formed when GM-CSF is treated and the macrophages are differentiated for 6 days according to the same experimental method. As a control group, macrophages differentiated with only M-CSF are used These cells are known to have similar properties to M2 macrophages.

Based on the above experimental method, M1-like macrophages were differentiated using GM-CSF, and gene expression of M1 macrophages was confirmed (FIG. 8). At this time, the results of the pelino-1 + / + group were set to 1 and other values were standardized accordingly. As a result, it was confirmed that the expression of nos2 (another name of inos), ptgs2 (another name of cox2), and cd86 were decreased in the macro cells in which pelino-1 was deleted. This has the same tendency as in the above experiment, and therefore, it has been proved through other experimental techniques that it plays an essential role in the M1 macrophage differentiation of pelino-1.

On the other hand, M2 gene expression of M2-like macrophages differentiated by M-CSF alone was not significantly different regardless of the expression of pelino-1 (FIG. 9). It has also been confirmed that pelino-1 is not largely involved in the differentiation into M2 macrophages.

Example 3. Analysis of phenotypic difference in obesity and diabetic model according to pelino-1 gene expression

In order to verify the role of pelino-1 in M1 macrophage differentiation in vivo, obesity and type 2 diabetes mellitus according to high fat diet were selected as disease animal models and experiments were conducted. Recently, studies on metabolic immunology have been actively conducted and it has been reported that M1 macrophages play an important role in the deterioration of the disease in the model.

In order to confirm the differences in phenotypic characteristics of obesity and diabetic models due to the expression of pelino-1, pelino-1 +/- expressing mice (Peli1 +/-) and pelino-1 expressing non- A high fat diet (HFD) or a normal chow diet (NCD) was administered to each of the pel1 - / - mice for 8 weeks, and then the following experiment was carried out .

Glucose intolerance was confirmed by glucose tolerance test to determine the degree of diabetes mellitus caused by the expression of pelino-1 (FIG. 10 (left)). It is known that when diabetes becomes worse, blood sugar increases and such a pattern persists for a long time. In the normal chow diet (NCD), glucose intolerance is similar regardless of the presence or absence of pelino gene expression. However, when diabetes was induced by high fat diet (HFD), a higher concentration of blood glucose was observed in the mouse expressing pelino-1, and this phenomenon was maintained for a long time. Thus, the pelino-1 gene is associated with glucose intolerance, one of the measures of diabetes mellitus.

Insulin resistance was confirmed by insulin resistance assay to determine the degree of diabetes mellitus caused by the expression of pelino-1 (Fig. 10 (right)). It is well known that insulin resistance occurs when diabetes becomes worse, and thus the effect of insulin-induced hypoglycemia is reduced. In the normal chow diet (NCD), insulin resistance appears to be similar regardless of the presence or absence of pelino gene expression. However, when diabetes was induced by high fat diet (HFD), a higher concentration of blood glucose was observed in the mouse expressing pelino-1, and this phenomenon was maintained for a long time. Thus, the pelino-1 gene plays a role in increasing insulin resistance, one of the measures of diabetes mellitus.

(ED: epididymal fat, epididymal adipose tissue, SC: subcutaneous fat, subcutaneous fat tissue, RP: retro-peritoneal fat) were obtained after euthanasia treatment of mice with high fat diets Fat and Post-peritoneal Adipose Tissue) The expression of pelino-1 was found to be positively correlated with the obesity index, and as a result, the weight of adipose tissue isolated from obesity-induced mice was measured separately , And the weight of each adipose tissue was also significantly decreased in the pelino-1-deleted mice.

After euthanasia of mice fed a normal or high fat diet for 8 weeks, epididymal adipose tissue was obtained and the ratio of macrophages and M1 or M2 macrophages in immune cells in adipose tissue was confirmed by flow cytometry (Fig. 12). The following markers were used: total macrophage: CD11b + F4 / 80 +; M1 macrophages: CD11b + F4 / 80 + CD11c + CD206-; M2 macrophages: CD11b + F4 / 80 + CD11c-CD206 +

In normal diet, the differentiation of macrophages in adipose tissue was not significantly related to the presence or absence of pelino - 1 gene. On the other hand, when the high fat diet was consumed, the ratio of total macrophages to M1 macrophages was decreased when pelino-1 was deleted. Therefore, in vitro experiments with the above bone marrow-derived macrophages have also been verified in vivo, once again confirming that pelino-1 plays an important role in the differentiation of M1 macrophages.

Euthanized mice fed a high fat diet for 8 weeks are sacrificed to obtain epididymal adipose tissue. Thereafter, RNA was extracted from adipose tissue to confirm expression of each gene (Fig. 13). As a result, it was confirmed that the expression of F4 / 80, a representative gene marker of macrophages, was significantly higher in adipose tissues of mice expressing pelino-1, which is consistent with the results of macrophage analysis by flow cytometry. In addition, flow cytometry revealed that M1 macrophages were significantly reduced in adipose tissues of pelino-1 deletion mice, and in agreement with these results, several M1 macrophages including inos, cox2, cd86, and il-12a Gene expression was significantly reduced in adipose tissues of pelino-1 gene deleted mice. It was confirmed that various M2 genes did not show a large difference in general, and thus the difference of M1 differentiation according to pelino-1 in adipose tissue was also verified through gene level in adipose tissue.

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (12)

A composition for diagnosing type 2 diabetes mellitus comprising a reagent for detecting pelino-1 marker.
The composition for diagnosing type 2 diabetes according to claim 1, wherein the specimen is at least one of adipose tissue, whole blood, serum, plasma, or whole blood.
3. The composition for diagnosing type 2 diabetes according to claim 1 or 2, wherein the detection reagent is a reagent capable of detecting the marker at a protein or nucleic acid level.
4. The method of claim 3, wherein the reagent detectable at the protein level is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a substrate, an aptamer, a receptor, a ligand or cofactor, A composition for diagnosing diabetes.
4. The composition for diagnosing type 2 diabetes according to claim 3, wherein the reagent which can be detected at the nucleic acid level is a reagent used for reverse transcription polymerase chain reaction, real-time RT-PCR, RNase protection assay, DNA chip or Northern blot.
Detecting the presence or amount of the pelino-1 marker from the sample; And
And a step of correlating the presence or the amount of the detected marker with the diagnosis or prognosis of the type 2 diabetes of the subject to be examined. In order to provide information necessary for diagnosis or prognosis of type 2 diabetes, A method for detecting a marker.
7. The method of claim 6, wherein the presence or amount of the marker is determined at the protein or nucleic acid level.
[8] The method of claim 7, wherein the associating step further comprises using non-marker clinical information including blood glucose, insulin concentration, or ratio of total macrophages to M1 macrophages in adipose tissue of the subject to be tested for type 2 diabetes In method.
7. The method of claim 6, wherein the associating step is to compare the detected amount or presence of the determined marker with the detection result for each marker determined in the normal control group to diagnose the second diabetes if it is decreased.
Providing a pelino-1 protein or gene;
Contacting the protein with a test substance; And
A screening method for a substance for treating type 2 diabetes mellitus comprising the step of comparing the expression of pelino-1 in a control group not in contact with the protein contacted with the test substance to select a substance that reduces the expression as a candidate substance .
11. The method of claim 10, wherein the pelino-1 protein or gene is provided in the form of a cell or an animal model expressing the pelino-1 protein or gene. 11. The method of claim 10, wherein the candidate agent inhibits the differentiation of M1 macrophages.

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