KR101789373B1 - Methods and kits for predicting development of diabetes - Google Patents

Abstract

The present invention relates to a method for predicting onset of diabetes mellitus and a kit for its prediction, which comprises the step of measuring the expression level of Pref-1 protein.

Description

METHODS AND KITS FOR PREDICTING DEVELOPMENT OF DIABETES [0002]

The present invention relates to a method for predicting the onset of diabetes mellitus, and more particularly to a method for predicting the onset of diabetes mellitus and a kit for the prediction of the level of expression of Pref-1 protein.

Diabetes mellitus is one of the most common chronic diseases, and diabetes patients in Korea account for about 12.4% of all adults over 30 years old, at least 4 million. As in the case of developed countries, the number of diabetic patients is gradually increasing in Korea as the living standard is improved and the lifestyle is westernized.

Diabetes is largely divided into type 1 diabetes (insulin dependent type) in which the insulin-secreting cells are destroyed and insulin levels are absolutely insufficient, and type 2 diabetes (insulin-dependent type) in which insulin resistance occurs in organs in which insulin acts About 90% of diabetic patients have type 2 diabetes. The causes of diabetes are genetic factors, increased age, obesity, taking medication, pregnancy, hypertension, hyperlipemia, etc. Prevention should be prevented through regular diet exercise routine.

Previously, family history, overweight, and lifestyle were used to predict the onset of type 2 diabetes. Recently, however, studies on biomarkers or markers that can identify people at high risk for type 2 diabetes .

In one study, the use of three markers, such as leukocyte count and albumin concentration in blood, indicating the presence or absence of inflammation throughout the expression of E-selectin, which suggests vascular endothelial dysfunction, was used early in the diagnosis of type 2 diabetes mellitus (73% . In another study, 14 metabolites related to the onset of type 2 diabetes were identified through analysis of 163 metabolite concentrations per patient using blood samples. It is true.

DISCLOSURE Technical Problem The present invention has been conceived to solve the above-mentioned problems, and it has been confirmed that the expression level of Pref-1 protein is correlated with progress of diabetes mellitus, and the present invention has been completed based on this. It is an object of the present invention to provide a method for predicting onset of diabetes mellitus and a prediction kit for measuring the expression level of Pref-1 protein.

However, the technical problem to be solved by the present invention is not limited to the above, 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 method for predicting the onset of diabetes and a kit for the prediction of the level of expression of Pref-1 protein.

The present invention provides a kit for predicting the onset of diabetes, wherein the subject is diagnosed that the expression level of the Pref-1 protein is less than a normal value, thereby diagnosing that the onset of diabetes is expected.

In one embodiment of the present invention, the subject is male and the normal value is 8.09 ± 3.10 ng / mL.

In another embodiment of the present invention, the subject is female and the normal value is 10.02 + - 3.02 ng / mL.

The present invention also relates to a method for screening a sample comprising: separating serum from a subject; And measuring the expression level of the Pref-1 protein from the serum; A method for predicting the incidence of diabetes mellitus.

It was confirmed that the expression level of Pref-1 protein in the pre-diabetes stage affected the onset of diabetes in the future through the prediction method according to the present invention. In particular, the expression pattern of Pref-1 protein in the serum before the onset of type 2 diabetes in women can predict future onset of diabetes.

FIG. 1 shows the results of confirming the level of Pref-1 present in the serum before the onset of the disease to confirm the function of Pref-1 as a biomarker that can predict the risk of diabetes in the pre-diabetes stage. FIG. 1A shows the normal group (NGT), the pre-diabetic group (Prediabetes), and the diabetes-onset group (DM), and FIG.
Figure 2 shows the changes in fasting plasma glucose (FPG) and function of the beta cells (HOMA-beta) and the degree of Pref-1 expression Of the respondents.
Figure 3 shows the partial correlation coefficients (r) between the changes in fasting plasma glucose and beta cell function and the level of Pref-1 expression after adjusting for age and body mass index (BMI).
FIG. 4 shows the difference in the degree of diabetes mellitus among the quartiles classified according to the degree of Pref-1 expression (Q1; minimum expression of Pref-1, maximum expression of Pref-1). The difference is shown by correcting the age, BMI and fasting plasma glucose (Model 1), further correcting the fasting insulin, the maximum blood pressure and the neutral density (Model 2), and performing the split logistic regression analysis.

The present inventors confirmed that the degree of Pref-1 expression before the onset of diabetes affects the onset of diabetes mellitus. Based on this finding, the present invention has been completed.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for predicting the onset of diabetes, which measures the level of expression of Pref-1 protein.

Diabetes mellitus is a chronic disease that can not be expected to be effective for short-term treatment. It is impossible to maintain ideal blood glucose control status for a long period of time with only a variety of drugs currently in use. Therefore, it is urgent to develop a new treatment method which can fundamentally heal diabetes. There is a growing interest in predicting or diagnosing diabetes before the onset of diabetes.

Previously, family history, overweight, and lifestyle were used to predict the incidence of type 2 diabetes. Recently, however, researchers have also been working on biomarkers as markers for early detection of people at high risk for type 2 diabetes .

In recent studies, the use of E-selectin, the number of leukocyte cells in the blood and the albumin concentration to predict the onset of type 2 diabetes or the metabolism associated with the onset of type 2 diabetes has been reported, It is not enough.

The definition of diabetes and pre-diabetes was based on the American Diabetes Association criteria. The fasting blood glucose level of diabetes mellitus is 126 mg / dL or more, and the fasting blood glucose level of the pre-diabetic state is 100 to 125 mg / dL. Diabetes is more than 200 mg / dL in the case of blood sugar for 2 hours after administration, and 140-199 mg / dL in the pre-diabetic state.

Pref-1 is an epidermal growth-like protein (EGF) -like factor present in the extracellular domain of the epidermal growth factor-like motif, Cloned in the 3T3-L1 cDNA library through screening of the expressed gene.

Pref-1 is generally expressed extensively in the born tissue, but its expression is limited in postnatal and adult tissues, and in the pancreas, Pref-1 is highly expressed in neonatal pancreatic tissue and pregnant rat tissues. Pref-1, which is present in hepatocyte progenitor cells, is known to be an important marker for high proliferation of hepatoblast. Pref-1 expressed myotube in damaged muscle tissue is involved in muscle cell regeneration. In these various tissues, it has been shown that they regulate proliferation and regeneration in common, but the role of Pref-1 in pancreatic function and diabetes has not been elucidated yet.

Thus, the present inventors have found that the expression pattern of Pref-1 in the pre-onset stage of NGT, pre-diabetes-fasting glucose insufficiency / impaired glucose tolerance (Prediabetes-IFG / IGT) The present invention has been accomplished based on the above findings and demonstrates the efficacy of Pref-1 as a marker for predicting the onset of diabetes mellitus in the pre-diabetic stage.

In one embodiment of the present invention, the serum is separated from the subject and the expression level of Pref-1 protein is measured by ELISA to determine the relationship between the degree of Pref-1 expression and the onset of diabetes mellitus Respectively.

In the present invention, "measurement of protein expression level" is a process for confirming the presence and expression level of a protein expressed in a marker gene for diabetes in a biological sample in order to predict the onset of diabetes, To identify the amount of protein. Methods for analysis include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis But are not limited to, tissue immuno staining, immunoprecipitation assays, complement fixation assays, FACS and protein chips.

In the present invention, the term "kit" can be used to measure the level of Pref-1 protein that differs in expression in subjects in whom diabetes can progress and in normal subjects. The kit according to the present invention distinguishes whether or not a diabetic can be progressed, so that a medical practitioner such as a doctor can predict the progress of diabetes, monitors the reaction of the subject, and enables treatment according to the result .

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.

An embodiment of the present invention was carried out by the following conditions and methods.

object

The data were analyzed using data from the Chungju Metabolic Disease Cohort (CMC) study conducted in the Chungju area, and approval from the Catholic University Clinical Examination Committee and approval from all subjects . A baseline analysis was conducted from 2003 to 2006 for 11,718 people. A second survey was conducted from 2,024 to 2,624 people, and a third survey was conducted from 2011 to 2014. The prevalence of type 2 diabetes mellitus (n = 43) or prediabetes (n = 345) and age, prevalence of diabetes mellitus (n = 345) (NGT, n = 389), which matched the sex, fasting plasma glucose level, were further selected for further analysis.

Measurement of various numerical values

Basal blood tests were performed after fasting for 12 hours and performed at the Neodin medical institute (Seoul, Korea). The values of Fasting Plasma Glucose (FPG) were measured by the hexokinase method and the serum insulin levels were measured using a radioimmunoassay kit (Izotop, Budapest, Hungary). (HOMA-beta): 20 x fasting insulin (mlU / L) / [fasting blood glucose nmol / L) -3.5 (HOMA-beta) ].

Serum Pref -1 protein level measurement

Blood samples were taken before and after the onset of disease in each group and serum was obtained by centrifugation to quantify Pref-1 present in the serum. Serum levels of Pref-1 protein were measured using a Dlk1, soluble (human) ELISA kit (AdipoGen, San Diego, Calif.).

Statistical processing

Statistical analysis was performed using SAS 9.2 software (SAS Institute Inc., Cary, NC, USA). Values for each item were calculated as mean, standard deviation (mean +/- SD), median (median; 25th-75th percentiles), or proportions. The variables that did not show normal distribution (insulin, HOMA-β) were applied to post logarithmic analysis. The relationship between Pref-1 values and other variables was calculated by partial correlation analysis, adjusted for age and BMI. To test the function of Pref-1 as a predictor of diabetes mellitus, relative risk was measured by a bivariate logistic regression model using the Proc GenMod procedure. The relative risk and the lowest percentile (95% confidence interval) were analyzed by setting the upper third quartile as the criterion category. A P value <0.05 was considered statistically significant.

Example 1 . In each pre-diabetic group, Pref Comparison of numerical values of -1

As shown in Fig. 1, the value of Pref-1 was NGT in women, 10.02 + 3.02; prediabetes, 9.48 ± 3.35; diabetes, 8.66 ± 3.29 ng / mL; ( P = 0.0151), while in males it was 8.09 ± 3.10; prediabetes, 7.96 ± 3.03; diabetes, 7.80 + - 2.61 ng / mL; ( P = 0.6323), indicating no significant difference (see FIG. 1).

Example 2 . Fasting plasma glucose  And beta cell function changes Pref -1 Correlation Comparison

As shown in FIG. 2, the increase in the fasting plasma glucose level during the follow-up period (ΔFPG ≧ 0) showed a lower Pref-1 than the decrease in ΔFPG <0, It was enemy. On the other hand, subjects with increased HOMA beta (ΔHOMA-β≥0) showed relatively higher Pref-1 values than those with decreased subjects (ΔHOMA-β <0) and were significant in women.

In addition, the correlation between the change in function of the fasting plasma glucose and beta cells and the Pref-1 level after correction of age and BMI is shown in Fig. As shown in FIG. 3, the correlation between the degree of Pref-1 expression and the fasting plasma glucose level was significantly negative only in women (r = -0.1272). On the other hand, the correlation between the degree of Pref-1 expression and beta cell function was significantly correlated (r = 0.1580, r = 0.1241) in both males and females.

Example 3 . Pre-diabetic Pref -1 level and the degree of diabetes incidence

As shown in FIG. 4, the correlation between low Pref-1 expression and diabetic development was significant in females. In the first quartile (Q1), the correlation between the second and fourth quartiles (Q2-Q4) And about 3 times higher correlation.

As described above, it was confirmed that Pref-1 protein is a marker or biomarker predictive of the onset of diabetes before the onset of diabetes.

Claims (7)

(a) measuring a fasting plasma glucose (FPG) level and a change in a homeostasis model assessment of beta-cell function (HOMA-beta) of a biological sample separated from a patient before the diabetes stage;
(b) measuring the level of Pref-1 protein expression of the sample; And
(c) comparing the correlation between the values measured in steps (a) and (b); / RTI &gt; and a method for predicting the onset of diabetes.
The method according to claim 1,
When the amount of fasting plasma glucose (FPG) changes in the sample separated from a female patient is increased,
Wherein the amount of Pref-1 protein expressed in the sample is 5.82 to 12.36 ng / mL
Methods for predicting the onset of diabetes.
The method according to claim 1,
When the amount of fasting plasma glucose (FPG) changes in the sample separated from a male patient is increased,
The Pref-1 protein expression amount of the sample is 4.75 to 10.25 ng / mL
Methods for predicting the onset of diabetes.
The method according to claim 1,
When the amount of change in the beta cell function value of the sample separated from the female patient is decreased,
And the Pref-1 protein expression amount of the sample is 5.93 to 12.55 ng / mL.
Methods for predicting the onset of diabetes.
The method according to claim 1,
When the amount of beta cell function change of the sample isolated from a male patient is decreased,
And the amount of Pref-1 protein expressed in the sample is 4.72 to 11.18 ng / mL.
Methods for predicting the onset of diabetes.
The method according to claim 1,
Characterized in that the diabetes is type 2 diabetes in women
Methods for predicting the onset of diabetes.
The method according to claim 1,
The Pref-1 protein expression level measurement is performed using any one of Western blotting, ELISA, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, complement fixation, FACS or protein chip methods
Methods for predicting the onset of diabetes.

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