CN110187123B - A biomarker for early diagnosis of diabetes and its application - Google Patents

Abstract

The biomarker for early diagnosis of diabetes and the application thereof, which are created by the invention, aim to explore the dynamic change of proinsulin under the condition of sugar load and the significance of proinsulin in early recognition of pancreatic beta cell function reduction, namely, provide a clinical index for more simply and effectively showing the early reduction of beta cell function and provide an early basis for preventing the occurrence of diabetes.

Description

A biomarker for early diagnosis of diabetes and its application

technical field

The invention and creation belong to the field of biotechnology, in particular to a biomarker for early diagnosis of diabetes and its application.

Background technique

The latest epidemiological survey results show that the prevalence of diabetes in my country is becoming more and more serious, and the prevalence of diabetes in people over 18 years old is nearly 11%. Although peripheral insulin resistance caused by genetic and environmental factors is an important cause of type 2 diabetes, pancreatic β-cell failure is a determinant of the development of diabetes, and the genome-wide association study (GWAS) also showed that , more than 80% of the genes related to type 2 diabetes are related to β cells, which further proves the key role of pancreatic β cell dysfunction in the pathogenesis of diabetes. Studies have shown that when overt type 2 diabetes occurs, patients lose at least 30-60% islet beta cells, and the number of beta cells decreases progressively with the duration of diabetes. Therefore, in clinical work, timely and effective evaluation of islet β-cell function is particularly necessary.

In current clinical work, oral glucose tolerance test (OGTT) + insulin release test is commonly used to evaluate insulin resistance and β-cell function. Commonly used evaluation indicators include homeostasis model (HOMA-IR, HOMA-B, HOMA-IS), insulin production index (IGI), insulin sensitivity index (Matsuda index), disposition index (DI), area under the insulin curve (AUC _ins ) ) and the recently proposed β-cell function correction index (MBCI).

The HOMA index is one of the most commonly used evaluation indicators in epidemiological surveys, and was first used in the UKPDS (United Kingdom Prospective Diabetes Study) study. HOMA integrates the relationship between fasting blood glucose and insulin, trying to reflect the degree of pancreatic β-cell damage and the sensitivity of target organs to insulin. We believe that basal blood glucose levels are regulated by insulin-dependent endogenous glucose output, and plasma insulin concentrations are dependent on islet β-cell responses to blood glucose concentrations. The advantage of the HOMA index is that it is simple and fast to calculate, but because it only involves fasting blood glucose and insulin levels, it has limitations in reflecting the reserve function of pancreatic β cells, so its accuracy has gradually been questioned in recent years.

The disposition index DI is the product of the insulin sensitivity index (Matsuda Index) and the insulin production index (IGI), which reflects the responsiveness of plasma insulin to blood glucose changes and the overall insulin sensitivity, so it is a better indicator of pancreatic β-cell function. index. Sonia Caprio et al. divided 60 obese adolescents with normal glucose tolerance (NGT) into three subgroups according to PBG2h levels (PBG2h < 100 mg/dL, PBG2h 100-119 mg/dL, PBG2h 120-139 mg/dL), and compared them with 21 obese IGT adolescents compared DI (DI30 and DI120) obtained in the hyperinsulinemic-euglycemic clamp test and found no statistically significant difference in these measures among adolescents in the NGT 120-139 mg/dL group compared with the IGT group ( p>0.05). According to Markku Laakso's research, among 6414 Finns who underwent OGTT test, both DI30 and DI120 showed a trend of decreasing with the increase of PBG2h. Before reaching the diagnostic criteria of IGT, DI was lower than that of PBG2h<5.0mmol/L. BMI decreased by more than 30%, and this change was independent of BMI. It can be found that in the normally considered normal population, the function of pancreatic islet β cells has been reduced in some people, and the DI index can better evaluate this function decline. However, because the calculation of DI index is very cumbersome, its application in clinical practice has limitations. Therefore, this research group tried to explore a new clinical detection index, which can more simply and directly reflect the early decline of β-cell function.

Impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) are both in the early stage of diabetes with impaired glucose regulation (IGR), but their insulin resistance and insulin secretion characteristics Not exactly the same. The study by Hanefeld.M et al. suggested that IGT patients have both the early and late phases of insulin secretion relative deficiency, while in IFG patients, insulin resistance is relatively severe, and insulin deficiency is not obvious. Among the newly diagnosed diabetic patients in my country, only those with elevated postprandial blood glucose without abnormal fasting blood glucose account for 50% of all newly diagnosed DM patients. Therefore, this application selects IGT patients as our research subjects.

According to WHO's 1999 diabetes diagnostic criteria, the diagnostic criteria for IFG are 6.1mmol/L≤fasting blood glucose (FBG)<7.0mml/L, and PBG<7.8mmol/L. However, in the diabetes diagnostic criteria released by ADA in 2003, the FBG of the diagnostic criteria of IFG has been lowered from 6.1mmol/L to 5.6mmol/L, so that when predicting the future risk of diabetes, the diagnostic criteria of FBG and PBG can be predicted. The meaning is basically close. This revision resulted in an increase in the IFG population, and some patients previously diagnosed with IGT will become the IGT+IFG population. The ADA expert association established a receiver operating curve based on data from an epidemiological survey of multiple population samples and believed that the optimal fasting blood glucose standard for predicting future diabetes should be different in different populations: in the Netherlands It should be 5.7 mmol/L, 5.4 mmol/L among Pima Indians, and 5.2 mmol/L among San Antonio populations. The above diagnostic criteria for abnormal fasting blood glucose all make the sensitivity and specificity of diagnosis close to 100%. In an epidemiological survey of 5,023 Pima Indians, people with fasting blood glucose between 5.7-6.9 mmol/L had a four-fold higher risk of developing diabetes in the next five years than those with fasting blood glucose < 5.7 mmol/L . Yang Zhaojun et al reviewed the data of 15564 OGTT cases in the database of the National Diabetes Prevention and Control Group in 1994, and compared the specificity and sensitivity of the two diagnostic criteria of 6.1mmol/L and 5.6mmol/L for the diagnosis of IFG, using 5.6mmol/L. When L is the diagnostic standard of IFG, its sensitivity and specificity can reach 61.9% and 63.9%, respectively, and the diagnostic index of the receiver operating curve (ROC) is the highest at 5.6 mmol/L (diagnostic index: 0.258), so they believe that 5.6 mmol/L is an appropriate diagnostic criterion for abnormal fasting glucose to predict the risk of developing DM. Yu Yang et al. also conducted an OGTT survey of 16,341 residents in rural Shandong Province and explored the diagnostic criteria for impaired fasting blood sugar, and their conclusions were similar to those of Yang Zhaojun et al. In order to minimize the impact of fasting blood glucose variability on the research results, the diagnostic criteria for diabetes promulgated by the ADA in 2016 were used in this application to exclude and enroll the test population.

Proinsulin (proinsulin, PI) is the precursor of insulin. The synthesis of insulin in β cells needs to go through three stages: preproinsulin, proinsulin and insulin. Several clinical studies have found that patients with type 2 diabetes mellitus and glucose tolerance (NGT) have been compared with peripheral serum levels of fasting proinsulin and 2h postprandial proinsulin in patients with type 2 diabetes mellitus, impaired glucose tolerance (IGT) and normal glucose tolerance (NGT). Both fasting and 2-h post-glucose-load proinsulin levels were higher in impaired patients than in healthy controls, and inappropriate increases in the proinsulin to insulin ratio (PI/I) were seen in patients with type 2 diabetes. However, although it has been found that pancreatic β-cell function has been reduced to varying degrees in people with normal blood sugar levels, little research has been done on the changes of proinsulin in the normoglycemic range. Therefore, we asked the question: Can proinsulin detect the tendency of islet beta function impairment earlier than the current blood glucose and insulin tests? In this regard, in young and middle-aged subjects who had not been diagnosed with diabetes before, we analyzed the dynamic changes of serum PI after administration of glucose load, and compared the levels of peripheral blood PI secretion under different postprandial glucose levels and insulin levels. Peak time, in order to find the dynamic change curve of proinsulin in the OGTT test with the increase of PBG2h before reaching the existing IGT diagnostic criteria.

Zethelius等人通过对874名50岁以上的人群进行的长达27年的随访，认为PI是冠状动脉性心脏病发病的独立危险因素。David J.Schneider等人发现PI可能通过上调内皮细胞的纤溶酶原激活物抑制剂-1(Plasminogen activator inhibitor-1ofendothelial cells，PAI-1)的表达而增加血管病变发生的风险。PAI-1是一种丝氨酸蛋白酶，能够降低纤溶系统的活性，增加血栓形成的风险。此外，PI还与血压(blood pressure，BP)的升高相关。因此，研究PI的合成和分泌特点除了能够为内分泌疾病治疗提供新思路外，对心血管疾病以及糖尿病心血管并发症的预防和治疗也具有指导意义。According to WHO statistics, patients who die from atherosclerotic vascular disease (ASVD) each year account for 50-80% of the total deaths of diabetic patients. In the past, it was thought that PI would increase the risk of cardiovascular disease, but with the advancement of insulin kit detection technology, the effect of the cross-action between PI and insulin polyclonal antibodies has been greatly reduced. The predictive significance of PI and insulin on the risk of disease can already be distinguished. More and more scholars believe that elevated PI, but not insulin, may increase the risk of atherosclerotic vascular disease.

Zethelius et al identified PI as an independent risk factor for coronary heart disease by following up to 27 years in 874 people over the age of 50. David J.Schneider et al. found that PI may increase the risk of vascular lesions by up-regulating the expression of plasminogen activator inhibitor-1 (PAI-1) in endothelial cells. PAI-1 is a serine protease that reduces the activity of the fibrinolytic system and increases the risk of thrombosis. In addition, PI was also associated with an increase in blood pressure (BP). Therefore, studying the synthesis and secretion characteristics of PI can not only provide new ideas for the treatment of endocrine diseases, but also have guiding significance for the prevention and treatment of cardiovascular diseases and cardiovascular complications of diabetes.

As a precursor of insulin, the secretion of PI may also be affected by insulin levels. Therefore, in addition to exploring the relationship between the dynamic changes of PI and PBG2h, it is also necessary to clarify whether the changes of PI are related to hyperinsulinemia (HI). In the OGTT + insulin release test, the diagnostic criteria for HI have not yet been unified. The most commonly used diagnostic criteria for HI in epidemiological studies are: fasting insulin ≥15mU/L or 2h postprandial insulin ≥80mU/L. This diagnostic criterion is simple to calculate, and can reflect the abnormal increase in insulin levels after glucose load to a certain extent. However, this "two-point" division also misses some individuals with high insulin peaks and 2-h insulin levels that fall within the normal range. Sahin, N.M. et al. studied the OGTT curve characteristics of obese adolescents with normal HOMA-IR, combined the diagnostic criteria of peak insulin, and defined HI patients as those who met one of the following three points: 1) Fasting insulin ≥ 18mU/L, 2) Peak insulin ≥150mU/L, 3) Insulin ≥75mU/L 2h after glucose load. This grouping method refers to more indicators in the OGTT test, and is more in line with the classic description of insulin secretion characteristics in the textbooks of Internal Medicine and Physiology. To minimize the effect of hyperinsulinemia on PI levels, diagnostic criteria combined with peak insulin levels were used in our study to divide NGT subjects into normoinsulinemic (NI) and hyperinsulinemic (HI), and Similarities and differences in PI secretion characteristics between groups at similar PBG2h levels and similar insulin levels will be compared, respectively.

SUMMARY OF THE INVENTION

The purpose of the invention is to provide a biomarker for early diagnosis of diabetes and its application, and to explore the dynamic changes of proinsulin during sugar load and its significance in early identification of pancreatic islet beta cell function decline. It is simpler and more effective to show the clinical indicators of early reduction of β-cell function, and to provide early basis for preventing the occurrence of diabetes.

The invention provides a biomarker for early diagnosis of diabetes, the marker is peripheral proinsulin, and the secretion of the peripheral proinsulin is delayed with the decrease of the secretion function of pancreatic beta cells.

The present invention also discloses the application of the above-mentioned biomarkers for early diagnosis of diabetes, including applications in developing and screening diabetes functional products.

Wherein, the functional product contains an active ingredient targeting peripheral proinsulin.

Preferably, the functional product is a kit.

The present invention shows through a large number of experimental studies that peripheral proinsulin can be used as a new biological marker, and plays an early warning role in the early detection of β-cell function damage.

Description of drawings

Figure 1A shows the changes in blood glucose in NGT1, NGT2 and IGT groups, *: the difference is statistically significant;

Figure 1B shows the changes of insulin in NGT1 group, NGT2 group and IGT group, *: the difference is statistically significant;

Figure 1C shows the changes of proinsulin in NGT1 group, NGT2 group and IGT group, *: the difference is statistically significant;

Figure 2A shows the changes of blood glucose in the IGT group, NI-NGT1 group, HI-NGT1 group, NI-NGT2 group, and HI-NGT2 group, *: the difference is statistically significant;

Figure 2B shows the changes of insulin in the IGT group, NI-NGT1 group, HI-NGT1 group, NI-NGT2 group, and HI-NGT2 group, *: the difference is statistically significant;

Figure 2C shows the changes of proinsulin in IGT group, NI-NGT1 group, HI-NGT1 group, NI-NGT2 group and HI-NGT2 group, *: the difference is statistically significant;

Figure 3A shows the changes of HOMA-IR in NGT1 group, NGT2 group and IGT group, a: Compared with NGT1 group, the difference is statistically significant; b: Compared with NGT2 group, the difference is statistically significant; ab: Compared with NGT1 group , compared with NGT2 group, the difference was statistically significant;

Figure 3B shows the changes of Matsuda Index in NGT1 group, NGT2 group and IGT group, a: Compared with NGT1 group, the difference is statistically significant; b: Compared with NGT2 group, the difference is statistically significant; ab: Compared with NGT1 group, Compared with the NGT2 group, the difference was statistically significant;

Figure 3C shows the changes of DI in NGT1 group, NGT2 group and IGT group, a: Compared with NGT1 group, the difference is statistically significant; b: Compared with NGT2 group, the difference is statistically significant; ab: Compared with NGT1 group, NGT2 group Compared with the groups, the difference was statistically significant;

Figure 3D shows the changes of IGI in NGT1 group, NGT2 group and IGT group, a: Compared with NGT1 group, the difference is statistically significant; b: Compared with NGT2 group, the difference is statistically significant; ab: Compared with NGT1 group, NGT2 group Compared with the groups, the difference was statistically significant.

Detailed ways

The present invention will be further described below with reference to specific embodiments.

This application adopts the diagnostic criteria for diabetes promulgated by the ADA in 2016 for the exclusion and inclusion of the test population. From June 2015 to November 2017, 116 healthy volunteers (age 20) with fasting blood glucose lower than 5.6 mmol/L < (100 mg/dL) and 2h blood glucose after glucose load were lower than 11.1 mmol/L (200 mg/dL). -45 years, 32 men and 84 women) participated in this study. All subjects denied the history of diabetes, hyperthyroidism, hypothyroidism, hypopituitarism, Cushing's syndrome and other diseases affecting glucose metabolism, and denied new myocardial infarction and cerebrovascular infarction within the past 6 months and other major diseases, and deny that there has been a major stressful event in recent life. All kinds of oral hypoglycemic drugs, glucocorticoids, thyroid hormones, insulin and other drugs that affect glucose metabolism were not used within one month before OGTT. Moreover, FBG of all subjects was <5.6mmol/L, and 2h blood glucose (PBG2h) after glucose load was <11.1mmol/L.

In order to improve the accuracy of the research results, the following Example 1 and Example 2 respectively conduct a group study on subjects from two dimensions of PBG2h level and insulin level.

Example 1

In this example, subjects are divided into groups according to the level of PBG2h, the name of this group is: PBG2h group, and is specifically divided into three groups: NGT1, NGT2 and IGT.

The PBG2h grouping criteria are shown in Table 1.

Table 1. PBG2h grouping criteria

Note: Conversion of blood sugar unit: 1mmol/L=18mg/dL

The basic information of the three groups in the PBG2h group showed that the age of the IGT group was significantly higher than that of the NGT1 group (p<0.01); the BMI, abdominal circumference and family history of diabetes of the IGT and NGT2 patients were significantly higher than those of the NGT1 group (p<0.01); The blood pressure in the NGT2 group was significantly higher than that in the NGT1 group (p<0.01) (see Table 2). That is to say, the risk of developing diabetes increased in the NGT1, NGT2 and IGT groups sequentially. The dynamic change curves of blood glucose and insulin after glucose load in each group (see Table 3) showed that the insulin secretion curve was a unimodal curve, and the NGT1, NGT2 and IGT groups reached their peaks at 1h, 1h and 2h, respectively, indicating that the insulin in the IGT group Delayed secretion, that is, delayed insulin secretion, indicates a decrease in islet β-cell function, which is consistent with current clinical research and theoretical knowledge.

This example further analyzes and compares the dynamic change characteristics of proinsulin after glucose load in each group of subjects. The results show that the proinsulin secretion curve is a unimodal morphological curve, and the NGT1, NGT2 and IGT groups reach the peak at 1 h, 2 h and 2 h, respectively. peaks, indicating delayed secretion of proinsulin in the NGT2 and IGT groups. The time to peak insulin secretion was consistent in NGT1 and NGT2, while NGT2 experienced a delay in proinsulin secretion and thus delayed secretion of proinsulin could precede the delay in insulin secretion and identify a decline in beta cell function (see Figure 1A, Figure 1B, Figure 1C).

Table 2 Subject characteristics of PBG2h group

Data are mean ± SD, *, with NGT1 group as control, p<0.05 was statistically significant.

Table 3 Statistical table of blood glucose, insulin and proinsulin during OGTT of subjects in different groups

The data in the above table are mean ± standard deviation;

*, with NGT1 group as control, p<0.05 is statistically significant;

以NGT2组为对照，p<0.05具有统计学意义；

Taking the NGT2 group as the control, p<0.05 was statistically significant;

以NGT1组、NGT2组为对照，p<0.05具有统计学意义。

Taking NGT1 group and NGT2 group as control, p<0.05 was statistically significant.

Example 2

In order to avoid the influence of insulin on the results, in this example, the subjects combined with insulin levels were regrouped on the basis of the PBG2h group in Example 1. The name of this group is: insulin group, which is specifically divided into: NI group (normal insulin group ) and HI group (high insulin group), among which, NI group is divided into two subgroups, NI-NGT1 group and NI-NGT2 group; HI group is divided into HI-NGT1 group, HI-NGT2 group and IGT group. subgroup.

The criteria for HI grouping were fasting insulin ≥18mU/L and/or 2h insulin ≥75mU/L and/or peak insulin ≥150mU/L.

The results showed that the proinsulin secretion curve of the above five subgroups was a unimodal curve, and the curves of NI-NGT1 and HI-NGT1 peaked at 1 h, while NI-NGT2, HI-NGT2 and IGT peaked at 2 h (see 2A, 2B and 2C), which are consistent with the results of Example 1. At the same time, the area under the curve of proinsulin gradually increased between the groups: NI-NGT1 group < NI-NGT2 group < IGT group, HI-NGT1 group < HI-NGT2 group < IGT group, the differences were statistically significant (p < 0.01). It was shown that the size of the area under the proinsulin curve can also indicate beta cell function. And the peak proinsulin of NI-NGT1 was the lowest among the 5 subgroups.

From the comparison of Example 1 and Example 2, it can be concluded that the delayed secretion of proinsulin is related to the level of PBG2h, and is not related to the presence or absence of HI.

Example 3

In order to further confirm that the function of pancreatic islet β-cells decreased sequentially in NGT1, NGT2 and IGT groups, in this example, by calculating and comparing the evaluation indexes of islet β-cell function in each group, including insulin production index (IGI), insulin sensitivity index (Matsuda index), Insulin resistance index (HOMA-IR), and disposition index (DI), which represents islet beta cell function (see Table 4). The results showed that IGI, which represents insulin production, decreased sequentially in NGT1, NGT2, and IGT groups, indicating that insulin production in the three groups decreased sequentially (see Figure 3A). The Matsuda index of insulin sensitivity decreased in NGT1, NGT2, and IGT groups sequentially, indicating The insulin sensitivity of the three groups decreased in turn (p<0.01) (see Figure 3B); the insulin resistance index HOMA-IR increased in the NGT1, NGT2 and IGT groups in turn, indicating that the insulin resistance of the three groups increased in turn (p<0.01) ( See Figure 3C); the disposition index DI, which represents pancreatic β-cell function, decreased sequentially in NGT1, NGT2, and IGT groups, indicating that β-cell function decreased sequentially in the three groups. The above indicators for evaluating β-cell function all reflected that the function of β-cells in the three groups decreased in turn, that is, the risk of diabetes increased in turn (p<0.01) (see Figure 3D).

Table 4 Statistical table of different indices of pancreatic β-cell function

The data in the above table are mean ± interquartile range;

*, with NGT1 group as control, p<0.05 is statistically significant;

以NGT2组为对照，p<0.05具有统计学意义；

Taking the NGT2 group as the control, p<0.05 was statistically significant;

以NGT1组、NGT2组为对照，p<0.05具有统计学意义。

Taking NGT1 group and NGT2 group as control, p<0.05 was statistically significant.

The conclusions of the above examples are as follows: (1) Before reaching the diagnostic criteria of IGT, the function of pancreatic islet β-cells has a tendency to decrease with the increase of PBG2h. (2) The proinsulin secretion curve of the non-diabetic population after glucose load is a single peak curve. (3) After administration of glucose load, when 120mg/dL<PBG2h<140mg/dL, the peak time of proinsulin secretion has been delayed, and it appeared earlier than the peak time of insulin. (4) The delayed secretion of proinsulin was correlated with the level of PBG2h, but not with the presence of HI. (5) Compared with the insulin detection in the conventional OGTT, the delayed secretion of proinsulin after glucose load is an early indicator of the decreased secretion function of pancreatic β cells.

The calculation formulas in the prior art applied in the above embodiments are summarized as follows:

Insulin resistance index HOMA-IR=fasting insulin×fasting blood glucose/22.5, fasting blood glucose: mmol/L, fasting insulin: mU/L;

Insulin production index (IGI) = (insulin ₃₀ - insulin ₀ )/(blood glucose ₃₀ - blood glucose ₀ ), blood glucose: mg/dL, insulin: mU/L;

Insulin sensitivity index Matsuda Index=10000/(fasting blood glucose × fasting INS × average blood glucose of OGTT × average insulin of OGTT) square root, blood glucose: mg/dL, insulin: mU/L;

Disposition index (DI) = insulin production index × insulin sensitivity index;

Area under the blood sugar curve = (fasting blood sugar + blood sugar ₃₀ ) × 0.5/2 + (blood sugar ₃₀ + blood sugar ₆₀ ) × 0.5/2 + (blood sugar ₆₀ + blood sugar ₁₂₀ )/2 + (blood sugar ₁₂₀ + blood sugar ₁₈₀ )/2, blood sugar : mg/dL;

Area under the insulin curve=(fasting insulin+ _insulin30 ×0.5/2+( _insulin30 + _insulin60 )×0.5/2+( _insulin60 + _insulin120 )/2+( _insulin120 + _insulin180 )/2, insulin: mU/L;

Area under the curve of proinsulin=(fasting proinsulin+proinsulin ₆₀ )/2+(proinsulin 1h+proinsulin 2h)/2+(proinsulin 2h+proinsulin 3h)/2, proinsulin: pmol/L;

Blood sugar/insulin, blood sugar: mmol/L, insulin: mU/L;

Blood sugar/proinsulin, blood sugar: mmol/L, proinsulin: pmol/L (1pmol/L=9.4pg/mL);

Proinsulin/insulin, proinsulin: pmol/L (1pmol/L=9.4pg/mL), insulin: pmol/L (1pmol/L=1mU/L×6.965).

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the within the scope of protection of the present invention.

Claims (3)

1. An application of a biomarker for early diagnosis of diabetes for a population with fasting blood glucose less than 5.6mmol/L and normal glucose tolerance in preparing a product for early diagnosis of diabetes is characterized in that the biomarker is a serum marker for early pancreatic islet beta cell dysfunction for a population with fasting blood glucose less than 5.6mmol/L and normal glucose tolerance, wherein the marker is peripheral proinsulin, and the peripheral proinsulin is secreted in a delayed manner along with the reduction of the secretion function of the pancreatic islet beta cells.

2. The use according to claim 1, wherein the product contains an active principle targeting peripheral proinsulin.

3. Use according to claim 2, wherein the product is a kit.

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