WO2020091237A1 - Biomarker for diagnosing obesity and use thereof - Google Patents

Abstract

Provided are: a composition and a kit for diagnosing obesity by using Ces1b and Cgref1, which are blood biomarkers; and a method for diagnosing obesity by using same. According to the present invention, obesity can be diagnosed at an early stage with high accuracy and sensitivity through a simple method by using blood, and information for preventing obesity-related metabolic diseases can be provided.

Description

Biomarker for obesity diagnosis and its use

A composition, a kit for diagnosing obesity, and a method for diagnosing obesity using the same.

Obesity is an important cause of metabolic diseases, and the incidence rate is increasing very much with the development of modern society. Obesity is caused by multiple causes, including genetic, cultural, and environmental factors. In particular, in modern times, the prevalence of obesity due to excessive calorie intake or high fat diet is increasing.

Obesity is very closely related to the development of various adult diseases, and as the obesity level increases, the prevalence of various diseases including diabetes, diabetes complications, cholelithiasis, hypertension, heart disease and stroke increases. Socially, the cost of treatment for the obese population is increasing, and the social overhead for these diseases is also astronomically increasing. Therefore, the initial diagnosis of obesity is essential.

Obesity can be diagnosed by various measures such as body mass index (BMI), body fat index, and waist circumference. However, since these diagnostic methods are diagnostic methods that rely only on limited physical characteristics, it is difficult to accurately diagnose obesity using only these scales.

Therefore, it is necessary to select an obesity-specific biomarker that can be used for initial diagnosis of obesity, has a simple diagnosis method, and has excellent diagnosis accuracy, based on a biological mechanism causing obesity.

Provided is a composition for diagnosing obesity.

Provided is a kit for diagnosing obesity.

It provides a diagnostic method of obesity or a method of providing information to diagnose obesity.

One aspect provides a composition for the diagnosis of obesity comprising an agent that measures the expression level of a protein or fragment thereof selected from the group consisting of Ces1b (Carboxylic ester hydrolase) and Cgref1 (Cell growth regulator with EF-hand domain 1).

The Ces1b may also be referred to as Carboxylic ester hydrolase or Carboxylesterase (CES) 1B. The Ces1b is one of carboxyl esterase 1 (CES1). CES1 can also be called ACAT, CE-1, CEH, CES2, HMSE, HMSE1, PCE-1, REH, SES1, TGH, or hCE-1. Ces1b can function as a serine esterase. The Ces1b is Uniprot No. It may include an amino acid sequence represented by P23141. The Ces1b is Uniprot No. Amino acid sequence represented by D3Z5G7 may be included.

The Cgref1 (Cell growth regulator with EF-hand domain 1) may also be referred to as Cell Growth Regulatory Gene 11 Protein (CGR11) or Hydrophobestin (Hydrophobestin). Cgref1 can function to mediate intercellular adhesion in a calcium-dependent manner. The Cgref1 is Uniprot No. Amino acid sequence represented by Q99674 may be included. The Cgref1 is Uniprot No. Amino acid sequence represented by Q8R1U2 may be included.

The fragment may be an immunogenic polypeptide as part of the protein.

The term "expression level" means the amount of protein or the amount of transcript. The expression level may be a relative proportion of protein or transcript. For example, an increase in the expression level may be an increase in the amount of protein or transcript compared to the negative control.

The agent may be an antibody or antigen-binding fragment thereof that specifically binds the protein or fragment thereof. The antibody can be a polyclonal antibody or a monoclonal antibody. The term "antibody" can be used interchangeably with the term "immunoglobulin". The antibody can be a polyclonal antibody or a monoclonal antibody. The antibody may be a full-length antibody. The antigen-binding fragment refers to a polypeptide comprising an antigen-binding site. The antigen-binding fragment may be a single-domain antibody, Fab, Fab ', or scFv. The antibody or antigen-binding fragment may be attached to a solid support. The solid support is, for example, a surface of a metal chip, plate, or well.

The agent may be a nucleic acid comprising a polynucleotide identical to or complementary to the polynucleotide encoding the protein or fragment thereof. The nucleic acid may be a primer or a probe. The primer or probe may be labeled with a fluorescent material, chemiluminescent or radioactive isotope on its terminal or inside.

The composition is haptoglobin (Haptoglobin: Hp), apolipoprotein C-II (Apolipoprotein C-II: Apoc2), apolipoprotein A-IV (Apolipoprotein A-IV: Apoa4), mannose-binding protein C (Mannose- binding protein C: Mbl2), angiotensin (Agt), apolipoprotein C-IV (Apopo4), leucine rich alpha 2 glycoprotein (Lrg1), and pigment epithelium-derived It may further include an agent for measuring the expression level of a protein or fragment thereof selected from the group consisting of Pigment epithelium-derived factor (PEDF).

The haptoglobin (Hp) may also be called BP, HP2ALPHA2, or HPA1S. Haptoglobin binds to free hemoglobin released from red blood cells in plasma with high affinity, and may function to inhibit oxidative activity of hemoglobin. The haptoglobin-hemoglobin complex can be removed by the reticuloendothelial system (mainly the spleen). Haptoglobin may be composed of two alpha chains and two beta chains linked by disulfide bonds. The haptoglobin is Uniprot No. It may include an amino acid sequence represented by P00738. The haptoglobin is Uniprot No. Amino acid sequence represented by Q61646 may be included.

The apolipoprotein C-II (Apolipoprotein C-II: Apoc2) is secreted into plasma and may be one of components of very low density lipoprotein (VLDL) and chylomicron. Apoc2 can activate lipoprotein lipase in capillaries and hydrolyze triglycerides to provide free fatty acids to cells. The Apoc2 is Uniprot No. Amino acid sequence represented by P02655 may be included. The Apoc2 is Uniprot No. Amino acid sequence represented by Q05020 may be included.

The apolipoprotein A-IV (Apolipoprotein A-IV: Apoa4) may be secreted by circulating on the surface of the chylomicron particle. Apoa4 can activate lecithin-cholesterol acyltransferase and cholesterylester transfer proteins. The Apoa4 is Uniprot No. Amino acid sequence represented by P06727 may be included. The Apoa4 is Uniprot No. Amino acid sequence represented by P06728 may be included.

The mannose-binding protein C (MBP-C or Mbl2) may also be referred to as Rhodo Colectin-1, or Mannan-binding lectin 1: MBP1. Mbl2 is a calcium-dependent lectin involved in innate immune response. Mbl2 can bind mannose, fucose and N-acetylglucosamine to microorganisms, activate the lectin complement pathway, and bind late apoptotic cells to promote predation by macrophages. The Mbl2 is Uniprot No. It may include an amino acid sequence represented by P11226. The Mbl2 is Uniprot No. Amino acid sequence represented by P41317 may be included.

The angiotensin (Agt) may also be called ANHU, SERPINA8, angiotensinogen, or hFLT1. Angiotensin is a peptide hormone that causes vasoconstriction and increased blood pressure. The angiotensin is Uniprot No. It may include an amino acid sequence represented by P01019. The angiotensin is Uniprot No. Amino acid sequence represented by P11859 or Q3UTR7.

The apolipoprotein C-IV (apoc4) is one of the apolipoprotein C family. Apoc4 can function in the metabolism of lipoproteins. The Apoc4 is Uniprot No. Amino acid sequence represented by P55056 may be included. The Apoc4 is Uniprot No. Amino acid sequence represented by Q61268 may be included.

The leucine rich alpha 2 glycoprotein (Lrg1) may also be referred to as HMFT1766. Lrg1 can be involved in protein-to-protein interactions, signal transduction, cell adhesion and development. Lrg1 may be involved in promoting angiogenesis by switching in the TGF beta signaling pathway in endothelial cells. The Lrg1 is Uniprot No. It may include an amino acid sequence represented by P02750. The Lrg1 is Uniprot No. Amino acid sequence represented by Q91XL1 may be included.

The pigment epithelium-derived factor (PEDF) may also be called serpin F1 (SERPINF1), EPC-1, OI12, OI6, PEDF, PIG35, or Serpin family F member 1. PEDF is a multifunctional secreted protein with anti-angiogenic, anti-tumor-forming and neurotrophic functions. The PEDF is Uniprot No. It may include an amino acid sequence represented by P36955. The PEDF is Uniprot No. Amino acid sequence represented by P97298 may be included.

The term "obesity (obesity)" refers to the state of excessive accumulation of fat tissue in the body. Obesity may be due to various factors such as eating habits such as overeating or high fat diet, genetic factors, other diseases, infections, lack of exercise, or social factors.

The term "diagnosis" refers to the determination of a disease name and may include obesity disease, disease condition, stage, etiology, presence of complications, prognosis, and relapse.

Another aspect provides a kit for diagnosing obesity comprising an agent that measures the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1.

The Ces1b, Cgref1, fragment, expression level, agent, obesity, and diagnosis are as described above.

The kit may further include a sample necessary for the diagnosis of obesity. The kit may include a substrate, a suitable buffer solution, a chromogenic enzyme, a fluorescently labeled secondary antibody, or a chromogenic substrate for immunological detection of a solid support, antibody or antigen-binding fragment. The kit may include a polymerase, a buffer, a nucleic acid, a coenzyme, a fluorescent substance, or a combination thereof for nucleic acid detection. The polymerase is, for example, Taq polymerase.

Another aspect is measuring the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1 in a biological sample isolated from a subject suspected of being obese; And comparing the measured expression level with the protein expression level of a normal control.

The Ces1b, Cgref1, fragment, expression level, agent, obesity, and diagnosis are as described above.

The method comprises measuring the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1 in a biological sample isolated from a subject suspected of being obese.

The subject can be a mammal, eg, a human, dog, cat, mouse, rat, rabbit, horse, sheep, cow, goat, or pig. The individual may be obese or suspected of being obese.

The biological sample refers to a sample obtained from the individual. The biological sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, papillary aspirate, lymph fluid, airway fluid, intestinal fluid, urogenital fluid, breast milk, lymphatic fluid, semen, cerebrospinal fluid, intratracheal Body fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof.

The measuring step may include incubating the biological sample and an antibody or antigen-binding fragment thereof that specifically binds a protein or a fragment thereof selected from the group consisting of Ces1b and Cgref1.

The measuring steps include electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), immunohistochemical staining, protein chip, immunoprecipitation, microarray, northern blotting, polymerase amplification reaction ( polymerase chain reaction (PCR), or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof. The PCR may be real-time PCR or reverse transcription PCR.

The method includes haptoglobin (Hp), apolipoprotein C-II (Apoc2), apolipoprotein A-IV (Apoa4), mannose-binding protein C (Mbl2), angiotensin (Agt), apolipolipid in the biological sample. The method may further include measuring the expression level of a protein or fragment thereof selected from the group consisting of protein C-IV (Apoc4), leucine rich alpha 2 glycoprotein (Lrg1), and pigment epithelial-derived factor (PEDF). The haptoglobin, Apoc2, Apoa4, Mbl2, angiotensin, Apoc4, Lrg1, and PEDF are as described above. Measuring the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1 in a biological sample, and selected from the group consisting of haptoglobin, Apoc2, Apoa4, Mbl2, angiotensin, Apoc4, Lrg1, and PEDF in the biological sample The step of measuring the expression level of the protein or fragment thereof can be performed individually, simultaneously, or sequentially.

The method includes comparing the measured expression level to the protein expression level of a normal control.

The term "normal control" can be used interchangeably with the term "negative control". The normal control group may be an obese individual or a healthy individual.

The method may further include determining that the subject is obese or has a high probability of being obese when the measured expression level of the protein or fragment thereof increases compared to the expression level measured in a normal control.

Another aspect is measuring the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1 in a biological sample isolated from a subject suspected of being obese; And it provides a method for providing information to diagnose obesity, comprising the step of comparing the measured expression level with the protein expression level of the normal control.

The method may provide information for preventing metabolic diseases associated with obesity, such as diabetes and diabetes complications, through the diagnosis of obesity.

According to the composition, kit, and method for diagnosing obesity using blood biomarkers Ces1b and Cgref1, it is possible to quickly diagnose obesity with high accuracy and sensitivity by using a simple method using blood, and to diagnose metabolic diseases associated with obesity. You can provide information to prevent it.

FIG. 1 is a graph analyzing proteins differentially expressed in the blood of the high-fat diet group and the normal diet group with a volcano plot (x-axis: log value of fold change, y-axis: p-value) -log value).

2 is a list of proteins significantly increased in the high-fat diet group compared to the normal diet group.

Figure 3 is a qRT-PCR graph confirming the expression level of Cgref1 in the abdominal fat tissue of the normal and high-fat diet group (***: p <0.001).

It will be described in more detail through the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Identification of obesity-related biomarkers

(1) Biomarker screening

C57BL / 6 male mice (8 weeks old, high fat diet: n = 3, normal diet: n = 3) were prepared and mice were fed a high fat diet (Rodent Diet with 60% Kcal Fat, Research Diet) or a normal diet (Rodent Diet with 10% Kcal Fat, Research Diet) for 16 weeks.

Serum protein was obtained from the prepared blood sample, and the obtained protein was digested with trypsin (Promega). The digested peptide sample was purified using a C ₁₈ column (Thermo Fisher Scientific Inc.). The purified peptide was processed using a mass spectrometer (Thermo Fisher Scientific Inc.) to calculate raw data according to the mass of the peptide. Using raw data, the proteins differentially expressed in the blood samples of the high-fat diet group and the blood samples of the normal diet group were analyzed by volcano plot. The results of the volcano floating analysis are shown in FIG. 1.

In the results of the volcano floating analysis, the protein that was significantly increased in the high fat diet group was screened compared to the normal diet group. Proteins with pV-value <0.05 and fold change> 1.2 were selected and the list is shown in FIG. 2.

As shown in Figure 2, Haptoglobin (Haptoglobin) was increased by about 12-fold in the high-fat diet group compared to the normal diet group, Apolipoprotein C-II is about 2.4 times, apolipoprotein A-IV Increased by about 2.1 times. In addition, Ces1b increased about 1.84-fold in the high-fat diet compared to the normal diet, and Cgref1 increased about 1.66-fold. Ces1b and Cgref1 are proteins that increase in a high fat diet that has not been previously reported, and it was confirmed that they can be used as a biomarker for obesity in blood.

(2) Validation of biomarkers

As described in Example 1 (1), mice (high fat diet: n = 5, normal diet: n = 5) were prepared and mice were fed a high fat diet or a normal diet for 16 weeks. Epididymal white adipose tissue (eWAT) of each mouse was obtained (5 tissues each).

Nucleic acid was isolated from the obtained tissue, and a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed using the following primer pairs.

Forward primer for Cgref1 amplification: 5'-ATAGAGTTCACGAAGCCGGAG-3 '(SEQ ID NO: 1)

Reverse primer for Cgref1 amplification: 5'-CTCGACCTAAAGGGAGCAGC-3 '(SEQ ID NO: 2)

Atpf1 was used as a reference gene for qRT-PCR. As a result of qRT-PCR, the relative expression level of Cgref1 relative to Atpf1 is shown in FIG. 3 (***: p <0.001).

As shown in Figure 3, Cgref1 was confirmed that the expression level increased by about 2.5 times in the high-fat diet group compared to the normal diet group.

Therefore, it was verified that Cgref1 is available as an obesity biomarker.

Claims (13)

A composition for the diagnosis of obesity comprising an agent for measuring the expression level of a protein or a fragment thereof selected from the group consisting of Ces1b (Carboxylic ester hydrolase) and Cgref1 (Cell growth regulator with EF-hand domain 1). The method according to claim 1, wherein the formulation The substance specifically binding to the protein or fragment thereof is an antibody, or an antigen-binding fragment thereof; or A composition comprising a polynucleotide that is identical to or complementary to a polynucleotide encoding the protein or fragment thereof. The composition according to claim 2, wherein the antibody is a polyclonal antibody or a monoclonal antibody. The composition according to claim 2, wherein the nucleic acid is a primer or a probe. The method according to claim 1, Haptoglobin (Haptoglobin: Hp), apolipoprotein C-II (Apolipoprotein C-II: Apoc2), apolipoprotein A-IV (Apolipoprotein A-IV: Apoa4), mannose-binding protein C ( Mannose-binding protein C: Mbl2), Angiotensin (Agt), Apolipoprotein C-IV: Apoc4, Leucine rich alpha 2 glycoprotein (Lrg1), and pigment epithelium -A composition further comprising an agent for measuring the expression level of a protein or fragment thereof selected from the group consisting of Pigment epithelium-derived factor (PEDF). A kit for diagnosing obesity comprising an agent that measures the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1. Measuring the expression level of a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1 in biological samples isolated from suspected obesity individuals; And Comprising the step of comparing the measured expression level with the protein expression level of the normal control, diagnostic method of obesity. The method of claim 7, wherein the individual is a human, dog, cat, mouse, rat, rabbit, horse, sheep, cow, goat, or pig. The method according to claim 7, wherein the biological sample is blood, plasma, serum, urine, mucus, saliva, tears, sputum, spinal fluid, pleural fluid, nipple suction, lymph fluid, airway fluid, intestinal fluid, genitourinary fluid, breast milk, lymphatic fluid, semen , Cerebrospinal fluid, intratracheal fluid, ascites, cystic tumor fluid, amniotic fluid, or a combination thereof. The method according to claim 7, wherein the step of measuring comprises incubating the biological sample with an antibody or antigen-binding fragment thereof that specifically binds a protein or fragment thereof selected from the group consisting of Ces1b and Cgref1. The method according to claim 7, wherein the measuring step is electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay: ELISA), immunohistochemical staining, protein chip, immunoprecipitation, microarray, Northern blotting, A method that is performed by polymerase chain reaction (PCR), or a combination thereof. The method according to claim 7, in the biological sample, haptoglobin (Hp), apolipoprotein C-II (Apoc2), apolipoprotein A-IV (Apoa4), mannose-binding protein C (Mbl2), angiotensin (Agt), Further comprising the step of measuring the expression level of a protein or fragment thereof selected from the group consisting of apolipoprotein C-IV (Apoc4), leucine rich alpha 2 glycoprotein (Lrg1), and pigment epithelial-derived factor (PEDF). How to be. The method of claim 7, further comprising determining that the individual is obese or has a high probability of being obese when the measured expression level of the protein or fragment thereof increases compared to the expression level measured in a normal control.

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