KR20240012623A - Use of biomarker ENPP7 for diagnosis and treatment of Diabetes - Google Patents
Use of biomarker ENPP7 for diagnosis and treatment of Diabetes Download PDFInfo
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- KR20240012623A KR20240012623A KR1020220088696A KR20220088696A KR20240012623A KR 20240012623 A KR20240012623 A KR 20240012623A KR 1020220088696 A KR1020220088696 A KR 1020220088696A KR 20220088696 A KR20220088696 A KR 20220088696A KR 20240012623 A KR20240012623 A KR 20240012623A
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- enpp7
- diabetes
- protein
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Abstract
본 발명은 바이오마커 ENPP7의 제2형 당뇨병의 진단 및 치료 용도에 관한 것으로, 당뇨 환자에서 ENPP7의 발현량이 감소함을 확인하였고, ENPP7 펩타이드 투여시 혈당 수준이 상승함을 확인한 바, ENPP7는 당뇨병을 진단, 예방, 개선 또는 치료에 유용하게 사용될 수 있다.The present invention relates to the use of the biomarker ENPP7 for diagnosis and treatment of type 2 diabetes. It was confirmed that the expression level of ENPP7 was decreased in diabetic patients, and the blood sugar level increased when ENPP7 peptide was administered. ENPP7 prevents diabetes. It can be useful for diagnosis, prevention, improvement or treatment.
Description
본 발명은 바이오마커 ENPP7의 당뇨병의 진단 및 치료 용도에 관한 것이다.The present invention relates to the use of the biomarker ENPP7 in the diagnosis and treatment of diabetes.
당뇨병 (diabetes mellitus)은 인슐린 (insulin) 분비 또는 인슐린 작용의 결함에 의해 나타나는 고혈당을 특징으로 하는 대사성 질환으로 인슐린이 부족하거나 표적세포에서 인슐린의 생물학적 효과가 감소함으로써 발생하게 되어 장기간 지속될 경우 신부전과 같은 합병증을 동반하는 만성 퇴행성 질환이다.Diabetes mellitus is a metabolic disease characterized by hyperglycemia caused by defects in insulin secretion or insulin action. It is caused by a lack of insulin or a decrease in the biological effect of insulin on target cells. If it persists for a long time, it can lead to kidney failure. It is a chronic degenerative disease accompanied by complications.
현재 급속한 경제발전에 따른 식생활의 변화로 당뇨병 유병률은 해마다 증가하여 국내의 경우 약 5 - 10%에 달하고 있으며, 2005년 통계 기준으로 당뇨병 환자는 400만 명을 넘어서 인구 100명 중 83명이며, 2025년에는 약 680만 명으로 늘어날 것으로 추산된다. 당뇨병은 전 세계적으로 3번째로 심각한 질병이며 우리나라에서도 사망원인 4위의 질병이고 다양한 합병증으로 인해 수명이 5 - 10년 정도 단축되게 된다.Currently, due to changes in dietary habits due to rapid economic development, the prevalence of diabetes is increasing every year, reaching approximately 5-10% in Korea. As of 2005 statistics, the number of diabetes patients exceeds 4 million, or 83 out of 100 people by 2025. It is estimated that the number will increase to about 6.8 million by 2018. Diabetes is the third most serious disease worldwide and the fourth leading cause of death in Korea. It shortens lifespan by 5 to 10 years due to various complications.
이러한 급격한 유병률의 증가는 영양 상태의 개선과 운동의 부족 등 환경적 요인의 변화가 가장 주된 원인인 것으로 추정된다. 우리나라의 경우, 인슐린 비의존형의 제2형 당뇨병 환자가 전체 당뇨병 환자의 90 - 95%를 차지하고 있으며, 선진국뿐만 아니라 개발 도상국의 사람들도 점차 신체 활동은 줄고 비만은 늘어나면서, 제2형 당뇨병의 발생이 무서운 속도로 증가하고 있다. It is presumed that this rapid increase in prevalence is mainly due to changes in environmental factors such as improvement in nutritional status and lack of exercise. In Korea, patients with non-insulin-dependent type 2 diabetes account for 90-95% of all diabetes patients, and as physical activity gradually decreases and obesity increases in people not only in developed countries but also in developing countries, the occurrence of type 2 diabetes is increasing. It is increasing at an alarming rate.
당뇨병은 크게 두 가지 유형으로 구분된다. 제1형 당뇨병 (type 1 diabetes mellitus, T1DM)은 혈액 내의 포도당 조절 호르몬인 인슐린의 분비 결핍으로 야기되며, 주로 10 - 20대의 젊은 연령층에서 발병되기 때문에 소아 당뇨병(juvenile diabetes)이라 불리기도 한다. 제2형 당뇨병 (type 2 diabetes mellitus, T2DM)은 주로 40대 이후에 발병되며, 우리나라 당뇨병 환자의 대부분을 차지한다. 제2형 당뇨병은 제1형 당뇨병과는 달리 성인형 당뇨병이라 불리며 발병 원인은 아직 명확히 밝혀져 있지 않으나, 유전적인 요인과 나이, 가족력, 과체중, 고혈압, 콜레스테롤 등의 환경적 요소가 함께 관여되어 발생하는 것으로 알려져 있다. 특히, 제2형 당뇨병은 특히 운동 부족, 비만 또는 스트레스 등에 의한 후천적 요인으로 인슐린의 분비 조절은 원활하나 인슐린이 제 기능을 하지 못하여 혈당 조절이 실패하는 경우 발생한다.Diabetes is largely divided into two types. Type 1 diabetes mellitus (T1DM) is caused by insufficient secretion of insulin, a hormone that regulates glucose in the blood, and is also called juvenile diabetes because it mainly occurs in young people in their teens and twenties. Type 2 diabetes mellitus (T2DM) mainly develops after the age of 40 and accounts for the majority of diabetes patients in Korea. Type 2 diabetes, unlike type 1 diabetes, is called adult-onset diabetes. The cause of its development is not yet clear, but it is caused by a combination of genetic factors, age, family history, overweight, and environmental factors such as high blood pressure and cholesterol. It is known that In particular, type 2 diabetes occurs when insulin secretion control is smooth due to acquired factors such as lack of exercise, obesity, or stress, but insulin does not function properly and blood sugar control fails.
기존의 당뇨병 진단에는 혈당 수치나 당화 혈색소의 수치로만 진단이 되지만, 이는 당뇨의 진행이 충분히 진행되고 나서의 임상학적 징후이고, 하나의 혈액학적 수치로는 진단이 정확하다고 볼 수 없다. 또한, 상기 진단 방법은 아직까지는 제2형 당뇨병의 치료를 위한 약물 표적 (target)을 제시하지 못하는 한계가 있고, 치료를 위해서는 식생활 습관 개선 및 기존 당뇨치료제를 처방받는 실정이다. Conventional diabetes is diagnosed only based on blood sugar levels or glycated hemoglobin levels, but these are clinical signs after diabetes has progressed sufficiently, and the diagnosis cannot be considered accurate based on a single hematological value. In addition, the above diagnostic method still has limitations in not being able to provide a drug target for the treatment of type 2 diabetes, and treatment requires improving eating habits and prescribing existing diabetes treatments.
ENPP7 (Ectonucleotide pyrophosphatase/phosphodiesterase family member 7, ENPP7)은 인간에서 ENPP7 유전자에 의해 인코딩되는 효소이고, 알칼리성 스핑고미엘린 포스포디에스테라제 (alkaline sphingomyelin phosphodiesterase, Alk-SMase) 또는 장내 알칼리성 스핑고미엘리나제 (intestinal alkaline sphingomyelinase)이며, ENPP 중에서 스핑고미엘린을 가수분해하는 유일한 ENPP로 알려져 있다.ENPP7 (Ectonucleotide pyrophosphatase/phosphodiesterase family member 7, ENPP7) is an enzyme encoded by the ENPP7 gene in humans and acts as alkaline sphingomyelin phosphodiesterase (Alk-SMase) or intestinal alkaline sphingomyelinase ( intestinal alkaline sphingomyelinase), and is known to be the only ENPP among ENPPs that hydrolyzes sphingomyelin.
종래의 선행기술로서 일본 등록특허 제5297799호에는 게놈 DNA 또는 RNA를 포함한 피험자의 생체 샘플로부터 ENPP1 유전자 하프로타입 (haplotype)을 이용하여 피험자가 비만 또는 2형 당뇨병을 발병하는 리스크가 증가하고 있는지를 판정하는 방법을 개시하고 있다. 또한, Brian D. Piccolo 등에 따르면 당뇨 진행에 따른 ENPP7의 전사 변화를 나타낸다는 것을 개시하고 있으나, ENPP7의 제2형 당뇨병의 진단 및 치료 용도에 대해서 개시하고 있지 않다 (Physiological Reports. 2021;9:e15102). 또한, Roderick C Slieker 등에 따르면, NogoR (RTN4R)가 고지방식이 마우스에서 포도당 제거 및 인슐린 감도를 향상시키는 효과를 개시하고 있다 (medRxiv 2021.04.22.21255625). 그러나, 상기 연구에서 제2형 당뇨병에 대해 실험적으로 검증하여 제시한 바이오마커는 NogoR (구조정보:PDBe-KBProteinPages (ebi.ac.uk))이며, 이는 본 발명의 ENPP7 (구조정보:PDBe-KBProteinPages(ebi.ac.uk))과 구조적으로 차이가 있다. 또한, 기능적인 측면에서 NogoR은 막 단백질로 신경돌기 성장 억제제 (Nogo)에 결합할 때, Rho kinase의 활성화를 통해 세포 성장을 억제하는 역할을 한다고 알려져 있지만, ENPP7은 스핑고미엘린 (sphingomyelin)을 세라마이드(ceramide)로 전환을 하는 기능을 한다고 알려져 있다. 따라서, 두 바이오마커는 제2형 당뇨병 관련 메커니즘에서 상이한 역할을 수행한다. As a conventional prior art, Japanese Patent No. 5297799 uses the ENPP1 gene haplotype from a subject's biological sample containing genomic DNA or RNA to determine whether the subject's risk of developing obesity or type 2 diabetes is increasing. A method for determining is disclosed. In addition, according to Brian D. Piccolo et al., it is disclosed that transcriptional changes in ENPP7 occur as diabetes progresses, but the use of ENPP7 for diagnosis and treatment of type 2 diabetes is not disclosed (Physiological Reports. 2021;9:e15102 ). In addition, according to Roderick C Slieker et al., NogoR (RTN4R) is disclosing the effect of improving glucose clearance and insulin sensitivity in high-fat diet mice (medRxiv 2021.04.22.21255625). However, the biomarker experimentally verified and presented for type 2 diabetes in the above study is NogoR (structural information: PDBe-KBProteinPages (ebi.ac.uk)), which is ENPP7 (structural information: PDBe-KBProteinPages) of the present invention. (ebi.ac.uk)) is structurally different. In addition, from a functional perspective, NogoR is a membrane protein and is known to play a role in inhibiting cell growth through activation of Rho kinase when it binds to neurite growth inhibitor (Nogo), but ENPP7 binds sphingomyelin to ceramide. It is known to have the function of converting to ceramide. Therefore, the two biomarkers play different roles in type 2 diabetes-related mechanisms.
이에, 본 발명자들은 당뇨병 특히, 제2형 당뇨병 진단 및 치료를 할 수 있는 바이오마커를 발굴하기 위해 노력한 결과, ENPP7를 바이오마커 후보군으로 도출하였고, ENPP7는 정상군에 비해 당뇨병 환자군에서 발현이 현저하게 감소하는 것을 확인하여 ENPP7를 통해 당뇨병 환자를 진단할 수 있음을 확인하였고, 동물모델에서 ENPP7를 투여하여 혈당이 낮아짐을 확인하여 ENPP7가 당뇨병을 치료 또는 개선할 수 있음을 확인함으로써 본 발명을 완성하였다.Accordingly, the present inventors made efforts to discover a biomarker that can diagnose and treat diabetes, especially type 2 diabetes, and derived ENPP7 as a biomarker candidate, and ENPP7 was significantly expressed in the diabetic patient group compared to the normal group. By confirming the decrease, it was confirmed that diabetic patients can be diagnosed through ENPP7, and by confirming that blood sugar was lowered by administering ENPP7 in an animal model, it was confirmed that ENPP7 can treat or improve diabetes, thereby completing the present invention. .
본 발명의 목적은 당뇨병 진단용 바이오마커 조성물을 제공하는 것이다.The purpose of the present invention is to provide a biomarker composition for diagnosing diabetes.
본 발명의 다른 목적은 당뇨병 진단용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for diagnosing diabetes.
본 발명의 또 다른 목적은 당뇨병 진단용 키트를 제공하는 것이다.Another object of the present invention is to provide a kit for diagnosing diabetes.
본 발명의 또 다른 목적은 당뇨병 진단에 필요한 정보를 제공하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method of providing information necessary for diagnosing diabetes.
본 발명의 또 다른 목적은 당뇨병의 예방 및 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing and treating diabetes.
본 발명의 또 다른 목적은 당뇨병의 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다.Another object of the present invention is to provide a health functional food composition for preventing or improving diabetes.
본 발명의 또 다른 목적은 당뇨병 치료제 후보물질의 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening diabetes treatment candidates.
상기 목적을 달성하기 위하여, In order to achieve the above purpose,
본 발명은 ENPP7를 포함하는 당뇨병 진단용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for diagnosing diabetes including ENPP7.
또한, 본 발명은 ENPP7의 mRNA 또는 단백질로 코딩되는 단백질 수준을 측정하는 제제를 포함하는 당뇨병 진단용 조성물을 제공한다.Additionally, the present invention provides a composition for diagnosing diabetes, including an agent for measuring the level of protein encoded by ENPP7 mRNA or protein.
또한, 본 발명은 상기 조성물을 포함하는 당뇨병 진단용 키트를 제공한다.Additionally, the present invention provides a kit for diagnosing diabetes including the composition.
또한, 본 발명은 (a) 환자로부터 분리된 시료에서 ENPP7의 mRNA 또는 단백질 수준을 측정하는 단계; 및 (b) 상기 측정된 mRNA 또는 단백질 수준을 정상 개체의 mRNA 또는 단백질 수준과 비교하는 단계; 를 포함하는 당뇨병 진단에 필요한 정보를 제공하는 방법을 제공한다.In addition, the present invention includes the steps of (a) measuring the mRNA or protein level of ENPP7 in a sample isolated from a patient; and (b) comparing the measured mRNA or protein level with the mRNA or protein level of a normal subject; Provides a method of providing information necessary for diagnosing diabetes, including.
또한, 본 발명은 ENPP7 펩타이드를 유효성분으로 포함하는 당뇨병의 예방 및 치료용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing and treating diabetes containing ENPP7 peptide as an active ingredient.
또한, 본 발명은 ENPP7 펩타이드를 유효성분으로 포함하는 당뇨병의 예방 또는 개선용 건강기능식품 조성물을 제공한다.Additionally, the present invention provides a health functional food composition for preventing or improving diabetes containing ENPP7 peptide as an active ingredient.
또한 본 발명은 (1) 후보물질을 ENPP7를 발현하는 분리된 세포주에 처리하는 단계; (2) 단계 1)의 처리된 세포주에서 상기 ENPP7의 mRNA 또는 단백질의 발현량을 측정하는 단계; (3) 단계 2)의 ENPP7의 mRNA 또는 단백질의 발현량이 후보물질을 무처리한 대조군 세포주와 비교하여 증가된 후보물질을 선별하는 단계; 를 포함하는 당뇨병 치료제 후보물질의 스크리닝 방법을 제공한다.In addition, the present invention includes the steps of (1) treating a candidate material to an isolated cell line expressing ENPP7; (2) measuring the expression level of the mRNA or protein of ENPP7 in the cell line treated in step 1); (3) selecting a candidate material whose expression level of ENPP7 mRNA or protein in step 2) is increased compared to a control cell line that was not treated with the candidate material; Provides a method for screening diabetes treatment candidates including.
본 발명은 바이오마커 ENPP7의 제2형 당뇨병의 진단 및 치료 용도에 관한 것으로, 당뇨 환자에서 ENPP7의 발현량이 감소함을 확인하였고, ENPP7 펩타이드 투여시 혈당 수준이 상승함을 확인한 바, ENPP7는 당뇨병을 진단, 예방, 개선 또는 치료에 유용하게 사용될 수 있다.The present invention relates to the use of the biomarker ENPP7 for diagnosis and treatment of type 2 diabetes. It was confirmed that the expression level of ENPP7 was decreased in diabetic patients, and the blood sugar level increased when ENPP7 peptide was administered. ENPP7 prevents diabetes. It can be useful for diagnosis, prevention, improvement or treatment.
도 1은 공개 전사체 데이터를 통한 제2형 당뇨병 신호전달 관련 유전자 바이오마커 도출 개념도이다.
도 2a는 제2형 당뇨병 신호 경로와 ceramide 신호 경로에 공통적으로 포함되는 유전자를 선별한 결과를 나타낸 도이다.
도 2b는 당뇨 환자군의 혈액 및 간 조직에서 공통적으로 발현량이 증가하거나 감소하는 유전자 중 제2형 당뇨병 신호전달 및 세라마이드 신호전달에 관련있는 유전자를 선별한 도이다.
도 2c는 혈액 조직 데이터에서의 ENPP7의 유전자 발현 수준을 나타낸 도이다.
도 2d는 간 조직 데이터에서의 ENPP7의 유전자 발현 수준을 나타낸 도이다.
도 3은 당뇨 환자군에서의 혈중 ENPP7 농도를 효소면역측정법 (ELISA)으로 확인한 결과를 나타낸 도이다.
도 4a는 렙틴 호르몬 결함이 있는 비만 모델 마우스 (C57BL/6J ob/ob)의 체중 변화를 나타낸 도이다.
도 4b는 렙틴 호르몬 결함이 있는 비만 모델 마우스 (C57BL/6J ob/ob)의 식이 섭취량(food intake) 변화를 나타낸 도이다.
도 5a는 렙틴 호르몬 결함이 있는 비만 모델 마우스 (C57BL/6J ob/ob)을 대상으로 0, 3, 6일차에 ENPP7 펩타이드를 투여하고 혈당을 측정한 결과를 나타낸 도이다.
도 5b는 렙틴 호르몬 결함이 있는 비만 모델 마우스 (C57BL/6J ob/ob)을 대상으로 6시간 공복 후 0, 4, 7일차에 ENPP7 펩타이드를 투여하고 혈당을 측정한 결과를 나타낸 도이다.
도 6a는 렙틴 호르몬 결함이 있는 비만 모델 마우스 (C57BL/6J ob/ob)을 대상으로 6시간 공복 후 ENPP7 펩타이드를 투여하고 1g/kg의 포도당을 복강 내 투여한 후 0, 15, 30, 60 및 120 분에 혈당을 측정한 결과를 나타낸 도이다.
도 6b는 도 6a의 실험에 대한 혈중농도곡선하 면적을 분석한 도이다.
도 7a는 렙틴 호르몬 결함이 있는 비만 모델 마우스 (C57BL/6J ob/ob)을 대상으로 6시간 공복 후 ENPP7 펩타이드를 투여하고 1U/kg의 인슐린을 복강 내 투여한 후 0, 15, 30, 60 및 120 분에 평균 혈당 강하 수치 (%)를 분석한 결과를 나타낸 도이다.
도 7b는 도 7a의 실험에 대한 혈중농도곡선하 면적을 분석한 도이다.Figure 1 is a conceptual diagram of deriving gene biomarkers related to type 2 diabetes signaling through public transcriptome data.
Figure 2a is a diagram showing the results of screening genes commonly included in the type 2 diabetes signaling pathway and the ceramide signaling pathway.
Figure 2b is a diagram showing a selection of genes related to type 2 diabetes signaling and ceramide signaling among genes whose expression levels are commonly increased or decreased in the blood and liver tissue of diabetic patients.
Figure 2c is a diagram showing the gene expression level of ENPP7 in blood tissue data.
Figure 2d is a diagram showing the gene expression level of ENPP7 in liver tissue data.
Figure 3 is a diagram showing the results of confirming the blood ENPP7 concentration in the diabetic patient group using enzyme-linked immunosorbent assay (ELISA).
Figure 4a is a diagram showing body weight changes in an obesity model mouse (C57BL/6J ob/ob) with a defect in the leptin hormone.
Figure 4b is a diagram showing changes in food intake of an obesity model mouse (C57BL/6J ob/ob) with a defect in the leptin hormone.
Figure 5a shows the results of administering ENPP7 peptide to an obese model mouse (C57BL/6J ob/ob) with a defect in the leptin hormone on days 0, 3, and 6 and measuring blood sugar.
Figure 5b is a diagram showing the results of administering ENPP7 peptide to an obese model mouse (C57BL/6J ob/ob) with a defect in the leptin hormone on days 0, 4, and 7 after a 6-hour fast and measuring blood sugar.
Figure 6a shows 0, 15, 30, 60 and 60 after administrating ENPP7 peptide and intraperitoneally administering 1 g/kg of glucose to leptin hormone defective obesity model mice (C57BL/6J ob/ob) after fasting for 6 hours. This diagram shows the results of measuring blood sugar at 120 minutes.
Figure 6b is a diagram analyzing the area under the blood concentration curve for the experiment of Figure 6a.
Figure 7a shows 0, 15, 30, 60 and 60 after administrating ENPP7 peptide and intraperitoneally administering 1U/kg of insulin to leptin hormone defective obesity model mice (C57BL/6J ob/ob) after fasting for 6 hours. This figure shows the results of analyzing the average blood sugar drop value (%) at 120 minutes.
Figure 7b is a diagram analyzing the area under the blood concentration curve for the experiment of Figure 7a.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 ENPP7를 포함하는 당뇨병 진단용 바이오마커 조성물을 제공한다.The present invention provides a biomarker composition for diagnosing diabetes including ENPP7.
본 발명의 바람직한 일 실시예에 있어서, 상기 당뇨병은 제2형 당뇨병이다. In a preferred embodiment of the present invention, the diabetes is type 2 diabetes.
또한, 본 발명은 ENPP7의 mRNA 또는 단백질 수준을 측정하는 제제를 포함하는 당뇨병 진단용 조성물을 제공한다.Additionally, the present invention provides a composition for diagnosing diabetes, including an agent for measuring the mRNA or protein level of ENPP7.
상기 mRNA의 수준을 측정하는 제제는 프라이머 또는 프로브를 포함할 수 있으나 이에 특별히 제한되지는 않는다.The agent for measuring the level of mRNA may include, but is not particularly limited to, a primer or probe.
상기 프라이머는 짧은 자유 3 말단 수화기 (free 3' hydroxyl group)를 가지는 핵산 서열로 주형 (template)과 상보적인 염기쌍 (base pair)을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응 (즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시될 수 있다.The primer is a short nucleic acid sequence having a free 3' hydroxyl group that can form a complementary base pair with the template and serves as a starting point for copying the template strand. It means sequence. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and a reagent for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.
상기 프로브는 표적 유전자와 상보적으로 결합할 수 있는 프로브가 될 수 있고, 상기 각 유전자와 상보적으로 결합할 수 있는 한, 상기 프로브의 뉴클레오티드 서열은 제한되지 않는다.The probe may be a probe that can bind complementary to a target gene, and the nucleotide sequence of the probe is not limited as long as it can bind complementary to each gene.
상기 mRNA 발현 수준을 측정하는 방법은 RT-PCR (Reverse transcription polymerase chain reaction), 경쟁적 RT-PCR (Competitive RT-PCR), 실시간 RTPCR (Real-time RT-PCR), RNase 보호 분석법 (RPA; RNase protection assay), 노던 블랏팅 (Northernblotting) 및 DNA 칩을 이용하지만, 이에 한정되는 것은 아니다.Methods for measuring the mRNA expression level include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (Competitive RT-PCR), real-time RT-PCR, and RNase protection assay (RPA). assay), Northern blotting, and DNA chip are used, but are not limited to these.
상기 단백질의 수준을 측정하는 제제는 상기 단백질에 특이적인 항체, 펩타이드, 앱타머 또는 화합물일 수 있다. The agent that measures the level of the protein may be an antibody, peptide, aptamer, or compound specific for the protein.
상기 항체는 단백질 또는 펩티드 분자의 항원성 부위에 특이적으로 결합할 수 있는 단백질성 분자를 의미한다. 이러한 항체는, 각 유전자를 통상적인 방법에 따라 발현벡터에 클로닝하여 상기 마커 유전자에 의해 코딩되는 단백질을 얻고, 얻어진 단백질로부터 통상적인 방법에 의해 제조될 수 있다. 상기 항체의 형태는 특별히 제한되지 않으며 폴리클로날 항체, 모노클로날 항체 또는 항원 결합성을 갖는 것이면 그것의 일부도 본 발명의 항체에 포함되고 모든 면역 글로불린 항체가 포함될 수 있다. 뿐만 아니라, 인간화 항체 등의 특수 항체를 포함할 수도 있다. 아울러, 상기 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며 Fab, F(ab'), F(ab') 2 및 Fv 등이 될 수 있다.The antibody refers to a protein molecule that can specifically bind to the antigenic site of a protein or peptide molecule. These antibodies can be produced by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by the marker gene, and from the obtained protein by a conventional method. The form of the antibody is not particularly limited, and as long as it is a polyclonal antibody, a monoclonal antibody, or has antigen-binding properties, a portion thereof may be included in the antibody of the present invention, and all immunoglobulin antibodies may be included. In addition, it may contain special antibodies such as humanized antibodies. Additionally, the antibodies include intact forms with two full-length light chains and two full-length heavy chains as well as functional fragments of the antibody molecule. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function and may include Fab, F(ab'), F(ab') 2, and Fv.
상기 펩타이드는 표적 물질에 대한 결합력 높은 장점이 있으며, 열/화학 처리시에도 변성이 일어나지 않는다. 또한 분자 크기가 작기 때문에 다른 단백질에 붙여서 융합 단백질로의 이용이 가능하다. 구체적으로 고분자 단백질 체인에 붙여서 이용이 가능하므로 진단 키트 및 약물전달 물질로 이용될 수 있다.The peptide has the advantage of high binding capacity to the target substance and does not undergo denaturation even during heat/chemical treatment. Additionally, because the molecule size is small, it can be used as a fusion protein by attaching it to another protein. Specifically, it can be used by attaching it to a polymer protein chain, so it can be used as a diagnostic kit and drug delivery material.
상기 앱타머는 단일 가닥 올리고 뉴클레오티드를 의미하는 것으로, 소정의 표적 분자에 대한 결합 활성을 갖는 핵산 분자를 말한다. 상기 앱타머는 그 염기 서열에 따라 다양한 3차원 구조를 가질 수 있으며, 항원-항체 반응과 같이 특정 물질에 대하여 높은 친화력을 가질 수 있다. 앱타머는 소정의 표적 분자에 결합함으로써 소정의 표적 분자의 활성을 저해할 수 있다. 앱타머는 항체와 동일하게 항원성 물질에 특이적으로 결합할 수 있으면서도, 단백질보다 안정성이 높고, 구조가 간단하며, 합성이 용이한 폴리뉴클레오티드로 구성되어 있으므로, 항체를 대체하여 사용될 수 있다.The aptamer refers to a single-stranded oligonucleotide and refers to a nucleic acid molecule that has binding activity to a given target molecule. The aptamer may have various three-dimensional structures depending on its base sequence and may have high affinity for a specific substance, such as in an antigen-antibody reaction. An aptamer can inhibit the activity of a certain target molecule by binding to it. Aptamers can specifically bind to antigenic substances in the same way as antibodies, but are composed of polynucleotides that are more stable than proteins, have a simpler structure, and are easier to synthesize, so they can be used as replacements for antibodies.
상기 단백질 수준을 측정하는 방법은 웨스턴 블랏 (Western blot), ELISA (enzyme linked immunosorbent asay), 방사선면역분석 (Radioimmunoassay; RIA), 방사면역확산법 (radioimmunodiffusion), 오우크테로니 (Ouchterlony) 면역 확산법, 로케이트 (rocket) 면역전기영동, 조직면역염색, 면역침전 분석법 (Immunoprecipitation assay), 보체고정분석법 (Complement Fixation Assay), FACS 및 단백질 칩을 이용하지만, 이에 한정되는 것은 아니다.Methods for measuring the protein level include Western blot, ELISA (enzyme linked immunosorbent assay), radioimmunoassay (RIA), radioimmunodiffusion, and Ouchterlony immunodiffusion. Rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip are used, but are not limited to these.
또한, 본 발명은 상기 조성물을 포함하는 당뇨병 진단용 키트를 제공한다.Additionally, the present invention provides a kit for diagnosing diabetes including the composition.
상기 키트는 RT-PCR (Reverse transcription polymerase chain reaction) 트, DNA 칩 키트, ELISA (Enzymelinked immunosorbent assay) 트, 단백질 칩 키트, 래피드 (rapid) 키트 또는 MRM (Multiple reaction monitoring) 키트를 포함할 수 있으나, 이에 제한되는 것은 아니다.The kit may include a reverse transcription polymerase chain reaction (RT-PCR) kit, a DNA chip kit, an ELISA (Enzymelinked immunosorbent assay) kit, a protein chip kit, a rapid kit, or a multiple reaction monitoring (MRM) kit. It is not limited to this.
또한, 본 발명은 (a) 환자로부터 분리된 시료에서 ENPP7의 mRNA 또는 단백질 수준을 측정하는 단계; 및 (b) 상기 측정된 mRNA 또는 단백질 수준을 정상 개체의 mRNA 또는 단백질 수준과 비교하는 단계; 를 포함하는 당뇨병 진단에 필요한 정보를 제공하는 방법을 제공한다.In addition, the present invention includes the steps of (a) measuring the mRNA or protein level of ENPP7 in a sample isolated from a patient; and (b) comparing the measured mRNA or protein level with the mRNA or protein level of a normal subject; Provides a method of providing information necessary for diagnosing diabetes, including.
상기 mRNA 또는 단백질 수준을 측정하는 방법은 상술한 바와 같다.The method for measuring the mRNA or protein level is as described above.
본 발명의 바람직한 또 다른 일실시예에 있어서, 상기 ENPP7 유전자의 mRNA 또는 단백질 수준이 정상 개체 보다 낮을 경우 당뇨병으로 진단하는 단계를 추가로 포함할 수 있다.In another preferred embodiment of the present invention, the step of diagnosing diabetes may be further included when the mRNA or protein level of the ENPP7 gene is lower than that of a normal individual.
또한, 본 발명은 ENPP7 펩타이드를 유효성분으로 포함하는 당뇨병의 예방 및 치료용 약학적 조성물을 제공한다.Additionally, the present invention provides a pharmaceutical composition for preventing and treating diabetes containing ENPP7 peptide as an active ingredient.
상기 ENPP7 단백질은 당업계에 알려진 어떠한 서열로 구성되는 폴리펩티드를 포함할 수 있고, 단백질의 기능에 영향을 미치지 않는 범위 내에서, 아미노산 잔기의 결실, 삽입, 치환 또는 이들의 조합에 의해서 상이한 서열을 가지는 아미노산의 변이체 또는 단편일 수 있다. 분자의 활성을 전체적으로 변경시키지 않는 단백질 또는 펩티드에서의 아미노산 교환은 당해 분야에 공지되어 있다. 경우에 따라서는 인산화 (phosphorylation), 황화 (sulfation), 아크릴화 (acrylation), 당화 (glycosylation), 메틸화 (methylation) 또는 파네실화 (farnesylation) 등으로 변형 (modification)될 수 있다. 이러한 변이체는 ENPP7 단백질 서열과 80％, 90％, 95％, 또는 그 이상의 상동성을 가질 수 있다. 본 발명의 일 실시예에서, 상기 ENPP7 펩타이드는 서열번호 1 또는 서열번호 2로 기재되는 아미노산 서열로 구성되는 펩타이드일 수 있다.The ENPP7 protein may include a polypeptide consisting of any sequence known in the art, and may have a different sequence due to deletion, insertion, substitution, or combination of amino acid residues, within the range that does not affect the function of the protein. It may be a variant or fragment of an amino acid. Amino acid exchanges in proteins or peptides that do not overall alter the activity of the molecule are known in the art. In some cases, it may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, or farnesylation. These variants may have 80%, 90%, 95%, or more homology to the ENPP7 protein sequence. In one embodiment of the present invention, the ENPP7 peptide may be a peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
상기 ENPP7 유전자는 당업계에 알려진 어떠한 서열로 구성되는 폴리뉴클레오티드를 모두 포함할 수 있고, 그 활성을 저하시키지 않는 하나 또는 그 이상의 치환, 삽입, 결실 및 그 조합을 갖는 변이체를 포함할 수 있다. 이러한 변이체는 본 발명의 ENPP7 유전자 서열과 80%, 90%, 95%, 98% 또는 그 이상의 상동성을 가질 수 있다. 본 발명의 일 실시예에서, 상기 ENPP7 유전자는 서열번호 3 또는 서열번호 4로 기재되는 염기서열로 구성되는 폴리뉴클레오티드일 수 있다.The ENPP7 gene may include all polynucleotides composed of any sequence known in the art, and may include variants having one or more substitutions, insertions, deletions, and combinations thereof that do not reduce its activity. These variants may have 80%, 90%, 95%, 98% or more homology to the ENPP7 gene sequence of the present invention. In one embodiment of the present invention, the ENPP7 gene may be a polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4.
상기 약학적 조성물은 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약제학적으로 허용 가능한 담체는 조성물을 생체 내에 전달하는데 적합한 것이면 특별히 제한되지 않으며, 예로서, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토덱스트린 용액, 글리세롤, 에탄올 또는 이들 성분 중 1 성분 이상을 혼합한 것일 수 있다. 이때, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.The pharmaceutical composition may include a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for delivering the composition in vivo, and include, for example, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, or one of these ingredients. It may be a combination of the above. At this time, other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
상기 조성물을 제제화할 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조된다.When formulating the composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
본 발명의 조성물은 경구제제 또는 비경구제제로 제형화 될 수 있다. 경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 하나 이상의 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스, 락토오스 및 젤라틴 등을 섞어 조제될 수 있다. 또한, 마그네슘 스티레이트, 탈크 같은 윤활제들도 첨가될 수 있다. 한편, 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 여기에는 습윤제, 감미제, 방향제, 보존제 등과 같은 부형제가 포함될 수 있다.The composition of the present invention may be formulated as an oral formulation or parenteral formulation. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, troches, etc. These solid preparations include at least one excipient in one or more compositions, such as starch, calcium carbonate, sucrose, It can be prepared by mixing lactose and gelatin. Additionally, lubricants such as magnesium styrate and talc may also be added. Meanwhile, liquid preparations include suspensions, oral solutions, emulsions, or syrups, which may include excipients such as wetting agents, sweeteners, fragrances, and preservatives.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제 등의 주사제가 포함될 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다.Preparations for parenteral administration may include injections such as sterilized aqueous solutions, non-aqueous solutions, suspensions, and emulsions. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate.
본 발명의 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구 투여될수 있으며, 비경구 투여는 피부 외용 또는 복강내 주사, 직장내 주사, 피하주사, 정맥주사, 근육내 주사 또는 흉부 내 주사 주입 방식 중 선택될 수 있다.The composition of the present invention can be administered orally or parenterally according to the desired method, and parenteral administration can be administered through external dermal or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection. can be selected
본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여된다. 이는 질환의 종류, 중증도, 약물의 활성, 약물 에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물 등에 따라 달라질 수 있다. 본 발명의 조성물은 단독 또는 다른 치료제와 병용하여 투여될 수 있다. 병용 투여시, 투여는 순차적 또 는 동시일 수 있다. 그러나, 바람직한 효과를 위해서, 본 발명에 따른 약학적 조성물에 포함되는 유효성분의 양 은 0.001 내지 10,000 mg/㎏, 구체적으로는 0.1 내지 5 g/kg 일 수 있다. 상기 투여는 하루에 1회일 수 있고, 수회로 나뉠 수도 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. This may vary depending on the type and severity of the disease, activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, etc. The composition of the present invention can be administered alone or in combination with other therapeutic agents. When administered in combination, administration may be sequential or simultaneous. However, for a desirable effect, the amount of the active ingredient included in the pharmaceutical composition according to the present invention may be 0.001 to 10,000 mg/kg, specifically 0.1 to 5 g/kg. The administration may be once a day, or may be divided into several times.
또한, 본 발명은 ENPP7 펩타이드를 유효성분으로 포함하는 당뇨병의 예방 또는 개선용 건강기능식품 조성물을 제공한다.Additionally, the present invention provides a health functional food composition for preventing or improving diabetes containing ENPP7 peptide as an active ingredient.
상기 ENPP7 펩타이드는 상술한 바와 같은 특징을 가질 수 있다. The ENPP7 peptide may have the characteristics described above.
또한, 본 발명은 (1) 후보물질을 ENPP7를 발현하는 분리된 세포주에 처리하는 단계; (2) 단계 1)의 처리된 세포주에서 상기 ENPP7의 mRNA 또는 단백질의 발현량을 측정하는 단계; (3) 단계 2)의 ENPP7의 mRNA 또는 단백질의 발현량이 후보물질을 무처리한 대조군 세포주와 비교하여 증가된 후보물질을 선별하는 단계; 를 포함하는 당뇨병 치료제 후보물질의 스크리닝 방법을 제공한다.In addition, the present invention includes the steps of (1) treating a candidate material to an isolated cell line expressing ENPP7; (2) measuring the expression level of the mRNA or protein of ENPP7 in the cell line treated in step 1); (3) selecting a candidate material whose expression level of ENPP7 mRNA or protein in step 2) is increased compared to a control cell line that was not treated with the candidate material; Provides a method for screening diabetes treatment candidates including.
상기 단계 1)의 후보물질은 펩티드, 단백질, 비펩티드성 화합물, 활성 화합물, 발효 생산물, 세포 추출액, 식물 추출액, 동물조직 추출액 및 혈장으로 이루어진 군으로부터 선택되는 하나 이상일 수 있다.The candidate material in step 1) may be one or more selected from the group consisting of peptides, proteins, non-peptide compounds, active compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
상기 단계 2)의 mRNA 또는 단백질의 발현 정도는 상술한 바와 같이 측정될 수 있다. The expression level of mRNA or protein in step 2) can be measured as described above.
본 발명의 구체적인 실시예 및 실험예에서, 본 발명자들은 제2형 당뇨병 관련 유전자 바이오마커를 도출하기 위해서 제2형 당뇨병 환자에 대한 공개 혈액 miRNA-Seq를 수집하였고, 혈액 및 간 조직 유래 유전자 발현 마이크로어레이 데이터로부터 miRNA 및 표적 유전자를 발굴하였고, 이를 이용하여 신호전달 경로 분석을 수행하였다 (도 1 참조). 또한 상기 선별한 제2형 당뇨병과 관련된 유전자 28개 중에서 대사물질과 관련된 세라마이드 (ceramide) 신호경로에 포함되는 유전자 8종(ENPP7, AKT1, AKT2, AKT3, NGFR, PIK3R1, PIK3R3 및 RELA)을 선별하여 혈액 조직 및 간조직에서의 발현량을 확인하였다 (도 2a 참조). 그 중에서 당뇨 환자의 혈액과 간 조직에서 공통적으로 유전자 발현량이 증가 또는 감소하는 유전자인 ENPP7과 AKT2를 도출하였으며, Genecards 데이터베이스를 통해 유전자 기능을 확인한 결과 ENPP7를 후보 바이오마커로 발굴하였다 (도 2b 참조). 상기 결과를 혈액 조직 및 간 조직 데이터에서의 ENPP7의 유전자 발현량을 프리즘 프로그램을 통해 도식한 결과, 정상인에 비해 당뇨환자의 ENPP7 유전자 발현량이 줄었음을 확인하였다 (도 2c 및 도 2d 참조). In specific examples and experimental examples of the present invention, the present inventors collected public blood miRNA-Seq for type 2 diabetes patients to derive type 2 diabetes-related gene biomarkers, and gene expression microorganisms derived from blood and liver tissue. MiRNAs and target genes were discovered from the array data, and signaling pathway analysis was performed using these (see Figure 1). In addition, among the 28 genes related to type 2 diabetes selected above, 8 genes (ENPP7, AKT1, AKT2, AKT3, NGFR, PIK3R1, PIK3R3, and RELA) included in the ceramide signaling pathway related to metabolites were selected. The expression level in blood tissue and liver tissue was confirmed (see Figure 2a). Among them, ENPP7 and AKT2, which are genes whose gene expression levels are commonly increased or decreased in the blood and liver tissue of diabetic patients, were identified, and as a result of confirming the gene function through the Genecards database, ENPP7 was discovered as a candidate biomarker (see Figure 2b). . As a result of plotting the above results with the ENPP7 gene expression level in blood tissue and liver tissue data using the Prism program, it was confirmed that the ENPP7 gene expression level was reduced in diabetic patients compared to normal people (see FIGS. 2C and 2D).
또한 본 발명자들은 정상인 및 당뇨병 환자군의 ENPP7 농도를 비교 및 분석한 결과, 당뇨병 환자의 혈중 ENPP7 농도가 정상인에 비해 약 2배 정도 감소하였음을 확인하였고 (도 3 참조), 이를 통해 혈중 ENPP7 농도를 측정하여 당뇨병의 진단에 이용할 수 있음을 확인하였다. In addition, the present inventors compared and analyzed the ENPP7 concentration of normal people and diabetic patients, and confirmed that the blood ENPP7 concentration of diabetic patients decreased by about two times compared to normal people (see Figure 3), and through this, the ENPP7 concentration in the blood was measured. It was confirmed that it can be used for the diagnosis of diabetes.
또한, 본 발명자들은 동물 실험을 통해 당뇨 질환의 진단 및 치료 가능성을 확인하였다. 이를 위해 렙틴 호르몬에 결함이 있는 비만 모델 마우스의 체중 및 식이 섭취량을 일정하게 조절하였다 (도 4a 및 도 4b 참조). 먼저, 무작위 포도당 검사에서 대조군과 비교하여 ENPP7 펩타이드를 투여한 실험군에서 혈당이 월등히 감소함을 확인하였고 (도 5a 참조), 이 후 6시간 공복 후 확인한 금식 포도당 검사 시 혈당수치 변화에서도 동일한 결과를 확인하여 (도 5b 참조), ENPP7의 혈당 상승 억제 효과를 알 수 있었다. 그 후, 복강 내 포도당 부하 실험을 수행하여 ENPP7 펩타이드 투여한 실험군에서 혈중 혈당 함량이 현저하게 낮음을 확인하였고 (도 6a 및 6b 참조), 복강 내 인슐린 부하 실험에서도 인슐린을 투여하였을 때 ENPP7를 투여한 실험군에서 혈중 혈당 함량이 현저하게 낮음을 확인하였다 (도 7a 및 7b 참조). 이를 통해 ENPP7의 펩타이드 투여함으로써 당뇨병을 치료 또는 개선할 수 있음을 확인하였다.Additionally, the present inventors confirmed the possibility of diagnosing and treating diabetic disease through animal experiments. For this purpose, the body weight and food intake of obese model mice defective in the leptin hormone were constantly controlled (see FIGS. 4A and 4B). First, in a random glucose test, it was confirmed that blood sugar was significantly reduced in the experimental group administered ENPP7 peptide compared to the control group (see Figure 5a), and the same result was also confirmed in the change in blood sugar level during a fasting glucose test after fasting for 6 hours. Thus (see Figure 5b), the effect of ENPP7 on suppressing the rise in blood sugar level was confirmed. Afterwards, an intraperitoneal glucose loading experiment was performed and it was confirmed that the blood glucose content in the experimental group administered ENPP7 peptide was significantly low (see Figures 6a and 6b), and in the intraperitoneal insulin loading experiment, when insulin was administered, ENPP7 was administered. It was confirmed that the blood glucose content in the experimental group was significantly low (see Figures 7a and 7b). Through this, it was confirmed that diabetes can be treated or improved by administering ENPP7 peptide.
이하, 본 발명을 하기 실시예 및 실험예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail through the following examples and experimental examples.
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해 한정되는 것은 아니다.However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following examples and experimental examples.
<실시예 1> 당뇨병 관련 바이오마커 도출<Example 1> Derivation of diabetes-related biomarkers
<1-1> 공개 전사체 데이터 및 신호전달 경로 분석을 통한 바이오마커 발굴<1-1> Discovery of biomarkers through analysis of public transcriptome data and signaling pathways
공개 miRNA-Seq 데이터 및 유전자 발현 마이크로어레이 데이터로부터 당뇨와 관련이 있는 유전자를 선별하였고, 신호전달 경로 분석을 통해서 당뇨발명에 영향을 줄 것으로 예상되는 바이오마커를 선별하였다.Genes related to diabetes were selected from public miRNA-Seq data and gene expression microarray data, and biomarkers expected to affect diabetes development were selected through signal transduction pathway analysis.
구체적으로, 제2형 당뇨병 환자에 대한 공개 혈액 miRNA-Seq (정상인 56명 및 당뇨환자 39명), 혈액 (정상인 8명 및 당뇨환자 9명) 및 간 조직 (정상인 7명 및 당뇨환자 10명) 유래 유전자 발현 마이크로어레이 (gene expression microarray) 데이터를 이용하여 통계적으로 유의하게 발현하는 99개의 miRNA 및 표적 유전자를 발굴하였다 (도 1). 제2형 당뇨병 환자에 대한 공개 혈액 miRNA-Seq 데이터 수집을 위해 SRA (Sequence Read Archive) 데이터베이스로부터 SRP151126과 SRP093728의 데이터의 fastq 파일을 fastq-dump 프로그램을 이용하여 다운로드 받았으며, FastQC 및 Cutadapt 프로그램을 통해 miRNA-Seq 데이터의 Quality Check 및 miRNA 크기로 재가공하였다. miRBase 및 miRdeep2 프로그램을 통해 miRNA-Seq 데이터의 정량화 및 어노테이션을 수행하였다. 혈액 및 간 조직 유래 유전자 발현 마이크로어레이 데이터 수집을 위해 GEO (Gene Expression Omnibus)로부터 GSE26168 (혈액 조직) 및 GSE23343 (간 조직) 데이터를 다운로드 받아 차등발현유전자 (differentially expressed gene, DEG) 분석에 사용하였다. DEG 분석을 위해서 miRNA-Seq 데이터는 edgeR 프로그램의 로그-우도비검정 (log-likelihood ratio test)을 사용하였고, 유전자 발현 마이크로어레이 데이터는 스튜던트의 T 검정 (student t-test)을 수행하였다. 발현량 차이 값은 Log2FoldChange를 이용하였다. DEG를 수행한 miRNA 및 유전자 중 P value < 0.05인 유전자에 대해 IPA (Ingenuity Pathway Analysis) 프로그램의 IPA core analysis 기능을 이용하여 신호전달 경로 분석을 수행하였고, 그 중 고전적인 신호경로 (canonical patwhay)를 이용하여 질환 온톨로지 (disease ontology) 분석 결과를 얻었다.Specifically, public blood miRNA-Seq for patients with type 2 diabetes (56 normal subjects and 39 diabetic patients), blood (8 normal subjects and 9 diabetic subjects), and liver tissue (7 normal subjects and 10 diabetic subjects). Using gene expression microarray data, 99 statistically significantly expressed miRNAs and target genes were discovered (Figure 1). To collect public blood miRNA-Seq data for patients with type 2 diabetes, fastq files of the data of SRP151126 and SRP093728 were downloaded from the SRA (Sequence Read Archive) database using the fastq-dump program, and the miRNA-Seq files were downloaded using the FastQC and Cutadapt programs. -Seq data was quality checked and reprocessed according to the size of the miRNA. Quantification and annotation of miRNA-Seq data were performed through the miRBase and miRdeep2 programs. To collect blood and liver tissue-derived gene expression microarray data, GSE26168 (blood tissue) and GSE23343 (liver tissue) data were downloaded from GEO (Gene Expression Omnibus) and used for differentially expressed gene (DEG) analysis. For DEG analysis, the log-likelihood ratio test of the edgeR program was used for the miRNA-Seq data, and Student's t-test was performed for the gene expression microarray data. Log2FoldChange was used to determine the difference in expression level. Signaling pathway analysis was performed using the IPA core analysis function of the IPA (Ingenuity Pathway Analysis) program for genes with a P value < 0.05 among the DEGs and genes for which DEG was performed. Among them, the classical signaling pathway (canonical pathhay) was analyzed. Using this, disease ontology analysis results were obtained.
그 결과, 도 1에 나타난 바와 같이, 통계적으로 유의하게 제2형 당뇨병 신호경로를 매개하는 28개 유전자 및 38개의 miRNA를 확인하였다. As a result, as shown in Figure 1, 28 genes and 38 miRNAs that statistically significantly mediate the type 2 diabetes signaling pathway were identified.
<1-2> 대사물질 조절과 연된되어 있는 바이오마커 발굴<1-2> Discovery of biomarkers linked to metabolite regulation
선별된 유전자 중 당뇨 환자의 혈액 조직 데이터 및 간 조직 데이터에서 공통적으로 발현되는 유전자 중 제2형 당뇨병 신호 경로와 관련이 있으면서 Ceramide 신호 경로에 관련있는 유전자를 선별하였다 (도 2a).Among the selected genes, genes related to the type 2 diabetes signaling pathway and the Ceramide signaling pathway were selected among genes commonly expressed in blood tissue data and liver tissue data of diabetic patients (Figure 2a).
구체적으로, IPA를 이용한 신호전달분석 결과 중 제2형 당뇨병과 대사물질 간의 연관성에 주목하여 제2형 당뇨병과 관련이 있으면서 대사물질과 관련된 신호경로인 Ceramide 신호경로에 공통적으로 포함되는 8개 유전자인 ENPP7 (Ectonucleotide pyrophosphatase/phosphodiesterase family member 7), AKT1 (RAC(Rho family)-alpha serine/threonine-protein kinase), AKT2, AKT3, NGFR (Nerve Growth Factor Receptor), PIK3R1 (Phosphoinositide-3-Kinase Regulatory Subunit 1), PIK3R3 및 RELA (nuclear factor NF-kappa-B p65 subunit)를 선별하여 혈액 조직 및 간조직에서의 발현량을 확인하였다. 선별된 유전자 중 당뇨 환자의 혈액 조직 데이터 및 간 조직 데이터에서 동시에 발현량이 증가하거나 감소한 유전자를 추가적으로 선별하였다.Specifically, among the results of signal transduction analysis using IPA, we focused on the relationship between type 2 diabetes and metabolites, and identified eight genes commonly included in the Ceramide signaling pathway, which is a signaling pathway related to type 2 diabetes and related to metabolites. ENPP7 (Ectonucleotide pyrophosphatase/phosphodiesterase family member 7), AKT1 (RAC (Rho family)-alpha serine/threonine-protein kinase), AKT2, AKT3, NGFR (Nerve Growth Factor Receptor), PIK3R1 (Phosphoinositide-3-Kinase Regulatory Subunit 1) ), PIK3R3, and RELA (nuclear factor NF-kappa-B p65 subunit) were selected to confirm their expression levels in blood and liver tissues. Among the selected genes, genes whose expression levels were simultaneously increased or decreased in the blood tissue data and liver tissue data of diabetic patients were additionally selected.
그 결과, 도 2b에 나타난 바와 같이, ENPP7과 AKT2가 당뇨 환자의 혈액과 간 조직에서 통게적으로 유의하게 발현 양상이 일치함을 확인하였다. As a result, as shown in Figure 2b, it was confirmed that the expression patterns of ENPP7 and AKT2 were significantly consistent in the blood and liver tissue of diabetic patients.
Genecards 데이터베이스를 통해 상기 유전자의 기능 정보를 확인한 결과, ENPP7는 알칼라인 스핑고미엘린 포스포디에스테라아제 (alkaline sphingomyelin phosphodiesterase)로서 스핑고미엘린 (Sphingomyelin)과 Ceramide 간의 가역적 전환에 매개하는 효소임을 확인하였고, 따라서 ENPP7을 당뇨의 진단 및 치료를 위한 후보 바이오마커로 발굴하였다.As a result of checking the functional information of the above gene through the Genecards database, it was confirmed that ENPP7 is an alkaline sphingomyelin phosphodiesterase, an enzyme that mediates the reversible conversion between sphingomyelin and ceramide. Therefore, ENPP7 was identified as an alkaline sphingomyelin phosphodiesterase. It was discovered as a candidate biomarker for the diagnosis and treatment of diabetes.
<1-3> 혈액 및 간 조직 데이터에서의 ENPP7의 유전자 발현량 감소 검증<1-3> Verification of reduced gene expression level of ENPP7 in blood and liver tissue data
혈액 및 간 조직 데이터에서의 ENPP7의 유전자 발현량을 프리즘 프로그램을 통해 도식화하였다. The gene expression level of ENPP7 in blood and liver tissue data was plotted using the Prism program.
그 결과, 도 2c에 나타난 바와 같이, 혈액 조직에서 ENPP7의 발현량이 당뇨환자에서 통계적으로 유의하게 감소함을 확인하였다 (Log2FoldChange= -2.27 및 P value= 0.005). 혈액 조직의 log2FoldChange 값은 -2.27로 대조군인 정상인이 당뇨환자보다 ENPP7의 유전자 발현량이 22.27배 많아서 정상인에 비해 당뇨환자의 유전자 발현량이 줄었음을 나타낸다. As a result, as shown in Figure 2c, it was confirmed that the expression level of ENPP7 in blood tissue was statistically significantly decreased in diabetic patients (Log2FoldChange= -2.27 and P value= 0.005). The log 2 FoldChange value of blood tissue was -2.27, indicating that the gene expression level of ENPP7 in normal controls was 2-2.27 times higher than in diabetic patients, indicating that the gene expression level in diabetic patients was reduced compared to normal people.
또한, 도 2d에 나타난 바와 같이, 간 조직에도 ENPP7의 발현량이 당뇨 환자에서 통계적으로 유의하게 감소함을 확인하였다 (Log2FoldChange=-0.69, P value=0.045). 간 조직의 log2FoldChange 값은 -0.69로 대조군인 정상인이 당뇨환자보다 ENPP7의 유전자 발현량이 20.69배 많아서 정상인에 비해 당뇨환자의 유전자 발현량이 줄었음을 나타낸다. In addition, as shown in Figure 2d, it was confirmed that the expression level of ENPP7 in liver tissue was statistically significantly decreased in diabetic patients (Log2FoldChange=-0.69, P value=0.045). The log 2 FoldChange value of liver tissue was -0.69, indicating that the gene expression level of ENPP7 in normal controls was 20.69 times higher than in diabetic patients, indicating that the gene expression level in diabetic patients was reduced compared to normal controls.
정상인과 비교하였을 때 당뇨 환자의 혈액 조직이 간 조직보다 ENPP7의 유전자 발현량이 더 많이 줄어들은 바, ENPP7 유전자를 혈액 조직을 용이하게 채취함으로써 제2형 당뇨병의 진단, 예방 또는 치료에 이용할 수 있다. Compared to normal people, the gene expression level of ENPP7 was reduced more in the blood tissue of diabetic patients than in the liver tissue, so the ENPP7 gene can be used for diagnosis, prevention, or treatment of type 2 diabetes by easily collecting blood tissue.
<실험예 1> 당뇨병 환자군에서의 ENPP7의 농도 분석<Experimental Example 1> Concentration analysis of ENPP7 in diabetic patient group
ENPP7 의 당뇨 진단 바이오마커로서의 가능성을 검증하기 위하여 정상인 및 제2형 당뇨병 환자의 혈액 테이터를 확보하여 효소면역측정법 (ELISA)을 진행하였다.To verify the potential of ENPP7 as a diabetes diagnostic biomarker, blood data from normal people and type 2 diabetes patients were obtained and enzyme-linked immunosorbent assay (ELISA) was performed.
구체적으로, 가천대 길병원의 바이오뱅크로부터 후향적 임상정보와 함께 혈액을 100 샘플 분양받았으며, 기관생명윤리위원회 (IRB)의 심의 및 승인을 거쳐 실험을 수행하였다(IRB 번호: GBIRB2020-338). 분양받은 100개의 샘플 중 정상인 (n=38) 및 당뇨병 환자 (n=36)를 대상으로 채취한 혈액에서 혈중 ENPP7 농도를 비교하였다. Specifically, 100 blood samples along with retrospective clinical information were received from the biobank of Gachon University Gil Medical Center, and the experiment was conducted after review and approval by the Institutional Bioethics Committee (IRB) (IRB number: GBIRB2020-338). Among the 100 samples distributed, the ENPP7 concentration in the blood was compared in blood collected from normal people (n=38) and diabetic patients (n=36).
ENPP7의 혈장 내 수준을 검사하기 위하여 시판 중인 Human ELISA 키트 (Cat:MBS723092, MyBioSource, Inc., USA)를 사용하여, 인간 ENPP7의 다중클론항체 (polyclonal antibody)로 코팅된 96 웰 플레이트 (well plate)에, 각 혈장 샘플을 희석없이 각 웰당 100 ㎕씩 로딩 (loading)한 후, 37°C에서 1시간 배양하였다. 배양이 끝난 플레이트를 3차례 세척한 후, 검출 항체 (detection antibody)를 로딩하였다. 37°C에서 1시간동안 배양하고 5차례 세척한 후, 100 ㎕ 기질용액 (substrate solution)을 로딩하여 실온에서 20분 동안 방치한 다음 450 ㎚에서 흡광도를 측정하였다. 이하 모든 결과는 평균±표준오차로 나타내었으며, 스튜던트 t 검정 (Student's t-test)이나 이원배치 분산분석 (Two-way ANOVA)으로 분석하였다. 통계분석은 Prism 버전 8.4.3 (GraphPad Software, San Diego, CA, USA)을 사용하였고, 유의수준은 0.05 미만으로 하였다.To test the plasma level of ENPP7, a 96-well plate coated with a polyclonal antibody of human ENPP7 was used using a commercially available Human ELISA kit (Cat:MBS723092, MyBioSource, Inc., USA). 100 ㎕ of each plasma sample was loaded into each well without dilution and incubated at 37°C for 1 hour. After the culture was completed, the plate was washed three times and then loaded with detection antibody. After culturing for 1 hour at 37°C and washing five times, 100 ㎕ substrate solution was loaded and left at room temperature for 20 minutes, and then the absorbance was measured at 450 nm. All results below are expressed as mean ± standard error and were analyzed by Student's t-test or two-way ANOVA. Statistical analysis was performed using Prism version 8.4.3 (GraphPad Software, San Diego, CA, USA), and the significance level was set at less than 0.05.
그 결과, 도 3에 나타난 바와 같이, 혈중 ENPP7 농도는 정상인에서는 192±22.84 pg/ml, 당뇨병 환자에서는 99.86±11.66pg/ml으로 확인되었다. 이는 당뇨병 환자의 경우 정상인에 비해 약 2배 정도 혈중 ENPP7 농도가 감소되었음을 나타낸다.As a result, as shown in Figure 3, the ENPP7 concentration in the blood was confirmed to be 192 ± 22.84 pg/ml in normal people and 99.86 ± 11.66 pg/ml in diabetic patients. This indicates that in diabetic patients, the concentration of ENPP7 in the blood decreased by approximately two times compared to normal people.
따라서, 혈중 ENPP7의 농도가 정상인 대비 현저히 감소한 경우 당뇨병이 발병한 것으로 진단할 수 있다. Therefore, when the concentration of ENPP7 in the blood is significantly reduced compared to normal, diabetes can be diagnosed.
<실험예 2> 동물 실험을 통한 당뇨 질환의 진단 및 치료 가능성 확인<Experimental Example 2> Confirmation of diagnosis and treatment possibility of diabetic disease through animal experiment
동물 실험을 통해 당뇨 질환의 진단 및 치료 가능성을 확인하기 위한 실험을 수행하였다. 본 발명에 사용한 비만 모델 마우스는 10주령의 수컷 C57BL/6J ob/ob 모델로서 렙틴 (leptin) 호르몬 결함이 있는 비만 모델 마우스 (계통-B6.Cg-Lepob/J, 제조사-Charles River Japan, 일련번호-JAX® Mice stock number:000632) 이다. An experiment was conducted to confirm the possibility of diagnosing and treating diabetes disease through animal experiments. The obesity model mouse used in the present invention is a 10-week-old male C57BL/6J ob/ob model with a defect in the leptin hormone (lineage-B6.Cg-Lep ob /J, manufacturer-Charles River Japan, series). Number-JAX® Mice stock number:000632).
<2-1> 마우스 실험 모델의 체중 및 식이 섭취량 변화 분석<2-1> Analysis of changes in body weight and food intake in mouse experimental model
ENPP7 펩타이드를 투여하지 않은 대조군 및 ENPP7 펩타이드를 투여한 실험군에 대해서 체중 및 식이 섭취량의 변화를 측정하였다.Changes in body weight and food intake were measured for the control group that did not administer ENPP7 peptide and the experimental group that administered ENPP7 peptide.
구체적으로, 실험 기간동안 모든 마우스에 제한이 없는 일반식이 (Cat: 5053, LabDiet)를 제공하였다. 매일 같은 시간에 피하주사를 통하여 대조군 (n=8)은 0.9%의 생리식염수를 투여하였고, 실험군 (ENPP7, n=8))은 재조합 ENPP7 펩타이드 (Cat: 50567-M08H, Sino Biological Inc.)를 최종농도 0.4μg/kg로 맞추어, 총 주사 투여량을 매일 새로이 측정되는 마우스 체중에 기반하여, '주사투여량(μl) = 마우스체중(g) X 2' 가 되도록 제조하여 투여하였다. 모든 체중 및 식이 섭취량은 3주 (21일)동안 ENPP7 펩타이드를 투여하기 전 매일 같은 시간 측정하여 기록하였다.Specifically, all mice were provided with a regular diet (Cat: 5053, LabDiet) with no restrictions during the experiment period. The control group (n=8) was administered 0.9% physiological saline through subcutaneous injection at the same time every day, and the experimental group (ENPP7, n=8) was administered recombinant ENPP7 peptide (Cat: 50567-M08H, Sino Biological Inc.). The final concentration was adjusted to 0.4 μg/kg, and the total injection dose was prepared and administered so that 'injection dose (μl) = mouse weight (g) All body weight and food intake were measured and recorded at the same time every day before ENPP7 peptide administration for 3 weeks (21 days).
그 결과, 도 4a 및 도 4b에 나타난 바와 같이, 최종 체중은 대조군에서 54.35±1.07g, 실험군 (ENPP7)에서 54.57±0.81g으로 측정되었으며, 총 식이섭취량은 대조군에서 104.2±6.72g, 실험군에서 102.2±2.47g으로 측정되었다. 따라서, 두 그룹 간에 체중 및 식이 섭취량의 유의미한 차이는 없음을 확인하였다.As a result, as shown in Figures 4a and 4b, the final body weight was measured as 54.35±1.07g in the control group and 54.57±0.81g in the experimental group (ENPP7), and the total dietary intake was 104.2±6.72g in the control group and 102.2g in the experimental group. It was measured as ±2.47g. Therefore, it was confirmed that there was no significant difference in body weight and dietary intake between the two groups.
<2-2> 마우스 실험 모델에서 ENPP7의 혈당 수준 감소 효과 확인<2-2> Confirmation of the effect of ENPP7 on reducing blood sugar levels in a mouse experimental model
상기 비만 모델 마우스에서 ENPP7의 투여가 혈당 수준에 미치는 영향을 분석하기 위해 무작위 포도당 검사 (Random glucose test) 및 금식 포도당 검사 (Fasting glucose test)를 실시하였다.Random glucose test and fasting glucose test were performed to analyze the effect of ENPP7 administration on blood sugar levels in the obesity model mice.
무작위 포도당 검사는 공복없이 ENPP7 펩타이드를 최종농도 0.4μg/kg로 피하투여 후 15분 뒤에 혈당계 (Accu-Chek Active; Roche Diagnostics)를 이용하여 마우스 꼬리에서 나온 혈액으로 총 0, 3, 6일차에 매회 같은 시간에 혈당시험지 (Accu-Chek Active Strip; Roche Diagnostics)로 혈당을 측정하였다. Random glucose tests were performed using blood from the tail of mice using a blood glucose meter (Accu-Chek Active; Roche Diagnostics) 15 minutes after subcutaneous administration of ENPP7 peptide at a final concentration of 0.4 μg/kg without fasting, every time on days 0, 3, and 6. At the same time, blood sugar was measured using a blood sugar test strip (Accu-Chek Active Strip; Roche Diagnostics).
그 결과, 도 5a에 나타난 바와 같이, 실험 시작 전 (0일)에 평균 혈당치는 대조군 (226.3±13.37mg/dl)과 ENPP7 펩타이드 투여한 실험군 (245.8±20.30mg/dl)에서 유의미한 차이가 없었다. 그러나, 실험시작 3일차의 평균 혈당치는 대조군에서 308.3±16.55mg/dl, ENPP7 펩타이드 투여한 실험군에서 257.4±11.44mg/dl로 실험군 (ENPP7)의 평균혈당치가 50.9±19.83mg/dl로 더 낮게 나타나 유의미한 혈당상승 억제 작용을 보였다. 또한, 실험시작 6일차에는 평균혈당치가 대조군은 351.6±10.19mg/dl, ENPP7 펩타이드 투여한 실험군은 301.6±8.91mg/dl을 나타내어 투여 3일차와 동일하게 실험군의 평균혈당치가 50.0±19.83mg/dl의 차이로 더 낮게 나타났다. As a result, as shown in Figure 5a, there was no significant difference in the average blood sugar level before the start of the experiment (day 0) between the control group (226.3 ± 13.37 mg/dl) and the experimental group administered ENPP7 peptide (245.8 ± 20.30 mg/dl). However, on the third day after starting the experiment, the average blood sugar level was 308.3 ± 16.55 mg/dl in the control group and 257.4 ± 11.44 mg/dl in the experimental group administered ENPP7 peptide. The average blood sugar level of the experimental group (ENPP7) was lower at 50.9 ± 19.83 mg/dl. It showed a significant effect of suppressing the rise in blood sugar levels. In addition, on the 6th day after the start of the experiment, the average blood sugar level in the control group was 351.6 ± 10.19 mg/dl and the experimental group administered ENPP7 peptide was 301.6 ± 8.91 mg/dl, the same as on the 3rd day of administration, the average blood sugar level in the experimental group was 50.0 ± 19.83 mg/dl. It appeared lower due to the difference in .
상기의 결과는 시간이 지날수록 비만이 가속화되어 혈당이 높아지는 당뇨 초기의 ob/ob마우스에서 ENPP7 펩타이드의 투여가 혈당 상승을 억제할 수 있음을 시사한다.The above results suggest that administration of ENPP7 peptide can suppress the rise in blood sugar in ob/ob mice in the early stages of diabetes, where obesity accelerates over time and blood sugar levels rise.
금식 포도당 검사는 6시간 동안 공복 시킨 마우스에 ENPP7 펩타이드를 최종농도 0.4μg/kg로 맞추어 피하투여 후 15분 뒤에 혈당계 (Accu-Chek Active; Roche Diagnostics)를 이용하여 마우스 꼬리에서 나온 혈액으로 총 0, 4, 7일차 매회 같은 시간에 혈당시험지 (Accu-Chek Active Strip; Roche Diagnostics)로 혈당을 측정하였다. For the fasting glucose test, ENPP7 peptide was administered subcutaneously at a final concentration of 0.4 μg/kg to mice that were fasted for 6 hours, and 15 minutes later, using blood from the tail of the mouse using a blood glucose meter (Accu-Chek Active; Roche Diagnostics), a total of 0, Blood sugar levels were measured using blood sugar test strips (Accu-Chek Active Strip; Roche Diagnostics) at the same time on days 4 and 7.
그 결과, 도 5b에 나타난 바와 같이, 실험 시작전 (0일)에 평균 공복혈당치는 대조군은 328.9±8.30mg/dl, 실험군 (ENPP7)은 302.6±11.21mg/dl로 유의미한 차이가 없었다. 그러나, 실험 시작 4일 차의 평균 공복혈당치는 대조군은 437.0±11.98mg/dl, ENPP7 펩타이드 투여한 실험군은 362.3±10.89mg/dl로, 실험군의 평균 공복혈당치가 74.8±17.46mg/dl 더 낮게 나타나 대조군에 비해 혈당상승이 억제됨을 확인하였다. 또한, 실험시작 7일차에는 대조군의 평균 공복혈당치가 461.6±8.65mg/dl이고, 실험군의 평균 공복혈당치가 395.0±19.58mg/dl로, 실험군의 평균 공복 혈당치가 66.6±17.46mg/dl 더 낮게 나타났다. As a result, as shown in Figure 5b, the average fasting blood sugar level before the start of the experiment (day 0) was 328.9 ± 8.30 mg/dl for the control group and 302.6 ± 11.21 mg/dl for the experimental group (ENPP7), with no significant difference. However, the average fasting blood sugar level on the 4th day after the start of the experiment was 437.0 ± 11.98 mg/dl in the control group and 362.3 ± 10.89 mg/dl in the experimental group administered ENPP7 peptide, showing that the average fasting blood sugar level of the experimental group was 74.8 ± 17.46 mg/dl lower. It was confirmed that the increase in blood sugar level was suppressed compared to the control group. In addition, on the 7th day of the experiment, the average fasting blood sugar level of the control group was 461.6 ± 8.65 mg/dl, the average fasting blood sugar level of the experimental group was 395.0 ± 19.58 mg/dl, and the average fasting blood sugar level of the experimental group was 66.6 ± 17.46 mg/dl lower. .
상기의 결과는 시간이 지날수록 비만이 가속화되어 혈당이 높아지는 당뇨 초기의 ob/ob마우스에서 ENPP7 펩타이드의 투여가 혈당 상승을 억제할 수 있음을 시사한다.The above results suggest that administration of ENPP7 peptide can suppress the rise in blood sugar in ob/ob mice in the early stages of diabetes, where obesity accelerates over time and blood sugar levels rise.
따라서, ENPP7 펩타이드는 혈당의 상승을 억제시켜 당뇨병 예방 또는 치료 효과가 있음을 확인하였다.Therefore, it was confirmed that ENPP7 peptide has an effect in preventing or treating diabetes by suppressing the rise in blood sugar levels.
<실험예 3> 비만 모델 마우스에서 포도당 부하 실험 결과 분석<Experimental Example 3> Analysis of glucose load experiment results in obese model mice
ENPP7 펩타이드 투여에 따른 포도당 내성능의 차이를 확인하기 위하여, 포도당 부하 실험 Glucose tolerance test, GTT)을 수행하였다. To confirm the difference in glucose tolerance ability according to ENPP7 peptide administration, a glucose tolerance test (GTT) was performed.
구체적으로, 실험 시작 후 19일 차에 6시간 동안 금식시킨 마우스에 ENPP7 펩타이드를 최종농도 0.4μg/kg로 피하투여 후 15분 경과 뒤에 포도당을 1g/kg의 농도로 맞추어 복강주사로 투여 [포도당 주사투여량 (μl) = 마우스 체중 (g) X 4]하여 0, 5, 15, 30, 60, 90 및 120분 동안 마우스 꼬리에서 나온 혈액으로 혈당계 (Accu-Chek Active; Roche Diagnostics)를 이용하여 혈당시험지 (Accu-Chek Active Strip; Roche Diagnostics)로 혈당을 측정하였다. Specifically, on the 19th day after the start of the experiment, ENPP7 peptide was administered subcutaneously at a final concentration of 0.4 μg/kg to mice that were fasted for 6 hours, and 15 minutes later, glucose was administered at a concentration of 1 g/kg by intraperitoneal injection [Glucose injection Dose (μl) = mouse body weight (g) Blood sugar was measured using a test strip (Accu-Chek Active Strip; Roche Diagnostics).
그 결과, 도 6a에 나타난 바와 같이, 포도당 투여 후 30분 후부터 실험군 (ENPP7)의 평균혈당치는 394.3±45.94mg/dl, 대조군의 평균 혈당치는 511.0±20.85mg/dl로 나타나, 실험군이 대조군에 비해 116.8±37.51mg/dl 유의미하게 더 낮게 관찰되어 ENPP7 펩타이드 투여에 의해 혈당상승이 억제됨을 확인하였다. 이러한 혈당 상승 억제 효과는 90분까지 1시간 가량 지속적으로 나타났다 (60분 평균혈당치: 대조군 509.1±26.95mg/dl, 실험군 391.1±22.64mg/dl, 평균 118.0±37.51mg/dl 차이, 90분 평균 혈당치: 대조군 537.9±19.19mg/dl, 실험군 422.9±20.19mg/dl, 평균 115.0±37.51mg/dl 차이). As a result, as shown in Figure 6a, 30 minutes after administration of glucose, the average blood sugar level of the experimental group (ENPP7) was 394.3 ± 45.94 mg/dl and the average blood sugar level of the control group was 511.0 ± 20.85 mg/dl, showing that the experimental group was higher than the control group. It was observed to be significantly lower at 116.8±37.51mg/dl, confirming that the increase in blood sugar level was suppressed by ENPP7 peptide administration. This effect of suppressing the rise in blood sugar levels continued for about an hour until the 90th minute (60-minute average blood sugar level: control group 509.1 ± 26.95 mg/dl, experimental group 391.1 ± 22.64 mg/dl, average difference of 118.0 ± 37.51 mg/dl, 90-minute average blood sugar level : Control group 537.9±19.19mg/dl, experimental group 422.9±20.19mg/dl, average difference 115.0±37.51mg/dl).
상기의 결과는 ENPP7 펩타이드 투여가 당뇨병 예방 및 치료에 효과가 있음을 시사한다.The above results suggest that ENPP7 peptide administration is effective in preventing and treating diabetes.
또한, 혈중농도 곡선하면적 (Total Area Under Curve, Total AUC) 분석을 수행하였다. In addition, total area under curve (Total AUC) analysis was performed.
구체적으로, 총 7회의 혈당을 측정하였으며 각 시간마다의 평균을 구하여 이를 통해 그래프의 총 면적으로 정의하여 Trapezoidal 방법으로 계산하였다.Specifically, blood sugar was measured a total of 7 times, and the average for each time was calculated, which was defined as the total area of the graph and calculated using the trapezoidal method.
그 결과, 도 6b에 나타난 바와 같이, 대조군에서는 Total AUC가 57484이고, 실험군 (ENPP7)은 46388으로 총 11096 ± 3093차이를 나타내 대조군보다 실험군 (ENPP7)에서 약 20% 낮은 혈당상승이 나타나는 것을 확인하였다.As a result, as shown in Figure 6b, the Total AUC in the control group was 57484, and the experimental group (ENPP7) was 46388, showing a total difference of 11096 ± 3093, confirming that the blood sugar rise was about 20% lower in the experimental group (ENPP7) than in the control group. .
상기의 결과는 ENPP7 펩타이드 투여가 당뇨병 예방 및 치료에 효과가 있음을 시사한다.The above results suggest that ENPP7 peptide administration is effective in preventing and treating diabetes.
<실험예 4> 비만 모델 마우스에서 인슐린 부하 실험 결과 분석<Experimental Example 4> Analysis of insulin load test results in obese model mice
ENPP7 펩타이드 투여에 따른 인슐린 저항성 차이를 확인하기 위하여, 인슐린 부하 실험 (Insulin tolerance test, ITT)을 수행하였다. To confirm the difference in insulin resistance according to ENPP7 peptide administration, an insulin tolerance test (ITT) was performed.
구체적으로, 실험 시작 후 20일 차에 6시간 동안 공복시킨 마우스에 ENPP7 펩타이드를 최종농도 0.4μg/kg로 피하투여 후 15분 경과 뒤에 인슐린 (recombinat human)을 1U/kg의 농도로 맞추어 복강주사로 투여하여 [인슐린주사 투여량(μl) = 마우스체중(g) X 2], 0, 15, 30, 60, 90 및 120분 동안 마우스 꼬리에서 나온 혈액을 혈당계 (Accu-Chek; Roche Diagnostics)를 이용하여 혈당시험지(Accu-Chek Active Strip; Roche Diagnostics)로 혈당을 측정하였다. Specifically, on the 20th day after the start of the experiment, ENPP7 peptide was administered subcutaneously at a final concentration of 0.4 μg/kg to mice that had fasted for 6 hours, and 15 minutes later, insulin (recombinant human) was administered intraperitoneally at a concentration of 1 U/kg. Administer [insulin injection dose (μl) = mouse body weight (g) Blood sugar was measured using a blood sugar test strip (Accu-Chek Active Strip; Roche Diagnostics).
그 결과, 도 7a에 나타난 바와 같이, 인슐린 투여 90 분 후 실험군 (ENPP7)의 혈당강하 수치(%)가 실험시작 직전 (100%)에 비해 실험군 (ENPP7)의 평균 50.44±5.48%, 대조군은 평균 70.44±3.02%로 실험군에서 혈당상승이 대조군에 비해 20.4±5.25% 억제되는 결과가 나타났다. 또한, 실험 직전 대비 120분에는 백분위 평균 혈당 강하 수치가 대조군은 60±4.45%, 실험군은 37.93±4.95%로 실험군이 22.08±5.25% 더 낮게 나타났다. 따라서 실험시작 후 120분까지 ENPP7 펩타이드의 투여로 인한 지속적이며 점진적인 혈당 상승의 억제가 이루어짐을 확인하였다.As a result, as shown in Figure 7a, the blood sugar lowering value (%) of the experimental group (ENPP7) 90 minutes after insulin administration was an average of 50.44 ± 5.48% for the experimental group (ENPP7) and an average of 50.44 ± 5.48% for the control group compared to (100%) just before the start of the experiment. The result was 70.44±3.02%, showing that the rise in blood sugar in the experimental group was suppressed by 20.4±5.25% compared to the control group. In addition, at 120 minutes compared to immediately before the experiment, the percentile average blood sugar drop was 60 ± 4.45% in the control group and 37.93 ± 4.95% in the experimental group, which was 22.08 ± 5.25% lower in the experimental group. Therefore, it was confirmed that the continuous and gradual increase in blood sugar level was suppressed by the administration of ENPP7 peptide until 120 minutes after the start of the experiment.
또한, 혈중농도 곡선하면적 (Total Area Under Curve, Total AUC) 분석을 수행하였다. In addition, total area under curve (Total AUC) analysis was performed.
구체적으로, 총 6회의 혈당을 측정하였으며 각 시간마다의 평균을 구하여 이를 통해 그래프의 총 면적으로 정의하여 Trapezoidal 방법으로 계산하였다.Specifically, blood sugar was measured a total of 6 times, and the average for each time was calculated, which was defined as the total area of the graph and calculated using the trapezoidal method.
그 결과, 도 7b에 나타난 바와 같이, 대조군에서는 Total AUC가 8249이고, 실험군 (ENPP7)은 6973으로 총 1276 ± 524.3 차이를 나타내 대조군보다 실험군 (ENPP7)에서 약 15.5%의 더 강력한 혈당상승 억제가 나타남을 확인하였다.As a result, as shown in Figure 7b, the Total AUC in the control group was 8249, and in the experimental group (ENPP7) it was 6973, showing a total difference of 1276 ± 524.3, showing a stronger inhibition of blood sugar rise by about 15.5% in the experimental group (ENPP7) than in the control group. was confirmed.
상기의 결과는 ENPP7 펩타이드 투여가 당뇨병 예방 및 치료에 효과가 있음을 시사한다.The above results suggest that ENPP7 peptide administration is effective in preventing and treating diabetes.
Claims (12)
A biomarker composition for diagnosing diabetes, characterized in that it contains ENPP7 (Ectonucleotide pyrophosphatase/phosphodiesterase family member 7).
상기 당뇨병은 제2형 당뇨병인 것을 특징으로 하는, 당뇨병 진단용 바이오마커.
According to paragraph 1,
A biomarker for diagnosing diabetes, characterized in that the diabetes is type 2 diabetes.
A composition for diagnosing diabetes, comprising an agent for measuring ENPP7 mRNA or protein levels.
The composition for diagnosing diabetes according to claim 3, wherein the agent for measuring the level of mRNA is a primer or probe that specifically binds to the gene.
The composition for diagnosing diabetes according to claim 3, wherein the agent for measuring the level of the protein comprises an antibody, peptide, aptamer, or compound specific for the protein.
A kit for diagnosing diabetes, comprising the composition of claim 3.
(b) 상기 측정된 mRNA 또는 단백질 수준을 정상 개체의 mRNA 또는 단백질 수준과 비교하는 단계;
를 포함하는 것을 특징으로 하는, 당뇨병 진단에 필요한 정보를 제공하는 방법.
(a) measuring the mRNA or protein level of ENPP7 in a sample isolated from a patient; and
(b) comparing the measured mRNA or protein level with the mRNA or protein level of a normal subject;
A method of providing information necessary for diagnosing diabetes, comprising:
상기 ENPP7 유전자의 mRNA 또는 단백질 수준이 정상 개체 보다 낮을 경우 당뇨병으로 진단하는 단계를 추가로 포함하는 것을 특징으로 하는, 당뇨병 진단에 필요한 정보를 제공하는 방법.
In clause 7,
A method of providing information necessary for diagnosing diabetes, characterized in that it additionally includes the step of diagnosing diabetes when the mRNA or protein level of the ENPP7 gene is lower than that of a normal individual.
A pharmaceutical composition for preventing and treating diabetes, comprising ENPP7 peptide as an active ingredient.
상기 ENPP7 펩타이드는 서열번호 1 또는 서열번호 2로 기재되는 아미노산 서열과 90% 이상의 상동성을 가진 아미노산 서열을 포함하는 것을 특징으로 하는, 약학적 조성물.
According to clause 9,
The ENPP7 peptide is a pharmaceutical composition, characterized in that it contains an amino acid sequence with more than 90% homology to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
A health functional food composition for preventing or improving diabetes, comprising ENPP7 peptide as an active ingredient.
(2) 단계 1)의 처리된 세포주에서 상기 ENPP7 mRNA 또는 단백질의 발현량을 측정하는 단계;
(3) 단계 2)의 ENPP7 mRNA 또는 단백질의 발현량이 후보물질을 무처리한 대조군 세포주와 비교하여 증가된 후보물질을 선별하는 단계;
를 포함하는 것을 특징으로 하는, 당뇨병 치료제 후보물질의 스크리닝 방법.(1) Processing the candidate material into an isolated cell line expressing ENPP7;
(2) measuring the expression level of ENPP7 mRNA or protein in the cell line treated in step 1);
(3) selecting a candidate material whose expression level of ENPP7 mRNA or protein in step 2) is increased compared to a control cell line that was not treated with the candidate material;
A method for screening diabetes treatment candidates, comprising:
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| KR1020220088696A KR20240012623A (en) | 2022-07-19 | 2022-07-19 | Use of biomarker ENPP7 for diagnosis and treatment of Diabetes |
| KR1020240078297A KR20240109228A (en) | 2022-07-19 | 2024-06-17 | Use of biomarker ENPP7 for diagnosis and treatment of Diabetes |
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| KR1020220088696A KR20240012623A (en) | 2022-07-19 | 2022-07-19 | Use of biomarker ENPP7 for diagnosis and treatment of Diabetes |
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