Metabolic profiling of mice brains and its application = 쥐 뇌의 대사체 프로파일링 및 그 응용

다국어 초록 (Multilingual Abstract)

High-fat diet causes the balance of energy homeostasis to be altered throughout the body and specifically causes inflammation of the brain. This reaction can cause brain damage and induce obesity. From previous studies, adipose tissue, plasma, urine, and liver from mice have been used for metabolite profiling during changes in energy intake, but the metabolic state has not been assessed in the brain. In the present experiments, brains from normal mice were used to establish the method for assessing brain metabolites and to identify the metabolites normally found in this tissue. After the method was established, brains from mice that were fed a high-fat diet for 4 weeks were used to identify the compounds that occur during inflammation and obesity. Also, brains of mice fasted for 24 hours were used to identify metabolites that potentially contribute to the regulation of energy metabolism in the brain. Gas chromatography time-of-flight mass spectrometry (GC-TOFMS) and a gas chromatography-flame ionization detector (GC-FID) were used to detect hydrophilic compounds and fatty acids from brain samples. Furthermore, to visualize differences in high-fat, low-fat, and fasting conditions, principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used. As a result, after short-term high-fat diet, C18:2 was increased in mouse brains. It is known that unsaturated fatty acids act as anti-inflammatory agents whereas saturated fatty acids induce inflammation in high-fat diets. In fasting mouse brains, 3-hydroxybutyric acid, which is generated when fat breaks down in the liver and with increased energy usage, was increased. Furthermore, it regulates food intake by sending signals to the hypothalamic area of the brain. In conclusion, C18:2 can act as an anti-inflammatory agent in the brain of obese mice. Furthermore, 3-hydroxybutyric acid can act as an energy source and regulate food intake in the brain during fasting conditions. These results provide a rationale for further studies on anti-inflammatory responses and regulation of the effects of feeding behavior on the brain.

국문 초록 (Abstract)

고지방 식단을 사용하면 에너지 항상성의 균형이 깨져 뇌의 염증이 발생한다. 이 반응은 뇌 손상을 일으켜 비만을 유발할 수 있다. 이전 연구에서 생쥐의 지방 조직, 혈장, 소변 및 간은 에너지 변화를 통해 대사 산물을 측정하는 데 사용되었지만 뇌를 사용한 사례는 없었다. 이번 실험에서는 일반 쥐 뇌를 사용하여 대사 성분을 검출하고, 뇌 손상을 주는 성분을 알기 위해 4주간 단기간 고지방식 먹이를 주어 비만을 유도한 생쥐를 사용했다. 반대로 뇌에서 에너지 대사의 조절에 잠재적으로 기여하는 대사 물질을 확인하기 위해 24 시간 절식한 생쥐의 뇌를 사용했다. 이번 실험에서 gas chromatography time-of-flight mass spectrometry (GC-TOFMS)과 gas chromatography flame ionization detector (GC-FID)를 사용 하여 대사 물질을 분석 하였다. 그리고 다변량 분석법인 주성분 분석 (PCA)과 부분 최소 제곱 회귀분석(PLS-DA)을 통해 고지방과 저지방의 차이, 절식한 쥐와 일반 사료를 먹인 쥐의 성분 차이를 시각화 하였다. 그 결과, 단기간에 비만이 유도된 쥐 뇌에서 C18:2가 증가하였다. 고지방식이로 인해 포화지방산이 염증을 유도하면, 불포화지방산이 항 염증반응 역할을 한다고 알려져 있다. 절식한 뇌에선 3-하이드로부티르산이 증가하고 젖산은 감소했다. 24시간을 굶겼을 때, 케톤 성분이 분비되는데, 이는 간에서 지방을 분해시켜 에너지로 사용할 때 나오는 성분이다. 또한 케톤 성분은 음식 섭취를 조절해주는 신호를 뇌로 전달하는 역할을 한다. 이러한 연구를 통해 고지방식이를 한 모델에서 C18:2가 많이 나오는 것을 알 수 있었고, 절식한 모델에서 케톤 성분이 분비 되는 것을 추측할 수 있었다.

목차 (Table of Contents)

• General Introduction 1
• Chapter 1. Quantification and identification of the mice brain composition by using GC-TOF/MS and GC-FID 2
• 1.1. Introduction 3
• 1.2. Materials and Methods 5
• 1.2.1. Materials 5
• 1.2.2. Mouse sample preparation 5
• 1.2.3. Quantification of amino acids 5
• 1.2.4. Quantification of fatty acids 6
• 1.3. Results and Discussion 8
• 1.3.1. Profiling of hydrophilic compounds using GC-TOFMS 8
• 1.3.2. Profiling of fatty acids using GC-FID 12
• Chapter 2. Metabolites profiling of mouse brain tissue following metabolic energy changes 16
• 2.1. Introduction 17
• 2.2. Materials and Methods 19
• 2.2.1. Animals 19
• 2.2.2. Feeding studies 19
• 2.2.3. Extraction and analysis of hydrophilic compounds 23
• 2.2.4. Tissue lipid extraction and analysis 24
• 2.2.5. Statistic analysis 24
• 2.3. Results and Discussion 26
• 2.3.1. Metabolic profiling of hydrophilic compounds and fatty acids in high fat diet mice brain 26
• 2.3.2. Principal component analysis of brains from fed high fat and low fat diets 31
• 2.3.3. Partial least-squares discriminant analysis in brains from high fat diet and low fat diet treated mice 35
• 2.3.4. Metabolic profiling of hydrophilic compounds and fatty acids in fasting mice brain 37
• 2.3.5. Principal component analysis in control and fasting mice brain 40
• 2.3.6. Partial least-squares discriminant analysis in control and fasting mice brain 42
• Reference 46
• 국문초록 50
• 감사의 글 52