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Effect of C. sporogenes-derived metabolites on the circadian rhythm of Bmal1 and Period2 expression

Effect of C. sporogenes-derived metabolites on the circadian rhythm of Bmal1 and Period2 expression
Translated Title
C. sporogenes 유래 대사산물이 생체시계 유전자 Bmal1 과 Period2 발현의 일주기 리듬에 미치는 영향
Kyojin Ku
DGIST Authors
Choe, Han Kyoung; Ku, Kyojin; Kim, Kyungjin
Han Kyoung Choe
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Circadian rhythm is an endogenous oscillation with an approximately 24-hour period in many physiological processes and behaviors. This daily oscillation is maintained by the molecular clock machinery with transcriptional-translational feedback loops mediated by clock genes including Bmal1 and Period2 (Per2). Recently, gut microbiome has been revealed to exert a significant impact on the circadian physiology and behaviors of its host. However, the ques-tion of how gut microbiome regulates the molecular clock remains unexplored. 3-(4-Hydroxyphenyl)propionic acid (4-OH-PPA) and 3-phenylpropionic acid (PPA) are major me-tabolites exclusively produced by Clostridium sporogenes (C. sporogenes), one of the most abundant genus ‘Clostridium’ in the small intestine and may function as unique chemical mes-sengers communicating with its host’s physiology. By using a real-time bioluminescence re-cording device, I examined whether C. sporogenes-derived metabolites can modulate the cir-cadian oscillation of mammalian molecular clock genes in Per2::Luc knockin mouse embry-onic fibroblast (MEF) and Bmal1:Luc MEF. Interestingly, 4-OH-PPA and PPA increased the amplitude of luciferase oscillation in both Per2::Luc and Bmal1:Luc in a dose-dependent manner when treated immediately after the nadir or the peak of their oscillatory rhythm. Be-sides, 4-OH-PPA increased the expression of Bmal1 in the transcript level. In addition, using ex vivo cultures of the suprachiasmatic nucleus (SCN), hippocampus, liver, and small intestine derived from Per2::Luc knockin mice, I demonstrated 4-OH-PPA increased the amplitude of Per2 circadian expression in the hippocampus, liver, and small intestine with a tendency to increase the amplitude of Per2 oscillation in the SCN, the MASTER clock. The period of Per2 oscillation was not changed in the organotypic cultures in contrast to that in MEFs. In sum-mary, C. sporogenes-derived metabolites can regulate the circadian rhythm by increasing the amplitude of both Bmal1 and Per2 oscillation.|일주기 리듬은 많은 생리 현상 및 행동에서 나타나는 약 24시간 주기의 내재성 리듬이다. 생체리듬은 Bmal1 및 Period2 (Per2)를 포함한 생체시계 유전자들이 형성하는 전사-번역 되먹임 조절 작용에 의해 유지된다. 최근 장내 미생물이 숙주의 일주기적 생리 및 행동에 중요한 영향을 미치는 것으로 밝혀졌지만, 어떠한 기작으로 분자 시계를 조절하는지는 알려져 있지 않다. 소장에 가장 많이 존재하는 장내 미생물 속(屬) 중 하나인 Clostridium에 속하는 Clostridium sporogenes (C. sporogenes)에 의해 생산되는 주 대사산물인 4-hydroxyl-phenylpropionic acid (4-OH-PPA), 3-phenylpropionic acid (PPA)는 숙주와 소통하는 화학적 매개자로 작용할 가능성이 있다. 본 연구에서는Per2::Luc혹은 Bmal1:Luc 이 knockin된 형질전환 쥐의 배아섬유아세포를 이용하여 C. sprorogenes 유래 대사산물이 포유류의 분자 시계의 일주기 진동을 조절할 수 있는지를 실시간 생체 인광 측정 기법으로 분석하였다. 4-OH-PPA와 PPA가 일주기 리듬의 최고점과 최저점 직후에 처리되었을 때, 농도 의존적으로 Bmal1과 Per2 일주기 발현의 진폭을 증가시켰고, 역전사 중합효소 연쇄반응을 통하여 분석한 결과 Bmal1의 발현은 4-OH-PPA 처리에 의해 증가하였다. 또한, Per2::Luc knockin 형질전환 쥐의 시교차상핵 (SCN), 해마, 간 및 소장의 체외 배양을 통하여 4-OH-PPA을 처리하였을 때, Per2 진동의 진폭을 상승시키는 경향을 보였다. 따라서, 본 연구를 통하여 C. sporogenes 유래 대사산물이 Bmal1과 Per2의 일주기 진동의 진폭을 증가시킴으로써 일주기 리듬을 변화시킬 수 있음을 시사하였다.
Table Of Contents
Ⅰ. INTRODUCTION 1 Ⅱ. MATERIALS AND METHODS 4 2-1. Cell culture 4 2-2. Organotypic primary slice culture 4 2-3. Real-time recording of bioluminescence in vitro and ex vivo 5 2-4. Reverse transcription quantitative polymerase chain reaction 6 2-5. Statistical analysis 7 Ⅲ. RESULTS 8 3-1. Effect of 4-OH-PPA and PPA on real-time Per2 and Bmal1 expression profiles in vitro 8 3-2. Effect of 4-OH-PPA on Per2 and Bmal1 mRNA level using reverse transcription quantitative polymerase chain reaction 10 3-3. Effect of 4-OH-PPA on circadian oscillation of Per2 gene in organotypic cultures of SCN, hippocampus, liver and small intestine ex vivo 11 Ⅳ. DISCUSSION 31 Ⅴ. REFERENCES 34
Brain and Cognitive Sciences
Related Researcher
  • Author Choe, Han Kyoung Laboratory of Animal Behavior and Circadian rhythm
  • Research Interests Modulation of neural circuit; Circadian regulation of behavior and perception; Neurotechnology
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Department of Brain and Cognitive SciencesThesesMaster

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