Name: activating autophagy by aerobic exercise in mice
Uploaded: 2017-02-03T14:00:00
Description: Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

We thank the Northwestern University Mouse Histology and Phenotyping Laboratoryfor technical support and assistance, and Noboru Mizushima (University of Tokyo) for providing GFP-LC3 transgenic mice. A. R. and C. H. were supported by the startup funds from Northwestern University and the grant from National Institutes of Health (DK094980).

동물과 관련된 모든 절차는 노스 웨스턴 대학 기관 동물 관리 및 사용위원회 (IACUC)의 승인 지침에 따라 수행 하였다. 1. 마우스 모델 <ol><li> 운동 훈련에 8~12주 된 마우스를 사용합니다. 생체 내에서 행사에 의한 자식 작용을 감지하기 위해 영상 검사, 생화학 적 분석을위한 C57BL / 6 마우스의 GFP-LC3 형질 전환 마우스 (C57BL / 6 배경)를 사용합니다. </li></ol> 2. 운동에 의한 ...

<ol>
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자식 작용은 에너지를 제공하고 세포질 구성 요소 나 손상된 세포 소기관의 리소좀 저하에 의해 세포 독성을 감소시키는 이화 과정이다. 자식 작용을 공부하는 것은 세포의 항상성의 조절 및 스트레스 반응의 메커니즘을 이해하는 것이 중요합니다. 새로운 모델과 방법론은 여러 병리학 적 과정 16, 17에 기여하는 방법을 손상 자식 작용 연구, 연구 분야 ...

자식 작용은 기아 저산소증 1, 2와 같은 다양한 스트레스 조건에 응답하여 유도 된 진화 적 보존 열화 경로이다. 자식 작용하는 동안 두 번 막 소포는, 불필요하거나 손상된 세포 내 구성 요소를 통합하고 분해 3 리소좀로 수송, autophagosomes을했다. 기저 자식 작용은 신경 퇴행, 종양 및 2 형 당뇨병 4, 5, 6을 포함하여 많은 질환에 연루되어 세포 기능 및 유기체 개발 및 손상된 기초 자식 작용을 위해 필수적이다. 가장 잘 알려진 생리 자식 작용의 유도는 기아이다. 그러나, 두 가지 제한 사항이 있습니다. 첫째, 기아는, 예를 들면, 효과적으로 동물에서 자식 작용을 유도하는 쥐의 음식 제한의 48 시간을 오랜 기간 소요대부분의 기관입니다. 둘째, 기아는 거의 때문에 뇌에서 상대적으로 안정적인 영양 공급, 뇌 자식 작용을 유도한다. 사실, 많은 약물이 혈액 뇌 장벽을 통과 할 수 없기 때문에, 저분자 유도제에 의해 자식 작용 유도를 검출하는 것이 곤란하다. 따라서, 더 좋은 질병 발병 자식 작용 활성의 기능을 분석하기 위해, 최근 운동 시간 7, 8, 9 단기간에 자식 작용을 유도하는보다 강력한 생리 방법임을 발견했다. 기아와 비교할 때, 자식 작용을 효과적으로 디딜 방아 빠른 30 분을 실행하여 유도된다. 따라서, 운동은 의료 혜택을 매개 질병을 예방 자식 작용의 메커니즘을 연구하는 편리하고 강력한 생리 학적 접근 방식이다. LC3 및 P62을 포함 자식 작용 활성의 검출을위한 여러 단백질 마커가있다. LC3 (미세 소관 - 관련 단백질 1A / 1B-빛 Chain 3) 세포질 단백질이다 (LC3-I는 자식 작용의 유도에 따라 PE (포스파티딜 에탄올 아민)에 결합된다)을 형성. PE-LC3 지질 화 (LC3-II 양식) autophagosomal 막에 채용하고 GFP로 표지 때 autophagosomes을 시각화 할 수있다. 현미경에서 autophagosomes의 구조를 반점하는 세포질에서의 전위는 자식 작용 유도의 표시입니다. P62 (예 유비퀴틴 단백질 등) 자식 작용 기판 용화물 수용체이고,도 autophagosomes에 포함된다. 단백질이 autophagosomes 함께 리소좀에서 분해되기 때문에, 그 농도는 자식 작용의 플럭스를 측정하는데 사용될 수있다. 여기에서 우리는 강제 운동 (러닝 머신)과 자발적 운동 (바퀴를 실행)을 포함한 유산소 운동에 의해 유발 된 다른 마우스 조직에서 자식 작용을 정량화하기 위해 이러한 마커를 사용하는 방법을 보여줍니다. 동일한 절차가 다른 유도제의 투여 후 생체 내 자식 작용의 측정에 적용 할 수있다.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Treadmill</td>
			<td style="width:204px;">Columbus Instruments</td>
			<td style="width:195px;">150-RM Exer 3/6</td>
			<td style="width:217px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mouse running wheel</td>
			<td style="width:204px;">Super Pet</td>
			<td style="width:195px;">100079365</td>
			<td>diameter 11.4 cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Odometer</td>
			<td style="width:204px;">Bell</td>
			<td style="width:195px;">DASHBOARD 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Syringe pump</td>
			<td style="width:204px;">KD Scientific</td>
			<td style="width:195px;">KDS100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fluorescence microscope</td>
			<td style="width:204px;">Nikon</td>
			<td style="width:195px;">Model: inverted microscope ECLIPSE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Cryostat</td>
			<td style="width:204px;">Leica</td>
			<td style="width:195px;">CM 1850UV</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Homogenizer</td>
			<td style="width:204px;">IKA</td>
			<td style="width:195px;">003737001 / Model: T10 Basic S1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Chloroquine</td>
			<td style="width:204px;">CAYMAN CHEMICAL COMPANY</td>
			<td style="width:195px;">14194</td>
			<td></td>
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			<td height="21" style="height:21px;width:247px;">Parafolmaldehye</td>
			<td style="width:204px;">SIGMA-ALDRICH</td>
			<td style="width:195px;">P6148</td>
			<td>Personal protection equipment required. This product may release formaldehyde gas, a chemical known to cause cancer</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Mounting media</td>
			<td style="width:204px;">Vector Laboratories</td>
			<td style="width:195px;">H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">p62 antibody</td>
			<td style="width:204px;">BD Biosciences</td>
			<td style="width:195px;">610833</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">LC3 antibody</td>
			<td style="width:204px;">Novus Biologicals</td>
			<td style="width:195px;">NB100-2220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2X Laemmli Sample Buffer</td>
			<td style="width:204px;">Bio-Rad Laboratories</td>
			<td style="width:195px;">161-0737</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">ImageJ</td>
			<td style="width:204px;">NIH</td>
			<td style="width:195px;"></td>
			<td></td>
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activating autophagy by aerobic exercise in mice

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced and targets various cargos, such as bulk cytosol, damaged organelles and misfolded proteins, for degradation in lysosomes. Resulting nutrient molecules are recycled back to the cytosol for new protein synthesis and ATP production. Upregulation of autophagy has beneficial effects against the pathogenesis of many diseases, and pharmacological and physiological strategies to activate autophagy have been reported. Aerobic exercise is recently identified as an efficient autophagy inducer in multiple organs in mice, including muscle, liver, heart and brain. Here we show procedures to induce autophagy in vivo by either forced treadmill exercise or voluntary wheel running. We also demonstrate microscopic and biochemical methods to quantitatively analyze autophagy levels in mouse tissues, using the marker proteins LC3 and p62 that are transported to and degraded in lysosomes along with autophagosomes.

이 프로토콜은 유산소 운동으로 마우스 조직에서 자식 작용을 유도하는 두 가지 방법을 설명합니다 순응의 두 일까지 진행 다중 차선 러닝 머신에 강제 운동의 90 분의 총; 또는 단일 보관 마우스를 사용하는 실행중인 휠에 자발적 운동 2 주. 각각의 운동 프로토콜에서, 우리는 다양한 기관에서 형광 현미경 및 웨스턴 블롯 분석에 의한 자식 작용 플럭스를 측정 할 수 있습?...

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Activating Autophagy by Aerobic Exercise in Mice

자식 작용의 활성화는 질병의 숫자의 예방에 유용하다. 생리 학적 접근 방법 중 하나는 생체 내에서 자식 작용은 운동이다 유도한다. 여기에서 우리는 유산소 운동으로 자식 작용을 활성화하고 마우스에서 자식 작용의 수준을 측정하는 방법을 보여줍니다.

마우스에 유산소 운동에 의해 자식 작용을 활성화

Autophagy is a lysosomal degradation pathway essential for cell homeostasis, function and differentiation. Under stress conditions, autophagy is induced ...

Research

JoVE Journal

Biology

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

에어로빅 곰팡이에 의해 제어 플랜트 조직 분해에 대한 한천 - 블럭 Microcosms

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

연구

생물학

Determining the Contribution of the Energy Systems During Exercise

운동하는 동안 에너지 시스템의 공헌을 결정

One of the most important aspects of the metabolic demand is the relative contribution of the energy systems to the total energy required for a given ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

마우스의 수력 학적 유전자 전달에 의한 단백질의 일시적 발현

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

Analysis of Autophagy in Penicillium chrysogenum by Using Starvation Pads in Combination With Fluorescence Microscopy

자식 작용의 분석에<em&gt; 페니 chrysogenum</em&gt; 형광 현미경과 조합 기아 패드를 사용하여

The study of cellular quality control systems has emerged as a highly dynamic and relevant field of contemporary research. It has become clear that cells ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Study of Protein-protein Interactions in Autophagy Research

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

근육 줄기 세포에서 Autophagy의 원위치 Immunofluorescent 얼룩

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

The Lactate Dehydrogenase Sequestration Assay ÃƒÆ’Ã‚Â¢ÃƒÂ¢Ã¢â‚¬Å¡Ã‚Â¬ÃƒÂ¢Ã¢â€šÂ¬Ã‚Â A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

젖 산 효소 격리 분석 결과-간단 하 고 신뢰할 수 있는 결정 하는 방법을 포유류 세포에서 대량 Autophagic 격리 활동

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

마우스로부터 단리되고 배양된 성숙한 골격근에서의 인슐린- 및 수축-자극된 글루코스 흡수의 측정

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...