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).

Todos os procedimentos envolvendo animais foram realizados de acordo com as diretrizes aprovadas pelo Comitê Animal Care Institucional e Use da Universidade Northwestern (IACUC). 1. mouse Models <ol><li> Use ratos 8-12 semanas de idade no treinamento físico. Para detectar autofagia induzida exercida in vivo, os ratinhos transgénicos usar GFP-LC3 (C57BL / 6, fundo) para estudos de imagiologia e murganhos C57BL / 6 para as análises bioquímicas. </li></ol> 2. Autophagy induzida...

<ol>
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A autofagia é um processo catabólico que fornece energia e reduz a citotoxicidade pela degradação lisossomal de componentes citoplasma ou organelas danificadas. Estudar a autofagia é importante compreender a regulação da homeostase celular e os mecanismos de resposta ao estresse. Novos modelos e metodologias estão a surgir no domínio da investigação 15, para estudar como autofagia deficiente contribui para inúmeros processos patológicos 16, <sup cla...

Autophagy é uma via de degradação evolutivamente conservada, a qual é induzida em resposta a diversas condições de stress tais como hipoxia fome e 1, 2. Durante a autofagia, vesículas de dupla membrana, chamada autofagossomas, incorporar componentes subcelulares desnecessários ou danificados e transportá-los para lisossomos para degradação 3. Autofagia basal é essencial para a função celular e desenvolvimento organismo, e autofagia basal prejudicada está implicada em muitas doenças, incluindo a neurodegeneração, tumorigénese e diabetes 4, 5, 6 do tipo 2. O indutor autofagia fisiológico mais conhecido é fome. No entanto, tem duas limitações principais. Em primeiro lugar, a fome leva um longo período para induzir eficazmente a autofagia em animais, por exemplo, 48 horas de restrição alimentar em ratosna maioria dos órgãos. Em segundo lugar, apenas induz inanição autofagia cérebro, devido a um fornecimento de nutrientes relativamente estável no cérebro. Na verdade, também é difícil de detectar indução autofagia por indutores de pequenas moléculas, como muitas drogas não pode passar a barreira hemato-encefálica. Assim, para analisar melhor a função de activação autofagia na patogênese da doença, descobrimos recentemente que o exercício é um método fisiológico mais potente para induzir autofagia num curto período de tempo de 7, 8, 9. Em comparação com a fome, a autofagia é efetivamente induzido pela esteira correndo tão rápido quanto 30 min. Assim, o exercício é uma abordagem conveniente e potente fisiológico para estudar o mecanismo de autofagia na mediação de benefícios para a saúde e prevenção de doenças. Existem vários marcadores proteicos para a detecção de actividade autofagia, incluindo LC3 e p62. LC3 (associada a microtúbulos de proteína 1A / 1B-luz cHain 3) é uma proteína citosólica (LC3-I formam) que é conjugado com PE (fosfatidiletanolamina) após indução autofagia. LC3 PE-lipidada (forma LC3-II) é recrutado para membranas autophagosomal e pode ser usado para visualizar autofagossomas quando marcado com GFP. A sua translocação para o citosol a partir de estruturas puntiformes de autofagossomas sob microscopia é uma indicação da indução autofagia. p62 é um receptor de carga para substratos autophagy (tais como proteínas de ubiquitinação), e é incorporado autofagossomas bem. Uma vez que a proteína é degradada em lisossomas, juntamente com autofagossomas, os seus níveis podem ser usadas para medir o fluxo de autofagia. Aqui nós mostramos como utilizar esses marcadores para quantificar a autofagia em diferentes tecidos de camundongos induzida por exercício aeróbico, incluindo exercício forçado (esteira) e exercício voluntário (rodas de correr). Os mesmos procedimentos podem também ser aplicados para a medição in vivo de autofagia após o tratamento de outros indutores.

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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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<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>
		</tr>
	</tbody>
</table>

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.

Este protocolo descreve dois métodos diferentes para induzir a autofagia em tecidos de camundongos por exercício aeróbico: um total de 90 min de exercício forçado em uma esteira multi-lane passou por dois dias de aclimatação; ou duas semanas de exercício voluntário em uma roda de corrida usados ​​por camundongos alojados individualmente. Em cada protocolo de exercício, podemos medir o fluxo autofagia por microscopia de fluorescência e análise western blot em vários órg?...

Watch this Scientific Journal Video about Activating Autophagy by Aerobic Exercise in Mice at JoVE.com

Activating Autophagy by Aerobic Exercise in Mice

Autophagy activação é benéfico na prevenção de uma série de doenças. Uma das abordagens fisiológicas para induzir autofagia in vivo é o exercício físico. Aqui nós mostramos como ativar a autofagia pelo exercício aeróbico e medir os níveis de autofagia em camundongos.

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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

Medição da absorção de glicose estimulada por insulina e contração em músculo esquelético maduro isolado e incubado de camundongos

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 ...