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 los procedimientos con animales se realizaron de acuerdo con las directrices aprobadas por el Comité Institucional de Cuidado y Uso de Animales de la Universidad de Northwestern (IACUC). 1. Los modelos de ratones <ol><li> Utilice los ratones 8-12 semanas de edad, en la práctica de ejercicio. Para detectar la autofagia inducida ejercido in vivo, utilizar los ratones transgénicos GFP-LC3 (C57BL / 6 antecedentes) para estudios de imagen y ratones C57BL / 6 para los análisis bioquímicos. </li><...

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La autofagia es un proceso catabólico que proporciona energía y reduce la citotoxicidad por la degradación lisosomal de los componentes del citoplasma o orgánulos dañados. El estudio de la autofagia es importante comprender la regulación de la homeostasis celular y los mecanismos de respuesta al estrés. Nuevos modelos y metodologías están emergiendo en el campo de investigación de 15, para estudiar cómo la autofagia alterada contribuye a numerosos procesos patol...

La autofagia es una vía de degradación evolutivamente conservado, que se induce en respuesta a diversas condiciones de estrés, tales como el hambre y la hipoxia 1, 2. Durante la autofagia, las vesículas de doble membrana, llamados autofagosomas, incorporan componentes subcelulares innecesarias o dañadas y los transportan hacia los lisosomas para la degradación 3. Autofagia basal es esencial para la función celular y el desarrollo organismo, y alteración de la autofagia basal está implicado en muchos trastornos, incluyendo neurodegeneración, la tumorigénesis y la diabetes 4, 5, 6 tipo 2. El más conocido inductor de la autofagia fisiológica es el hambre. Sin embargo, tiene dos limitaciones importantes. En primer lugar, el hambre toma un largo período de inducir la autofagia con eficacia en animales, por ejemplo, 48 horas de restricción de alimentos en ratonesen la mayoría de órganos. En segundo lugar, el hambre apenas induce la autofagia cerebro, debido a un suministro de nutrientes relativamente estable en el cerebro. De hecho, también es difícil de detectar la inducción de la autofagia por inductores de molécula pequeña, como muchos fármacos no pueden pasar la barrera hematoencefálica. Por lo tanto, para analizar mejor la función de activación de la autofagia en la patogénesis de enfermedades, recientemente hemos descubierto que el ejercicio es un método fisiológico más potente para inducir la autofagia en un corto período de tiempo 7, 8, 9. En comparación con el hambre, la autofagia se induce de manera efectiva mediante la ejecución de cinta de correr tan rápido como 30 minutos. Por lo tanto, el ejercicio es un enfoque fisiológico conveniente y potente para estudiar el mecanismo de la autofagia en la mediación de beneficios para la salud y la prevención de enfermedades. Hay varios marcadores de proteínas para la detección de la actividad de la autofagia, incluyendo LC3 y p62. LC3 (proteína asociada a los microtúbulos 1A / 1B-c luzhain 3) es una proteína citosólica (LC3-I de forma) que está conjugado con PE (fosfatidiletanolamina) a la inducción de la autofagia. LC3 lipidada-PE (forma LC3-II) es reclutado en las membranas de autophagosomal y se puede utilizar para visualizar autofagosomas cuando están marcados con GFP. Su translocación desde el citosol a punctata estructuras de autofagosomas bajo microscopía es una indicación de la inducción de la autofagia. p62 es un receptor de carga para sustratos autofagia (tales como proteínas de ubiquitinación), y se incorpora en autofagosomas también. Dado que la proteína se degrada en los lisosomas, junto con autofagosomas, sus niveles se pueden utilizar para medir el flujo la autofagia. Aquí mostramos cómo utilizar estos marcadores para cuantificar la autofagia en diferentes tejidos de ratón inducidos por el ejercicio aeróbico, incluyendo ejercicio forzado (cinta) y el ejercicio voluntario (con ruedas de rodamiento). Los mismos procedimientos se pueden aplicar también a la medición in vivo de la autofagia después del tratamiento de otros inductores.

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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>
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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.

Este protocolo describe dos métodos diferentes para inducir la autofagia en tejidos de ratón de ejercicio aeróbico: un total de 90 minutos de ejercicio en una cinta rodante forzada de varios carriles precedida por dos días de aclimatación; o dos semanas de ejercicio voluntario en una rueda de funcionamiento utilizado por los ratones alojados individualmente. En cada protocolo de ejercicio, podemos medir el flujo autofagia mediante microscopía de fluorescencia y análisis de Western...

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

Activating Autophagy by Aerobic Exercise in Mice

activación autofagia es beneficioso en la prevención de una serie de enfermedades. Uno de los enfoques fisiológicos para inducir la autofagia in vivo es el ejercicio físico. Aquí mostramos cómo activar la autofagia mediante el ejercicio aeróbico y medir los niveles de autofagia en ratones.

La activación de la autofagia mediante ejercicio aeróbico en ratones

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

Research

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

Bloque de agar-microcosmos de descomposición controlada de Tejidos Vegetales de hongos aerobios

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

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Determining the Contribution of the Energy Systems During Exercise

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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

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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

Estudio de las interacciones proteína-proteína en la autofagia investigación

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

Tinción Inmunofluorescente In Situ de Autofagia en Células Madre Musculares

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

El ensayo de secuestro del lactato deshidrogenasa, Es un método Simple y confiable para determinar la actividad de autofagia secuestro a granel en células de mamíferos

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

Medición de la absorción de glucosa estimulada por insulina y contracción en músculo esquelético maduro aislado e incubado de ratones

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 ...